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Mutation in VMAT2 Causing a Pediatric Neurotransmitter Disease
by
Jennifer Rilstone
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Institute of Medical Science University of Toronto
© Copyright by Jennifer Rilstone 2015
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Mutation in VMAT2 Causing a Pediatric Neurotransmitter Disease
Jennifer Rilstone
Doctor of Philosophy
Institute of Medical Science University of Toronto
2015
Abstract
This thesis describes a new disease encompassing infantile-onset movement disorder (including
severe parkinsonism and nonambulation), mood disturbance, autonomic instability, and
developmental delay that was identified in eight cousins of a consanguineous Bedouin family in
Saudi Arabia. The autosomal recessive disease was hypothesized to be a disorder of monoamine
neurotransmission, and evidence supporting its causation by a mutation in SLC18A2 (which
encodes the vesicular monoamine transporter 2 [VMAT2]) was acquired by single nucleotide
polymorphism (SNP) genotyping, homozygosity analysis, and exome sequencing. VMAT2
translocates dopamine and serotonin into synaptic vesicles and is essential for motor control,
stable mood, and autonomic function. The loss of VMAT2 function was further confirmed
through biochemical assay of serotonin uptake. Consistent with a defect in vesicular monoamine
transport, treatment of the patients with levodopa was associated with worsening, whereas
treatment with direct dopamine agonists was followed by immediate ambulation, near-complete
correction of the movement disorder, and resumption of development. Understanding the
underlying mechanism of this disorder extends the spectrum of known pediatric neurotransmitter
diseases, serves as the first demonstration of mutation in VMAT2 causing a human phenotype,
and thereby provides new insight into the role of VMAT2 in monoamine homeostasis. This
thesis additionally demonstrates the utility of implementing genomic diagnosis in the clinic, with
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respect to providing simple and effective treatments in a timely manner to improve outcomes for
patients with rare inborn errors of metabolism.
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Acknowledgments I would first like to thank my supervisor, Dr. Berge Minassian, for his consistent support
throughout this degree, and more importantly—infinite patience. He taught me to think foremost
about the patients and their families, and that perspective motivated me when the work itself was
a struggle.
I would also like to thank the members of my advisory committee, Dr. Lucy Osborne and Dr.
Stephen Scherer, for timely strategic guidance and their support—particularly with the decision
to submit to NEJM and the decision to write.
I would additionally like to thank the members of the Minassian laboratory. Thank you to Peter
Wang and Xiaochu Zhao for their incredible technical expertise. I’d also like to thank the
summer students who worked with me over the years for giving me the opportunity to teach and
for demonstrating enthusiasm for this project: Tarek Abdelhalim, John Bilbily, Ari Damla, and
Alex Bilbily.
Support and encouragement through completion of this thesis were appreciated, with particular
thanks due to all those who routinely asked me whether I was done yet.
Finally, I’d like to thank my parents, David and Karen Rilstone, for being unendingly supportive
through a nonlinear path to a place in which I’m settled and happy. I’d also like to thank Katie
Mercer, mostly for existing, also for being curious and driven and inspiring me through example
to pursue new opportunities with confidence.
This work was supported by a Vanier Canada Graduate Scholarship from the Natural Sciences
and Engineering Research Council of Canada (NSERC).
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Contributions The intellectual content of this thesis, including the planning and direction of experiments, are
attributable to the author.
In Chapter 2, clinical characterization of the disease was performed in collaboration with Dr.
Reem Alkhater. As described throughout the text of the chapter, Dr. Alkhater identified this
family in clinical practice, performed neurological examinations and coordinated clinical
investigations (including the collection of urine and cerebrospinal fluid), collected written
informed consent for participation in the study, and collected blood samples from members of
the extended family for genetic analyses. Dr. Alkhater maintained contact with the family and
their physicians, providing treatment guidance and obtaining follow-up information. Ethical
approval for the study, including blood collection, was sought by the author. Genetic analyses of
candidate genes for pediatric neurotransmitter disorder were performed by the author.
Interpretations of CSF and urine analyses and the underlying pathophysiology of the disorder
were determined through discussions among Dr. Alkhater, Dr. Berge Minassian, and the author.
In Chapter 3, all genetic investigations were conceived and coordinated by the author. As
described in the methods section of the chapter, single nucleotide polymorphism analysis was
contracted through the University of Helsinki, and whole exome sequencing was performed by
The Center for Advanced Genomics at the Hospital for Sick Children. All mutation screening,
linkage and homozygosity analyses, and analyses of exome data were performed by the author,
as well as database searches and bioinformatic analyses of the described variant.
In Chapter 4, all functional analyses were conceived and performed by the author.
Acknowledgement is due to Dr. Robert Edwards and members of his laboratory for providing
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guidance in performing the serotonin uptake assays, some of which were completed by the
author in a visit to his laboratory.
In Chapter 5, all future experiments have been conceived and planned by the author. Cloning
steps and the construction of cell lines described in this chapter were performed by the author,
with gratitude to Xiaochu Zhao for troubleshooting and completing the final step of the mouse
construct, and to the aforementioned summer students for assistance with cell culture
maintenance and PCR.
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Table of Contents
Acknowledgments ................................................................................................................................ iv
Contributions .......................................................................................................................................... v
Table of Contents .................................................................................................................................vii
List of Tables .......................................................................................................................................... xi
List of Figures ...................................................................................................................................... xiii
List of Abbreviations ...................................................................................................................... xviii
Chapter 1 Literature Review ............................................................................................................. 1
1 Movement Disorder and Monoamine Deficiency ................................................................ 1
1.1 Pathophysiology ..................................................................................................................................... 1 1.2 The Biogenic Amines ............................................................................................................................ 4 1.3 Parkinson’s Disease .............................................................................................................................. 9 1.4 Pediatric Neurotransmitter Diseases ........................................................................................... 10
1.4.1 GTP Cyclohydrase I (GTPCH1) Deficiency ......................................................................................... 12 1.4.2 Sepiapterin Reductase (SR) Deficiency .................................................................................................. 15 1.4.3 6-Pyruvoyl Tetrahydropterin Synthase (PTPS) Deficiency ............................................................. 15 1.4.4 Dihydropteridine Reductase (DHPR) Deficiency ............................................................................... 16 1.4.5 Pterin-4α-Carbinolamine (PCD) Deficiency ......................................................................................... 16 1.4.6 Tyrosine Hydroxylase (TH) Deficiency ................................................................................................. 17 1.4.7 Aromatic Amino Acid Decarboxylase (AADC) Deficiency ............................................................ 18 1.4.8 Dopamine Transporter Deficiency Syndrome (DTDS) ..................................................................... 18 1.4.9 Dopamine β-Hydroxylase Deficiency ..................................................................................................... 19 1.4.10 Secondary neurotransmitter disorders and related diseases ........................................................... 19 1.4.11 Phenotypic Spectrum of Pediatric Neurotransmitter Disorders ................................................... 20 1.4.12 Diagnosis of Monoamine Deficiencies ................................................................................................ 21
2 Vesicular Monoamine Transporters ..................................................................................... 23
2.1 VMAT Isolation ..................................................................................................................................... 24 2.2 VMAT Structure .................................................................................................................................... 24 2.3 VMAT Biochemistry ............................................................................................................................ 25 2.4 Role of VMAT2 in Biogenic Amine Physiology .......................................................................... 27
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2.4.1 Vmat2 Knockout Mice ................................................................................................................................ 28 2.4.2 Vmat2 Heterozygous Mice ....................................................................................................................... 29 2.4.3 Constitutive Overexpression of Vmat2 in Mice ............................................................................... 30 2.4.4 Selective Deletion of Vmat2 in Serotonergic Neurons .................................................................. 31 2.4.5 Selective Expression of Vmat2 in Noradrenergic Neurons ........................................................ 32
2.5 Role of VMAT2 in Disease Processes ............................................................................................ 33 2.5.1 VMAT2 and Parkinson’s Disease ........................................................................................................... 33 2.5.2 VMAT2, Neuropsychiatric Phenotypes, and Drugs of Abuse ..................................................... 36 2.5.3 Genetic Variation in VMAT2 .................................................................................................................... 38
3 Thesis Overview ........................................................................................................................... 39
Chapter 2 Clinical Characterization of a New Pediatric Neurotransmitter Disease ... 42
1 Introduction................................................................................................................................... 42
2 Methods ........................................................................................................................................... 43
2.1 Patients .................................................................................................................................................... 43 2.2 General Investigations ....................................................................................................................... 43 2.3 Clinical Measurement of Neurotransmitter Metabolites ...................................................... 43 2.4 AADC Enzyme Test .............................................................................................................................. 44 2.5 Candidate Gene Sequencing ............................................................................................................. 44
3 Results ............................................................................................................................................. 48
3.1 Patient History and Neurological Examination ........................................................................ 48 3.2 Investigations ........................................................................................................................................ 49 3.3 Family Structure .................................................................................................................................. 53 3.4 Genetic Screening of Candidate Genes ......................................................................................... 55 3.5 Drug Response ...................................................................................................................................... 56 3.6 Summary of Clinical Features and Differential Diagnosis .................................................... 59
4 Discussion....................................................................................................................................... 63
Chapter 3 Identification of Disease-Causing Genetic Variant ............................................. 69
1 Introduction................................................................................................................................... 69
2 Methods ........................................................................................................................................... 70
2.1 Patient Samples .................................................................................................................................... 70 2.2 Genotyping of Single Nucleotide Polymorphisms .................................................................... 70
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2.3 Homozygosity Analysis ...................................................................................................................... 70 2.4 Linkage Analysis .................................................................................................................................. 71 2.5 Mutation Screening ............................................................................................................................. 72 2.6 Whole-Exome Sequencing ................................................................................................................ 75 2.7 TaqMan SNP Genotyping Assay ...................................................................................................... 76
3 Results ............................................................................................................................................. 76
3.1 Homozygosity Analysis ...................................................................................................................... 76 3.2 Linkage Analysis .................................................................................................................................. 79 3.3 Characterization of Genes in the Locus ........................................................................................ 82 3.4 Whole-Exome Sequencing of the Proband .................................................................................. 86 3.5 Identification of VMAT2 Variant as a Causative Candidate .................................................. 86 3.6 Controls ................................................................................................................................................... 89
4 Discussion....................................................................................................................................... 89
Chapter 4 Functional Characterization of the Disease-Causing Variant ......................... 95
1 Introduction................................................................................................................................... 95
2 Methods ........................................................................................................................................... 95 2.1 Construct Design and Site-Directed Mutagenesis .................................................................... 95 2.2 Cell Culture and Transfection.......................................................................................................... 96 2.3 Serotonin Uptake Assay..................................................................................................................... 97 2.4 Western Blotting .................................................................................................................................. 97 2.5 Sucrose Gradient Centrifugation .................................................................................................... 98
3 Results ........................................................................................................................................... 101
3.1 Steady-State Protein Levels of Transfected Protein Unaffected by P387L Variant in
Heterologous System .................................................................................................................................. 101 3.2 Highly Reduced Transport Activity of VMAT2 p.P387L in Heterologous System ...... 104 3.3 Measurable Residual Transport Activity of VMAT2 p.P387L Protein ........................... 107 3.4 Subcellular Localization of Protein in Heterologous System ............................................ 109
4 Discussion..................................................................................................................................... 111
Chapter 5 General Discussion and Future Directions .......................................................... 113
1 Introduction................................................................................................................................. 113
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2 Identification of the VMAT2 p.P387L Mutation: Impact on Knowledge of
Monoamine Physiology .................................................................................................................. 114
2.1 Future Directions and Experiments ........................................................................................... 116 2.1.1 Subcellular Localization of VMAT2 p.P387L ................................................................................. 116 2.1.2 Additional Biochemical Characterization of VMAT2 p.P387L................................................ 120 2.1.3 Development of a Mouse Model Expression Vmat2 p.P390L ................................................. 121
3 VMAT2 Mutation in PND: Impact on Understanding of PND Pathogenesis ........... 135 3.1 Future Directions and Experiments ........................................................................................... 136
3.1.1 18F-DOPA and 11C-DTBZ PET scans .................................................................................................... 136 3.1.2 Screening of Patient Cohorts to Identify Novel SLC18A2 Variants ...................................... 137
4 VMAT2 Mutation: Impact on Approaches to PND Diagnosis ...................................... 138
4.1 Future Directions and Experiments ........................................................................................... 139 4.1.1 Development of Methodology to Measure Biogenic Amines Directly in CSF ................... 139 4.1.2 Development of VMAT2 Platelet Activity Assay .......................................................................... 140 4.1.3 Use of Next-Generation Sequencing for Genetic Diagnosis of Rare Monogenic Diseases
141 4.1.4 Knowledge Translation Challenges and Strategies for PND Diagnosis .............................. 145
5 Concluding Statement .............................................................................................................. 149
References ........................................................................................................................................... 150
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List of Tables Table 1 Causative genes and modes of inheritance for monoamine neurotransmitter diseases 11
Table 2 Metabolites of biogenic amine neurotransmitters and pterin profiles in the
cerebrospinal fluid of patients with pediatric neurotransmitter diseases ...................................... 23
Table 3 Primer sequences for the amplification of exons of candidate genes associated with
known pediatric neurotransmitter diseases ................................................................................... 45
Table 4 Serum metabolic screen revealed no abnormalities ...................................................... 49
Table 5 Cerebrospinal fluid and urine neurotransmitters and their metabolites measured in a
younger affected sibling of the proband reveal decreased monoamines and elevated metabolites
in urine, but not in cerebrospinal fluid. Values outside reference ranges are presented in bold. . 52
Table 6 Pediatric neurotransmitter disease–associated genes screened in the proband revealing
no putative mutations .................................................................................................................... 56
Table 7 Age at initation of dopamine agonist affects disease course ........................................ 58
Table 8 Clinical features common to all affected individuals in the pedigree ........................... 59
Table 9 Clinical features of the disease organized by category ................................................. 60
Table 10 Comparison of clinical features of the present disease with those of closely related
monoamine deficiency syndromes ................................................................................................ 62
Table 11 Comparison of metabolite profiles in cerebrospinal fluid for pediatric
neurotransmitter diseases, including VMAT2 deficiency ............................................................ 65
Table 12 Primer sequences for amplification of exons of candidate genes located at 10q26 ... 73
Table 13 Primer sequences for amplification of SLC18A2 exons ............................................. 75
Table 14 Genes of known neuronal function or localization present in the disease-associated
bomozygous region ....................................................................................................................... 83
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Table 15 Sequence variants identified among eight candidate genes in the disease-associated
locus by direct sequencing of exons ............................................................................................. 85
Table 16 Primers to amplify SLC18A2 cDNA sequence for subcloning into pcDNA3.1 ......... 96
Table 17 Oligonucleotides to introduce cytosine to thymine substitution at position 1160 of
SLC18A2 using site-directed mutagenesis .................................................................................... 96
Table 18 Primers for the integration of the HA epitope at Arg94 of VMAT2 using site-direted
mutagenesis ................................................................................................................................. 119
Table 19 Primer sequences to amplify a probe for Slc18a2 exon 13 in mouse ....................... 130
Table 20 Primer sequences to amplify the upstream and downstream arms of the gene-targeting
construct 130
Table 21 Oligonucleotide sequences for site-directed mutagenesis of gene-targeting construct
to introduce the p.P390L variant in the expressed protein .......................................................... 130
Table 22 Sequencing primer sequences for gene-targeting construct ..................................... 130
Table 23 Primer sequences for generating labelled probes to identify the neomycin selection
cassette by Southern blot ............................................................................................................ 131
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List of Figures Figure 1 Schematic diagram of the direct and proposed indirect pathways of the basal ganglia;
(a) direct/striatonigral pathway, (b) indirect/striatopallidal pathways. The overall effect on motor
thalamus of pathways in (b) are equivalent, and opposite in sign to (a). ........................................ 3
Figure 2 Monoaminergic neurotransmission exists as a balance of several critical processes:
biosynthesis, packaging, release, degradation, and reuptake. VMAT2, vesicular monoamine
transporter 2. ................................................................................................................................... 7
Figure 3 Common and parallel pathways for the synthesis and degradation of biogenic amines.
AADC, aromatic amino acid decarboxylase; BH4, tetrahydrobiopterin cofactor; COMT,
catechol-O-methyltransferase;; DAT, plasma membrane dopamine transporter;; DβH, dopamine β-
hydroxylase; 5-HIAA, 5-hydroxyindoleacetic acid; HVA, homovanillic acid; 5-HTP, 5-
hydroxytryptophan; MAO, monoamine oxidase; MHPG, 3-methoxy-4-hydroxyphenylglycol;
3-OMD, 3-O-methyldopa; qBH2, quinonoid dihydrobiopterin; PNMT, phenylethanolamine
N-methyltransferase; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase; VMA,
vanillylmandelic acid; VMAT2, vesicular monoamine transporter 2. ........................................... 8
Figure 4 Synthesis and regeneration of tetrahydrobiopterin—the conversion of BH4 to PCBD is
coupled to the generation of dopamine or serotonin. AR, aldose reductase; BH4,
tetrahydrobiopterin; DHPR, dihydropteridine reductase; GTPCH, GTP cyclohydrolase 1; H2NP3,
dihydroneopterin triphosphate; PCBD, tetrahydrobiopterin-α-carbinolamine; PCD, pterin-4α-
carbinolamine dehydratase; 6-PTP, 6-pyruvoyltetrahydropterin; qBH2, quinonoid
dihydrobiopterin; PTPS, 6-pyruvoyltetrahydropterin synthase; SP, sepiapterin; SR, sepiapterin
reductase. 14
Figure 5 T2-weighted magnetic resonance images of proband at age 14 revealed no
abnormalities. ................................................................................................................................ 50
Figure 6 Family structure of kindred demonstrates autosomal recessive mode of inheritance
and consanguineous pedigree structure. Proband is labelled V:6. Cerebrospinal fluid and urine
neurotransmitter analyses were performed on individual V:10. Black, affected; white,
unaffected; square, male; circle, female; diamond, spontaneous abortion. Numbers in squares or
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circles indicate number of male or female offspring, respectively. From New England Journal of
Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular
Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical
Society. Reprinted with permission. ............................................................................................ 54
Figure 7 Single nucleotide polymorphism alleles for all genotyped family members in the
homozygous region uniquely shared by affected family members. Black, affected; white,
unaffected; square, male; circle, female. From New England Journal of Medicine, Rilstone JJ,
Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its
Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with
permission. .................................................................................................................................... 78
Figure 8 Multipoint LOD scores estimated across chromosome 10 ............................................. 80
Figure 9 Multipoint LOD scores estimated across chromosomes 3 and 16 ................................. 81
Figure 10 Genes present within the homozygous region shared by all affected individuals in the
family. C10orf82, chromosome 10 open reading frame 82; CACUL 1, CDK2-assocaited cullin
domain 1; CASC2, cancer susceptibility candidate 2 (non-protein coding); CCDC172, coiled-coil
domain containing 172; EIF3A, eukaryotic translation factor 3 subunit A; EMX2, empty
spiracles homeobox 2; ENO4, enolase family member 4; FAM204A, family with sequence
similarity 204 member A; GFRA1, GDNF family receptor alpha 1; GRK5, G protein–coupled
kinase 5; HSPA12A, heat shock 70 kDa protein 12A; KCNK18, potassium channel subfamily K
member 18; KIAA1598, Shootin1; MIR, microRNA; PDZD8, PDZ domain containing 8;
PNLIP, pancreatic lipase; PNLIPRP, pancreatic lipase–related protein; PRDX3, peroxiredoxin 3;
PRLHR, prolactin-releasing hormone receptor; RAB11FIP1, RAB11 family interacting protein 2
(class I); SFXN4, sideroflexin 4; SLC18A2, solute carrier family 18 member 2; SNORA19,
small nucleolar RNA H/ACA box 19; VAX1, ventral anterior homeobox 1. Image from UCSC
Genome Browser [http://genome.ucsc.edu] .................................................................................. 84
Figure 11 Electropherogram depicting sequence variation in unaffected and affected family
members. Top panel, unaffected sibling with wild-type genomic sequence; middle panel,
asymptomatic parent (IV:3) possesses the SLC18A2 c.1160C→T variant in heterozygous form;;
lower panel, proband (V:6) is homozygous for SLC18A2 c.1160C→T. From New England
xv
Journal of Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin
Vesicular Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts
Medical Society. Reprinted with permission. .............................................................................. 88
Figure 12 Predicted structure of VMAT2 comprises 12 transmembrane domains, a large
lumenal loop including four proposed glycosylation sites, and both N-terminal and C-terminal
cytoplasmic regions. Proline residue 387 is located immediately adjacent to the insertion of
transmembrane domain X. From New England Journal of Medicine, Rilstone JJ, Alkhater RA,
Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368,
543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission. .......... 93
Figure 13 Multiple sequence alignment of VMAT2 in the region of the p.P387L variant. TM9
and TM10 represent sequences associated with transmembrane domains 9 and 10, respectively.
Residues that differ from human VMAT2 sequence are indicated in gray. Amino acid position
387 is indicated by an asterisk. Homo, homo sapiens (human); Patient, proband; Pan, Pan
troglodytes (chimpanzee); Macaca, Macaca mulatta (Rhesus macaque); Mus, Mus musculus
(mouse); Rattus, Rattus norvegicus (rat); Canis, Canis familiaris (dog); Bos, Bos taurus (cow);
Monodelphis, Monodelphis domestica (opossum); Gallus, Gallus gallus (chicken); Tetraodon,
Tetraodon nigroviridis (pufferfish); Danio, Danio rario (zebrafish); Drosophila, Drosophila
melanogaster (fruit fly); C. elegans, Caenorhabditis elegans (nematode); hVMAT1, human
VMAT1 isoform. From New England Journal of Medicine, Rilstone JJ, Alkhater RA,
Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368,
543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission. .......... 94
Figure 14 Visualization of ATP by absorbance at 260 nm to illustrate sucrose gradient
linearity 99
Figure 15 Consistent fractionation demonstrated by absorbance at 280 nm of parallel sucrose
gradient fractionations ................................................................................................................ 100
Figure 16 Markers of cellular compartments in sucrose gradient fractions ............................. 101
Figure 17 Parallel transient transfections of wild-type and p.P387L VMAT2 in Cos7 cells, and
vector-transfected control. GAPDH, protein loading control. .................................................... 102
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Figure 18 Comparison of banding pattern observed for wild-type and p.P387L VMAT2
expressed transiently in Cos7 cells. unt, untransfected; WT, wild-type. .................................... 104
Figure 19 Vesicular uptake of tritiated serotonin by the wild-type and p.P387L human VMAT2
transiently expressed in a heterologous Cos7 cell system. Inset: Expression of VMAT2 in lysates
used for uptake assay. 5-HT, serotonin; wt, wild-type. From New England Journal of Medicine,
Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport
Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society.
Reprinted with permission. ......................................................................................................... 106
Figure 20 Vesicular uptake of tritiated serotonin by wild-type and p.P387L human VMAT2
transiently expressed in a heterologous Cos7 cell system after 10 minutes with and without the
addition of the specific VMAT inhibitor reserpine (10 µM). From New England Journal of
Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular
Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical
Society. Reprinted with permission. .......................................................................................... 108
Figure 21 Sucrose gradient centrifugation of transiently transfected Cos7 lysates ................. 110
Figure 22 Expression of wild-type and p.P387L VMAT2 in stable clones of the Cos7 cell line;
band at 37 kDa represents GAPDH loading control. .................................................................. 118
Figure 23 Expression of wild-type and p.P387L VMAT2 in stable clones of the MN9D cell line
120
Figure 24 Schematic of incorporation of mouse gene targeting construct into mouse genome.
Upper panel, wild type mouse genomic sequence of Slc18a2 exons 11–15 and flanking intronic
sequences. Middle panel, mouse genomic sequence after incorporation of the gene-targeting
construct by homologous recombination with the long arms of the construct in embryonic stem
cells. Slc18a2 c.1169C→T mutation, coding for the P390L amino acid substitution, is present in
exon 13. Lower panel, mouse genomic sequence after expression of Cre recombinase and
excision of the selection cassette. PGKneobpA, neomycin selection cassette; loxP, recombination
sites targeted by Cre recombinase. .............................................................................................. 125
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Figure 25 Gene targeting construct for the introduction of the c.1169C→T mutation into
Slc18a2. AmpR, ampicillin resistance gene; F1_origin, origin of replication; loxP, recombination
site for Cre recombinase; NeoR/KanR, neomycin and kanamycin resistance gene (of the
PGKneobpA selection cassette); NheI, HindIII, restriction sites for insertion of downstream arm
of genomic sequence for recombination; NotI, SacII, restriction sites for insertion of upstream
arm of genomic sequence for recombination. Gray boxes, mouse intronic sequence of Slc18a2;
black boxes, mouse exonic sequence of Slc18a2. ....................................................................... 127
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List of Abbreviations
AADC aromatic amino acid decarboxylase
AR aldose reductase
ATP adenosine triphosphate
BH4 tetrahydrobiopterin
CHO Chinese hamster ovary
CIHR Canadian Institutes of Health Research
COMT catechol-O-methyltransferase
CP cerebral palsy
CSF cerebrospinal fluid
DA dopamine
DAT plasma membrane dopamine transporter
DβH dopamine β-hydroxylase
DDC encodes AADC
DHPR dihydropteridine reductase
DMEM Dulbecco’s modified Eagle medium
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DOPAC 3,4-dihydroxyphenylacetic acid
DRD dopa-responsive dystonia
DTBZ dihydrotetrabenazine
DTDS dopamine transporter deficiency syndrome
E epinephrine
EEG electroencephalogram
ELISA enzyme-linked immunosorbant assay
ER endoplasmic reticulum
ES embryonic stem
GAPDH glyceraldehyde 3-phosphate dehydrogenase
GPi globus pallidus internus
GCH1 encodes GTPCH1
GTP guanosine-5'-triphosphate
GTPCH1 GTP cyclohydrolase I
5-HIAA 5-hydroxyindoleacetic acid
HIS histamine
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H2NP3 dihydroneopterin triphosphate
HPLC-EC high-performance liquid chromatography with electrochemical detection
5-HT serotonin
5-HTP 5-hydroxytryptophan
HVA homovanillic acid
kDa kilodalton
LC locus coerulus
LC-MS/MS liquid chromatography tandem mass spectrometry
LDCV large dense-core vesicles
LOD logarithm of odds
MAO monoamine oxidase
MFS major facilitator superfamily
MHPG 3-methoxy-4-hydroxyphenylglycol
MPP+ 1-methyl-4-phenylpyridinium; active metabolite of MPTP
MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
MRI magnetic resonance imaging
xxi
NBIA neurodegeneration with brain iron accumulation
NE norepinephrine
NHLBI National Heart, Lung, and Blood Institute
3-OMD 3-O-methyldopa
PBS phosphate-buffered saline
PCBD tetrahydrobiopterin-α-carbinolamine
PCBD encodes PCD
PCD pterin-4α-carbinolamine dehydratase
PD Parkinson’s disease
PET positron emission tomography
PKU phenylketonuria
PLP pyridoxal phosphate
PND pediatric neurotransmitter disorder
PNMT phenylethanolamine N-methyltransferase
PNPO pyridoxamine 5’-phosphate oxidase
6-PTP 6-pyruvoyltetrahydropterin
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PTPS 6-pyruvoyl tetrahydropterin synthase
PTS encodes PTPS
PVDF polyvinylidene fluoride
qBH2 quinonoid dihydrobiopterin
QDPR encodes DHPR
SCA spinocerebellar ataxia
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SERT serotonin plasma membrane transporter
SLC6A3 encodes DAT
SLC18A2 encodes VMAT2
SNP single nucleotide polymorphism
SNpc substantia nigra pars compacta
SNR substantia nigra pars reticulata
SP sepiapterin
SPR encodes SR
SR sepiapterin reductase
xxiii
SSV small synaptic vesicles
TBS-T 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20
TfR transferrin receptor
TH tyrosine hydroxylase
TMD transmembrane domain
TPH tryptophan hydroxylase
VAChT vesicular acetylcholine transporter
VMA vanillylmandelic acid
VMAT vesicular monoamine transporter
VTA ventral tegmental area
1
Chapter 1 Literature Review
1 Movement Disorder and Monoamine Deficiency Movement disorders are a large group of diseases in which patients suffer impairment of
the planning, control or execution of movement (Klein, 2005). The clinical spectrum of
movement disorders includes, but is not limited to, ataxia, blepharospasm, dysphonia,
dystonic disorders, gait disorders, Huntington’s disease, myoclonus, Parkinson’s disease,
spasticity, tardive dyskinesia, tics and Tourette syndrome, and tremor (Klein, 2005).
They are clinically, pathologically, and genetically heterogeneous. Genes for many are
already known (Klein, 2005). Despite this, the pathogenic mechanisms of the vast
majority of these movement disorders are still being elucidated.
For example, the prototypical movement disorder, Parkinson’s disease, is characterized
by neurodegeneration of the dopamine-secreting neurons of the substantia nigra pars
compacta (SNpc), and a deficit of dopamine in the brain (see Section 1.3). This results in
bradykinesia (slowing of physical movement), rigidity, postural instability, and rest
tremor. The molecular mechanisms leading to this pathology are still unclear despite the
discovery of 11 distinct genes causing monogenic forms of the disease, as well as a
number of associations with additional genes and loci associated with sporadic forms of
the disease (Singleton et al., 2013).
1.1 Pathophysiology The underlying pathophysiology of Parkinson’s disease and related movement
disorders involves misregulation of a series of structures comprising the basal ganglia.
2
The basal ganglia function in the fine modulation of motor activity. Input from the cortex
is received by the striatum (input nucleus), which in turn regulates the output nuclei—the
substantia nigra pars reticula (SNR) and globus pallidus internus (GPi). The output
nuclei tonically inhibit the motor thalamus, and therefore motor activity. By current
models, this occurs through the structures of the basal ganglia via two possible
pathways—the direct (striatonigral) or indirect (striatopallidal) pathway (Figure 1)
(Miller, 2008). Inhibitory striatal neurons can be identified as members of either of these
two pathways by the presence of peptide markers (substance P or enkephalin,
respectively).
3
Figure 1 Schematic diagram of the direct and proposed indirect pathways of the basal ganglia; (a) direct/striatonigral pathway, (b) indirect/striatopallidal pathways. The overall effect on motor thalamus of pathways in (b) are equivalent, and opposite in sign to (a).
4
The role of dopamine in the basal ganglia is in regulation of the striatum. Dopaminergic
neurons originate in the SNpc, and project their axons to innervate and regulate the
striatum via D1- and D2-class receptors. At physiological concentrations, dopamine is
sequestered in vesicles of dopaminergic neurons and undergoes regulated exocytotic
release at the synapse. However, it has been demonstrated that at higher, pathological
concentrations, unsequestered cytosolic dopamine can cause toxicity with the formation
of reactive oxygen species generated by dopamine auto-oxidation or monoamine oxidase
(MAO) metabolism (Cubells et al., 1994, Graham, 1978, Hastings et al., 1996). This
natural dopamine toxicity is a suggested mechanism underlying the progressive
nigrostriatal neurodegeneration in Parkinson’s disease (see Section 1.3).
1.2 The Biogenic Amines The biogenic amines (also referred to as monoamines) are a class of neurotransmitter that
are synthesized enzymatically from amino acids. Serotonin is derived from tryptophan,
and is the basis of the serotonergic system. The catecholamines (dopamine,
norepinephrine, and epinephrine) are derived from tyrosine. Dopamine is the basis of the
dopaminergic system, and norepinephrine and epinephrine underlie the adrenergic
system. Histamine is derived from histidine, and is present in neurons of the
hypothalamus. The biogenic amines are modulatory in nature (acting on a timescale of
seconds to minutes), have mixed effects, and are subject to significant crosstalk.
The dopaminergic system comprises the nigrostriatal pathway that projects from the
SNpc to the striatum (caudate and putamen), and the mesolimbic and mesocortical
projections from the ventral tegmental area (VTA) to the nucleus accumbens and cerebral
5
cortex, respectively. There are also dopaminergic projections comprising the
tuberoinfundibular pathway.
The serotonergic system comprises widespread projections from the dorsal raphe nuclei
to many regions of the brain, including the neocortex, amygdala, hippocampus, thalamus,
hypothalamus, striatum, cerebellum, brain stem, and spinal cord.
The noradrenergic system also involves widespread projections from the locus coeruleus
to the neocortex, amygdala, hippocampus, thalamus, hypothalamus, tectum, cerebellar
cortex, visceral cranial nuclei, and spinal cord. Both norepinephrine and epinephrine also
function peripherally in sympathetic ganglion cells.
Histamine is present in the hypothalamus, where it plays an integral role in
neurometabolic functions (e.g., wakefulness, appetite regulation, response to pain, and
immunological response).
Monoaminergic neurotransmission exists as a balance of several critical processes:
biosynthesis, packaging, release, degradation, and reuptake. First, transmitters are
synthesized from their amino acid precursors in the cytosol. Next, they are accumulated
into synaptic vesicles through the action of transporters that are driven by the proton
gradient. Further biochemical transformation occurs within the synaptic vesicle in the
case of norepinephrine. Neurotransmission occurs by way of regulated exocytotic release
from the synaptic vesicles into the synaptic cleft in response to physiologic stimuli. At
the synapse, the transmitter interacts with target receptors on the postsynaptic cell to
mediate signal transduction. Transmitter at the synapse is then cleared by both
degradation and reuptake processes. Specific monoamine metabolizing enzymes are
6
present to inactivate the transmitter. Reuptake into the presynaptic cell and glia occurs
through plasma membrane monoamine transporters that are driven by Na+ and Cl–
gradients. This model of monoamine neurotransmission and homeostasis is illustrated in
Figure 2. The biochemical pathways for the synthesis and degradation of the biogenic
amines are presented in Figure 3.
7
Figure 2 Monoaminergic neurotransmission exists as a balance of several critical processes: biosynthesis, packaging, release, degradation, and reuptake. VMAT2, vesicular monoamine transporter 2.
8
Figure 3 Common and parallel pathways for the synthesis and degradation of biogenic amines. AADC, aromatic amino acid decarboxylase; BH4, tetrahydrobiopterin cofactor; COMT, catechol-O-methyltransferase; DAT, plasma membrane dopamine transporter;; DβH, dopamine β-hydroxylase; 5-HIAA, 5-hydroxyindoleacetic acid; HVA, homovanillic acid; 5-HTP, 5-hydroxytryptophan; MAO, monoamine oxidase; MHPG, 3-methoxy-4-hydroxyphenylglycol; 3-OMD, 3-O-methyldopa; qBH2, quinonoid dihydrobiopterin; PNMT, phenylethanolamine N-methyltransferase; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase; VMA, vanillylmandelic acid; VMAT2, vesicular monoamine transporter 2.
9
1.3 Parkinson’s Disease Parkinson’s disease (PD) is a common, adult-onset neurodegenerative disease
characterized by the selective and progressive loss of neuronal subtypes—in particular,
the nigrostriatal dopaminergic pathway. PD is primarily characterized by the
deterioration of motor function, evidenced as bradykinesia, rigidity, postural instability,
and rest tremor. In addition, there has been increased recognition of the nonmotor
manifestations of PD. These include anosmia, depression, anxiety, sweating, dyspnea,
orthostatic hypotension, constipation, pain, genitourinary problems, sexual dysfunction,
and sleep disorders (Witjas et al., 2002). These nonmotor symptoms may be grouped
into domains: cognitive and psychiatric, autonomic, and sensory/pain. Many of the
nonmotor manifestations of PD precede the motor symptoms of the disease, and certainly
emerge upon disease progression. Furthermore, the nonmotor symptoms of PD are
reported to have a greater impact on patient quality of life (Barone et al., 2009).
Evidence of the degeneration of other monoaminergic neuronal types (serotonergic
neurons in the raphe nuclei and noradrenergic neurons in the locus coeruleus), as well as
cholinergic neurons in the basal forebrain and midbrain tegmentum, establish a broader
monoaminergic deficiency phenotype in PD (Hirsch et al., 2003).
The etiology and pathogenesis of PD have been the subject of considerable study and
reflect significant complexity. PD is primarily sporadic, although multiple monogenic
forms of the disease exist (Singleton et al., 2013). Familial and sporadic forms of the
disease have nearly identical motor phenotypes. Furthermore, toxins such as 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) can selectively destroy dopaminergic neurons
and acutely cause parkinsonism—providing evidence that environmental exposures may
10
play a role in the pathogenesis of PD. Notably, the strongest risk factor for PD is
advancing age, possibly implicating the aging process in PD pathogenesis.
Current thinking regarding PD pathogenesis favors a mixture of pathogenic mechanisms.
Multiple avenues of investigation have identified common pathways that may underlie
the disease. These include oxidative stress, protein aggregation, defects in the ubiquitin–
proteasome pathway, autophagy, and alterations in mitochondrial function. The
pathologic hallmark of PD is Lewy bodies—inclusion bodies comprising α-synuclein and
other protein deposits—and there has been some evidence of cell-to-cell transfer of these
deposits that may be associated with the spread of the disease to other neuronal types
(Steiner et al., 2011).
1.4 Pediatric Neurotransmitter Diseases The pediatric neurotransmitter diseases, by contrast, are a group of early onset rare
diseases attributable to a disturbance in neurotransmitter metabolism with causative
defects in enzymes that are involved in the biosynthesis, degradation, or membrane
transport of transmitter. Known disorders specifically of the biogenic amine
neuromediators (dopamine, norepinephrine, epinephrine, and serotonin) comprise defects
in nine enzymes and one transporter—each presenting in early childhood with symptoms
and signs referable to the affected neurotransmitter. Deficiency in dopamine results in
movement disorder, including parkinsonism and dystonia; deficient norepinephrine and
epinephrine cause sympathetic autonomic dysfunction; and serotonin deficiency leads to
sleep disturbance and psychiatric disease. The monoamine neurotransmitter disorders of
known genetic etiology are summarized in Table 1 Causative genes and modes of
inheritance for monoamine neurotransmitter diseases.
11
Table 1 Causative genes and modes of inheritance for monoamine neurotransmitter diseases
Disorder Causative Gene Mode of Inheritance Autosomal dominant GTP cyclohydrolase I deficiency (Dopa-responsive dystonia or Segawa disease)
GCH1 Autosomal dominant
Autosomal recessive GTP cyclohydrolase I deficiency
GCH1 Autosomal recessive
Sepiapterin reductase deficiency
SPR Autosomal recessive
6-Pyruvoyltetrahydropterin synthase deficiency
PTS Autosomal recessive
Dihydropteridine reductase deficiency
QDPR Autosomal recessive
Pterin-4α-carbinolamine dehydratase deficiency
PCD Autosomal recessive
Tyrosine hydroxylase deficiency
TH Autosomal recessive
Aromatic L-amino acid decarboxylase deficiency
DDC Autosomal recessive
Pyridoxal phosphate-dependent epilepsy (PLP-DE)
PNPO Autosomal recessive
Dopamine transporter deficiency syndrome
SLC6A3 Autosomal recessive
The multiple deficits of PNDs overlap substantially with the combination of
dopaminergic motor dysfunction and the domains of nonmotor symptoms in PD.
Similarly, evidence of degeneration of other monoaminergic neuronal types (serotonergic
neurons in the raphe nuclei and noradrenergic neurons in the locus coeruleus), as well as
cholinergic neurons in the basal forebrain and midbrain tegmentum have established the
involvement of multiple monoaminergic systems in PD (Hirsch et al., 2003). However,
whereas PND are disorders of infancy and childhood, PD is a disease of aging. It thus
12
reflects distinct underlying pathogenic mechanisms. Similarly, the manifestations of
PNDs are expected to differ from those of PD because the monoaminergic dysfunction
occurs during critical periods of neurological development.
1.4.1 GTP Cyclohydrase I (GTPCH1) Deficiency
The longest recognized monoamine neurotransmitter disorder is autosomal dominant
GTP cyclohydrolase I (GTPCH1) deficiency that was first described in the 1970s
(Segawa et al., 1976), and is more commonly referred to as dopa-responsive dystonia
(DRD) or Segawa disease. DRD results from a defect in tetrahydrobiopterin (BH4)
synthesis—which is a necessary cofactor for the rate-limiting step in dopamine synthesis
(tyrosine hydroxylase conversion of tyrosine to L-DOPA). These patients exhibit
dystonic spasms with diurnal fluctuation that begin in the first decade of life. The disease
is associated with short stature. Adult-onset cases have also been described, in which the
primary features are writer’s cramp, torticollis, or generalized rigid hypertonus with
tremor, but not postural dystonia (Segawa, 2011). The disorder is extremely responsive
to treatment with levodopa in combination with a decarboxylase inhibitor (carbidopa).
The disease is caused by heterozygous mutations in GCH1 at 14q22.1–q22.2 (Ichinose et
al., 1994). More than 100 mutations have been discovered. The autosomal dominant
mutations are dominant negative, as a result of an inability to properly form the native
homodecameric form of the protein. Less than 10% of identified mutations are found in
homozygous or compound heterozygous form, causing an autosomal recessive form of
the disease (Thony and Blau, 2006).
Several other disorders of tetrahydrobiopterin synthesis or regeneration present as
pediatric movement disorder, including defects in 6-pyruvoyl tetrahydropterin synthase
13
(PTPS; PTS), sepiapterin reductase (SR; SPR), dihydropteridine reductase (DHPR;
QDPR), and pterin-4α-carbinolamine dehydratase (PCD; PCBD) (Longo, 2009). The
pathway for tetrahydrobiopterin synthesis and regeneration is presented in Figure 4.
Tetrahydrobiopterin is an essential cofactor for tyrosine hydroxylase and tryptophan
hydroxylase, as well as phenylalanine hydroxylase, nitric oxide synthases, and glyceryl-
ether monooxygenase. Despite its necessity for phenylalanine hydroxylase activity,
fewer than 2% of patients with phenylketonuria have mutations in PTS, QDPR, GCH1, or
PCBD (Longo, 2009). Laboratory diagnosis of tetrahydrobiopterin deficiency is based
on newborn screening for phenylketonuria, urine or dried blood profiling of pterins, and
the measurement of DHPR enzyme activity in blood.
14
Figure 4 Synthesis and regeneration of tetrahydrobiopterin—the conversion of BH4 to PCBD is coupled to the generation of dopamine or serotonin. AR, aldose reductase; BH4, tetrahydrobiopterin; DHPR, dihydropteridine reductase; GTPCH, GTP cyclohydrolase 1; H2NP3, dihydroneopterin triphosphate; PCBD, tetrahydrobiopterin-α-carbinolamine; PCD, pterin-4α-carbinolamine dehydratase; 6-PTP, 6-pyruvoyltetrahydropterin; qBH2, quinonoid dihydrobiopterin; PTPS, 6-pyruvoyltetrahydropterin synthase; SP, sepiapterin; SR, sepiapterin reductase.
15
1.4.2 Sepiapterin Reductase (SR) Deficiency
SR deficiency, like GTPCH1 deficiency, is not associated with hyperphenylalaninemia
but phenylalanine loading tests are often positive. The disease is caused by mutations in
the SPR gene at 2p13, and at least 19 mutations have been identified (Thony and Blau,
2006). Symptoms arise in the first decade of life, including psychomotor retardation,
dystonia, oculogyric crises, choreoathetosis, hypotonia, spasticity, tremor, ataxia,
parkinsonism, seizures, temperature instability, hypersalivation, microcephaly, and
irritability, as well as psychiatric symptoms. These symptoms exhibit diurnal variation,
as with GTPCH1 deficiency. Alternatively, a mild form of the disease with mild motor
symptoms has been reported, associated with a splicing defect leading to a missense
mutation with residual protein function (Arrabal et al., 2011). Metabolite profiles in CSF
reveal a disturbance of dopamine, norepinephrine, and serotonin synthesis. SR activity
can be measured clinically in skin fibroblasts.
1.4.3 6-Pyruvoyl Tetrahydropterin Synthase (PTPS) Deficiency
PTPS deficiency is the most common disorder of BH4 metabolism (Leuzzi et al., 2010),
caused by mutations in the PTS gene at 11q22.3–23.3. More than 50 mutations have
been described (Thony and Blau, 2006). It exists as both severe and peripheral forms.
The severe form manifests in the first few months of life with delayed developmental
milestones and movement disorder, as well as hyperphenylalaninemia and risk of
premature birth and low birth weight. The peripheral form involves
hyperphenylalaninemia but minor or no disturbance in biogenic amine neurotransmitters,
as measured by their metabolites in CSF and a normal neurological course. Genotype–
phenotype correlation is evident in that mutations with high residual enzyme activity
16
cause the peripheral form of the disease, whereas mutations causing gross disturbances to
protein function (such as frameshift mutations leading to protein truncation or altered
protein zinc binding or oligomerization) lead to the severe form of the disease (Brasil et
al., 2011).
1.4.4 Dihydropteridine Reductase (DHPR) Deficiency
DHPR deficiency is the most severe of the disorders of pterin metabolism, caused by
mutations in the QDPR gene at 4p15.31. DHPR deficiency reflects a defect in the
regeneration of tetrahydrobiopterin. It has neonatal onset, or onset in early infancy.
Patients exhibit feeding difficulties, bulbar dysfunction, hypersalivation, and
microcephaly. They also exhibit delayed motor and cognitive milestones, truncal and
limb hypertonia, dyskinesia, tremor, dystonia, choreoathetosis, and seizures. These
patients are at risk of sudden death. There are often white matter abnormalities and basal
ganglia calcifications visible by MRI (Longhi et al., 1985, Woody et al., 1989). The
severity of this particular defect in biopterin metabolism is thought to be related to the
concomitant accumulation of q-dihydrobiopterin and its inhibitory effects on AADC,
tyrosine hydroxylase, tryptophan hydroxylase, and phenylalanine hydroxylase, as well as
depletion of folate in the brain related to the absence of DHPR and the accumulation of
q-dihydrobiopterin. A mild form of the disease affects only serotonin metabolism, and is
caused by two specific protein mutations (G151S and F212C) (Blau et al., 1992).
1.4.5 Pterin-4α-Carbinolamine (PCD) Deficiency
PCD deficiency is also a defect in the regeneration of tetrahydrobiopterin resulting from
mutations in PCBD at 10q22. It is associated with a mild hyperphenylalaninemia that
17
normalizes after a few months of life. There is usually no neurotransmitter phenotype,
but transient neonatal hypotonia has been reported in some patients (Thony et al., 1998).
1.4.6 Tyrosine Hydroxylase (TH) Deficiency
A deficiency of tyrosine hydroxylase is a progressive and lethal encephalopathy with
poor prognosis, involving dystonia and other extrapyramidal movement disorder
symptoms. It is a rare autosomal recessive disorder caused by mutations in TH at
11p15.5, and reported in less than 40 patients worldwide thus far. Importantly, mutations
reflecting a complete loss of function (such as premature truncations) have not been
observed in homozygous form, suggesting that a complete lack of tyrosine hydroxylase is
incompatible with life (Willemsen et al., 2010). There are two overlapping subtypes:
type A, a progressive extrapyramidal movement disorder (hypokinetic–rigid syndrome
with dystonia) with onset in infancy or childhood; and type B, a complex encephalopathy
with onset in the neonatal period or early infancy (Willemsen et al., 2010). Type A
comprises approximately 70% of patients who present in infancy with a parkinsonian
phenotype of hypokinesia, bradykinesia, dystonia, and rigidity. There can be diurnal
variation in the dystonia. An onset in the first year is associated with some mild
cognitive impairment. Some patients develop onset of symptoms later (within the first 5
years), with gait instability and difficulty walking. Type B disease has an onset within
the first 3 months of life with hypotonia and severe parkinsonism, in combination with
mental retardation, dystonia, oculogyric crises, myoclonus, tremor, dyskinesia, and
ptosis. Autonomic features (sweating, temperature instability, hyperpyrexia, and
drooling) are also observed. Seizures have also been reported in these patients. Notably,
18
MRI investigations are usually normal, though some white matter abnormalities and
increased extracerebral CSF spaces have been observed in more severe cases.
1.4.7 Aromatic Amino Acid Decarboxylase (AADC) Deficiency
AADC deficiency disorder, for which there have been less than 100 patients diagnosed
worldwide (Brun et al., 2010), results from mutations in the aromatic amino acid
decarboxylase gene (DDC; also known as dopa decarboxylase) at 7p12.3–12.1. These
patients present with hypotonia in the first six months of life, and further develop
extrapyramidal movement disorder symptoms including dystonia, chorea, blepharospasm,
bulbar dysfunction, and myoclonus. They further exhibit developmental delay,
irritability, sleep disturbances, autonomic manifestations, temperature instability,
irritability, and nasal congestion. Most patients exhibit cognitive impairment. AADC
activity can be assayed clinically in plasma. MRI is generally normal, although some
nonspecific changes have been reported (Brun et al., 2010). Patients generally do not
respond to treatment.
Briefly, pyridoxal phosphate is an essential cofactor for AADC. Deficiency in the
synthesis of this cofactor resulting from mutations in the gene encoding pyridoxamine 5’-
phosphate oxidase (PNPO at 17q21.32) leads to prenatal seizures an a severe, potentially
fatal, anticonvulsant-resistant neonatal encephalopathy (Mills et al., 2005). CSF
metabolite profiles mimic those of AADC deficiency.
1.4.8 Dopamine Transporter Deficiency Syndrome (DTDS)
Whereas the pediatric disorders described thus far reflect deficiencies in the synthesis of
one or more biogenic amine neurotransmitters, dopamine transporter (DAT) deficiency
19
syndrome (DTDS) is a defect in monoamine transport. Children with homozygous or
compound heterozygous mutations in SLC6A3 (coding for DAT) at 5p15.3 present with a
complex motor disorder in early infancy that comprises hyperkinetic symptoms,
hypokinesia, or a mixed phenotype that involves progressive dystonia, axial hypotonia,
and parkinsonism (Kurian et al., 2011b, Kurian et al., 2009). Eye movement disorder
(ocular flutter, saccade initiation failure, eyelid myoclonus, and oculogyric crises) is also
observed. Patients also exhibit sleeping difficulties, orthopedic complications, and
cardiorespiratory insufficiency. The mean life expectancy for patients with DTDS is 13.6
years.
Defects in presynaptic dopamine reuptake reduce presynaptic dopamine stores and an
accumulation of synaptic dopamine that is susceptible to catabolism. This is reflected by
increased HVA concentrations in the CSF. Increased synaptic dopamine may also lead to
downregulation of postsynaptic receptor expression (Blackstone, 2009).
1.4.9 Dopamine β-Hydroxylase Deficiency
Dopamine β-hydroxylase deficiency is characterized as a primary autonomic failure
stemming from the inability to convert dopamine to norepinephrine or epinephrine
(Senard and Rouet, 2006).
1.4.10 Secondary neurotransmitter disorders and related diseases
Dopamine and serotonin depletion can occur secondary to certain other neurological
disorders, including perinatal asphyxia, disorders of folate metabolism, phenylketonuria,
Lesch-Nyhan disease, mitochondrial disorders, epilepsy and infantile spasms,
20
opsoclonus-myoclonus, pontocerebellar hypoplasia, leukodystrophies, Rett’s syndrome,
and certain neuropsychiatric disorders (Kurian et al., 2011a).
Additionally, there are several other rare disorders that present in the first decade of life
and involve a combination of dystonia and parkinsonism complicated by pyramidal tract
involvement (Pearl, 2013, Schneider and Bhatia, 2010). These include Wilson’s disease;;
parkinsonism associated with mutations in parkin (PARK2), PINK1 (PARK6), and DJ1
(PARK7); x-linked dystonia-parkinsonism/Lubag (DYT3); rapid-onset dystonia-
parkinsonism (DYT12); DYT16 dystonia; neurodegeneration with brain iron
accumulation (NBIA, including PANK2 and PLA2G6/PARK14); neuroferritinopathy;
Kufor-Rakeb disease; SENDA syndrome; and autosomal recessive spastic paraplegia
with thin corpus collosum (SPG11).
1.4.11 Phenotypic Spectrum of Pediatric Neurotransmitter Disorders
A phenotypic continuum has been proposed for GTPCH1 deficiency and extended to all
pediatric monoamine biosynthetic deficiencies (see Table 3 in Pons, 2009). This
spectrum illustrates the variability in the presentation of pediatric neurotransmitters
disorders that reflects a range of severities stemming from a common underlying
pathophysiology. The severity of presentation depends upon the effect of the mutation on
enzyme function and the importance of the affected enzyme to biogenic amine
availability, on the extent of monoamine deficiency, on the relative involvement of each
of the biogenic amines, and on the age of onset and subsequent effect on neurological
development. Additional variability in presentation is observed between patients with
common defects.
21
1.4.12 Diagnosis of Monoamine Deficiencies
In addition to clinical signs, several tests may be useful to the diagnosis of pediatric
neurotransmitter disorder. Because tetrahydrobiopterin is also a cofactor for
phenylalanine hydroxylase, some monoamine deficiencies include peripheral
hyperphenylalanemia that may be identified in serum or in neonatal screening. These
include autosomal recessive GTP cyclohydrolase deficiency, pterin-carbinolamine
dehydratase deficiency, dihydropteridine reductase deficiency, and pyruvoyl-
tetrahydropterin synthase deficiency. In the absence of peripheral hyperphenylalanemia,
an oral phenylalanine loading test, in which the ratio of phenylalanine to tyrosine is
measured in serum after loading, may be useful to identify defects in tetrahydrobiopterin
metabolism (Bandmann et al., 2003, Opladen et al., 2010).
Genetic testing of associated genes is reasonably simple, but may not reveal noncoding
mutations with functional relevance, and the functional relevance of novel mutations may
be unclear. Enzyme activity tests are available for GTPCH activity in fibroblasts, SR
activity in fibroblasts, DHPR activity in blood spots, and AADC activity in plasma.
These tests are not considered routine, but may be useful in cases of negative genetic
screening.
Urine metabolite (HVA, 3-OMD, 5-HIAA, VMA; see Figure 3) and pterin profiles
(biopterin and neopterin; see Figure 4) may also aid diagnosis. However, as a
measurement of metabolites and pterins in the periphery, urine results are not conclusive.
An abnormal pterin profile in the urine may help distinguish diseases with peripheral
22
hyperphenylalanemia from phenylketonuria (PKU; phenylalanine hydroxylase
deficiency).
The most conclusive available diagnostic is CSF neurotransmitter metabolite and pterin
profiles (see Table 2). Metabolite levels are indirect measures of each neurotransmitter,
as well as an indication of turnover. There are, however, several difficulties with this
analysis that prohibit routine testing. CSF collection requires lumbar puncture, with
inherent risks. CSF neurotransmitter analysis must be performed in specialized
laboratories, of which there are few. CSF is labile, and special collection procedures are
necessary, including immediate centrifugation, snap freezing, and preservatives.
Metabolites exhibit diurnal variation, and there is a rostrocaudal gradient of metabolite
and pterin concentrations that requires precise labeling of collected fractions and careful
interpretation of results. In addition, there is variation in concentrations with age, so
reference ranges must be defined in age-matched controls.
23
Table 2 Metabolites of biogenic amine neurotransmitters and pterin profiles in the cerebrospinal fluid of patients with pediatric neurotransmitter diseases Affected Enzyme
HVA 5-HIAA HVA/5-HIAA MHPG 3-OMD Pterin profile
Disorders of BH4 synthesis (recessive)
decrease decrease normal decrease normal abnormal
GTP cyclohydrolase (dominant)
decrease normal normal normal normal abnormal
Tyrosine hydroxylase
decrease normal normal decrease normal normal
AADC decrease decrease normal decrease increase normal PNPO decrease decrease normal decrease increase normal Dopamine β-hydroxylase
increase normal normal decrease normal normal
Dopamine transporter (Kurian et al., 2009, Kurian et al., 2011b)
increase normal increase NR NR normal
AADC = L-aromatic amino acid decarboxylase; BH4 = tetrahydrobiopterin; GTP = guanosine-5'-triphosphate;; NR = not reported;; PNPO = pyridoxamine 5’-phosphate oxidase; Note: Data in table derived from Hyland 2008, except where otherwise noted.
2 Vesicular Monoamine Transporters The vesicular monoamine transporters are responsible for the efficient uptake of cytosolic
monoamines into storage vesicles. A 10,000-fold concentration of monoamines (up to
0.5 M) can be achieved by active transport. This transport against the concentration
gradient is driven by the transmembrane pH and electrochemical gradient generated by
the vesicular H+-ATPase in the synaptic membrane.
Two closely related vesicular monoamine transporters, VMAT1 and VMAT2, have been
characterized. The VMAT1 protein is primarily expressed in neuroendocrine cells,
including chromaffin and enterochromaffin cells, and localized to large dense core
24
vesicles. The VMAT2 protein is primarily expressed in monoaminergic neurons of the
central nervous system, as well as the sympathetic nervous system, mast cells, platelets,
β cells of the pancreas, and histaminergic cells of the gut. Both VMAT1 and VMAT2 are
expressed in chromaffin cells of the adrenal medulla (Erickson et al., 1996).
2.1 VMAT Isolation The VMATs were initially isolated and characterized from chromaffin granules.
The SLC18A2 gene encoding VMAT2 was initially identified from a PC12 cDNA library
for its ability to confer resistance to the neurotoxin MPP+ in MPP+-sensitive Chinese
hamster ovary (CHO) cells (Liu et al., 1992). MPTP (of which MPP+ is the active
metabolite) is a neurotoxin that, in humans and primates, as well as other mammals,
produces a very specific phenotype—parkinsonism characterized by oxidative damage
and progressive neurodegeneration in the substantia nigra region of the brain. It is
thought that MPTP represents a greatly accelerated model of natural dopamine toxicity.
2.2 VMAT Structure Both VMAT1 and VMAT2 are acidic glycoproteins with an apparent endogenous
molecular weight of 70 kDa, and they share 63% overall amino acid identity (the related
vesicular acetylcholine transporter, VAChT, has 34% amino acid identity). The predicted
secondary structure of the VMATs comprises 12 transmembrane domains (TMDs)
(Erickson and Eiden, 1993). This includes a large lumenal loop near the N-terminus with
four putative N-linked glycosylation sites. Both the C- and N-termini of the protein are
predicted to be cytoplasmic. These regions are the most variable with respect to species
conservation. The lumenal loop and C- and N-termini are both subject to the greatest
25
sequence divergence between VMAT1 and VMAT2. The VMATs are part of the broader
DHS12 superfamily of multidrug transporters of the major facilitator superfamily (MFS)
of secondary transporters that include drug-resistance proteins (toxin-extruding
antiporters), sugar uniporter proteins, H+ symporters, antiporters of organic phosphate
esters, and bacterial permeases (Marger and Saier, 1993).
2.3 VMAT Biochemistry The biochemistry of the VMATs has been extensively reviewed (Schuldiner et al., 1995,
Wimalasena, 2011, Eiden and Weihe, 2011, Henry et al., 1994). The inward transport of
each cytosolic monoamine is coupled to the efflux of two protons. The first proton is
proposed to alter transporter conformation to generate a high-affinity amine-binding site
on the cytosolic side. The second proton is proposed to generate a second conformational
change to move the amine from the cytosol to the vesicle lumen (Parsons, 2000).
The VMATs have several natural substrates: serotonin (5-HT), dopamine (DA),
norepinephrine (NE), and epinephrine (E). VMAT2 additionally transports histamine
(HIS). VMAT2 has a consistently higher affinity for all monoamine substrates than
VMAT1, though the rank order of affinity is the same for both isoforms: serotonin,
dopamine, epinephrine (Peter et al., 1994). Other substrates, including MPP+ and
methamphetamine show a similar pattern. The greatest difference in affinity, however, is
for histamine: Km of 3 µM for VMAT2 and 436 µM for VMAT1. Common structural
features among native and non-native substrates and inhibitors of VMAT include a
positive charge and aromatic ring. Hydroxyl, methoxy, and amino substituents in the ring
improve affinity, whereas negative charge in the molecule reduces affinity
26
(Schuldiner et al., 1995). Specificity for a wide variety of substrates demonstrates
significant plasticity in the binding site.
In synaptic vesicles, uptake is dependent upon the magnitude of the pH and
electrochemical gradients maintained by the vesicular H+-ATPase, as well as the
cytoplasmic concentration of transmitters, the transporter density on the vesicle
membrane, and the composition of the extravesicular media (Pothos et al., 2000, Sulzer
and Pothos, 2000). The stoichiometry of transport (net transport out of the vesicle of two
H+ but only one positive charge) leads to a greater dependency on pH than on membrane
potential.
There are two commonly utilized VMAT inhibitors, reserpine and tetrabenazine.
Biochemical study has revealed that these inhibitors function by two different
mechanisms. The binding of reserpine is modulated by the proton gradient, as is
substrate binding (Darchen et al., 1989), as well as substrate concentration. It is thus
suggested that reserpine interacts with the substrate-binding site of VMAT. However,
tetrabenazine binding is not affected by the proton gradient or substrate concentration
(except at concentrations greater than 100 × Km) and therefore likely binds a unique site
of the VMAT protein. Because tetrabenazine inhibits the binding of reserpine, it is
further suggested that it binds a distinct conformation of the VMAT protein than that
bound by reserpine and monoamine substrates. Notably, VMAT1 is less sensitive to
tetrabenazine than VMAT2 (Peter et al., 1994).
27
2.4 Role of VMAT2 in Biogenic Amine Physiology Although classically attributed to postsynaptic mechanisms such as receptor number and
sensitivity, evidence that receptors are not saturated under physiological conditions
provides a role for the dynamic regulation of quantal size to affect synaptic transmission.
A “quantum” is defined as a single vesicle filled with transmitter (Katz, 1971). As
reviewed by Edwards (Edwards, 2007), VMAT2 is the key determinant of vesicle filling
for serotonin and catecholamines, and therefore a mediator of quantal size for
catecholaminergic and serotonergic neurotransmission. The activity of VMAT2 may
therefore be a rate-limiting step in monoamine production and release, and its regulation
is thus relevant to the pathophysiology of perturbations in monoamine neurotransmission.
Indeed, the overexpression of VMAT2 increases quantal size (Pothos et al., 2000).
VMAT2 function depends on the cytosolic concentrations of monoamine, the transport
mechanism itself, and nonspecific leakage of monoamine across the vesicle membrane.
Low cytosolic concentrations of monoamine (possibly reflective of cytosolic toxicity) are
compensated by the relatively high affinity of VMAT2 (low Km). Furthermore, the rapid
depletion of vesicular monoamine stores in chromaffin granules with the use of reserpine
suggests substantial nonspecific leakage (Kozminski et al., 1998), which further
emphasizes and explains the importance of VMAT2 to maintaining quantal size.
In addition to its role in synaptic release, VMAT2 has also been found to localize to a
subpopulation of vesicles that undergo regulated exocytotic release in dendrites,
conferring somatodendritic release of retrograde signals (Li et al., 2005).
28
The physiological consequences of VMAT2 deletion and hypomorphism have been
explored through a number of mouse models.
2.4.1 Vmat2 Knockout Mice
Complete deletion of Slc18a2 (Vmat2-/-) in mice resulted in elimination of exocytotic
release and dramatic reduction of monoamine stores in the brain. Vmat2-/- mice survive
only a few days after birth (Fon et al., 1997, Wang et al., 1997). They have extremely
low monoamine levels relative to wild-type animals (DA, 1.5%; NE, 6%; 5-HT, 1%), and
consequently a severe impairment in monoaminergic signaling that is most likely the
underlying cause of postnatal lethality. The pups feed poorly and move little prior to
dying within the first postnatal week. However, they are born in normal Mendelian ratios
(~1/4) and there are no gross differences in brain morphology. There was also no
difference in TH immunohistochemistry between wild-type and Vmat2-/- mice, indicating
that vesicular monoamine transport (and subsequent exocytotic release) are not required
for normal development of the mesostriatal projections.
In vitro cell cultures derived from mesostriatal cells of Vmat2-/- mice exhibited effectively
no release of dopamine after K+-induced depolarization, demonstrating the requirement
of vesicular transport for exocytotic release. The biogenesis and cycling of empty
synaptic vesicles were unaffected by Vmat2 deletion (Croft et al., 2005).
The motor and feeding phenotype can be partially rescued with amphetamine (Fon et al.,
1997), demonstrating that reverse efflux of monoamines through plasma membrane
transporters can mimic regulated exocytotic release with temporary success. This effect
is most likely related to dopamine. The profound deficit in monoamine levels indicates
29
an increased ratio of degradation to synthesis, underscoring the importance of vesicular
storage to this balance. Inhibition of monoamine degradation by MAO also rescues
behavior and improves survival (Fon et al., 1997).
Detailed histology of the early postnatal cerebral cortex shows an increase in
developmental programmed cell death in these mice, which is partially rescued by MaoA
inhibition (Vmat2-MaoA double knockout mice) with increased serotonin levels
(Stankovski et al., 2007).
2.4.2 Vmat2 Heterozygous Mice
Mice that are heterozygous for Vmat2 (Vmat2+/-) exhibit a 50% reduction in Vmat2
expression, and normal viability and physiology compared with wild-type littermates.
They also exhibit significant reductions in monoamines (DA, 42%; NE, 23%; 5-HT,
34%) (Fon et al., 1997), demonstrating the importance of vesicular storage capacity to
monoamine content. In vitro cell cultures derived from mesostriatal cells of Vmat2+/-
mice exhibited approximately half (46.8%) the dopamine released from cells of wild-type
mice after K+-induced depolarization, demonstrating the role of vesicular transport in
determining quantal size (Fon et al., 1997). Notably, Vmat2+/- mice are sensitized to
direct and indirect DA agonists such as cocaine and amphetamine (Wang et al., 1997),
and are significantly more vulnerable to MPTP and methamphetamine toxicity
(Takahashi et al., 1997, Gainetdinov et al., 1998, Fumagalli et al., 1999).
Phenotypically, the heterozygotes exhibit moderately elevated heart rate and blood
pressure relative to wild-type littermates (Takahashi et al., 1997). Behaviorally, they
exhibit no difference in locomotor activity, passive avoidance habit, or stress response
30
(Takahashi et al., 1997). However, they do express a depressive phenotype comprising
locomotor retardation, anhedonia, immobility in forced swim and tail suspension test, and
increased sensitivity to learned helplessness (Fukui et al., 2007). The forced swim and
tail suspension phenotypes were ameliorated by antidepressants with serotonergic,
dopaminergic, and noradrenergic mechanisms (fluoxetine, bupropion, and imipramine,
respectively), suggesting involvement of multiple monoamine systems in the depressive
phenotype (Fukui et al., 2007).
Additionally, the heterozygous mice were reported to prolonged QT syndrome and an
increased rate of sudden death at 2–4 months of age (Fukui et al., 2007, Itokawa et al.,
1999).
2.4.3 Constitutive Overexpression of Vmat2 in Mice
Evidence of the dose dependence of the Vmat2 phenotype was recently complemented by
a study investigating the effects of constitutive Vmat2 overexpression. BAC transgenic
C57BL/6 mice were generated that possess three additional copies of the complete
Slc18a2 gene and flanking regions (Lohr et al., 2014). These mice exhibit 3.5-fold higher
Slc18a2 mRNA levels, and 3-fold higher Vmat2 protein levels, compared with wild type
littermates. The mice exhibited increases both in vesicular uptake and in vesicular
capacity, corresponding to increased striatal vesicle volume and increased vesicle size.
Resultingly, the mice exhibited increased striatal dopamine content, synaptic dopamine
release, and extracellular dopamine.
The locomotor and behavioral phenotypes of the mice also corresponded to these
increases in dopamine neurotransmission—the mice showed increased locomotor activity
31
and reduced anxiety-like and depressive-like behaviors (measured by marble-burying
assay and forced-swim test, respectively)—although these measures were likely also
confounded by serotonergic and noradrenergic effects. Finally, these mice with increased
Vmat2 expression had demonstrably reduced susceptibility to MPTP neurotoxicity, as
measured by loss of striatal dopamine terminal markers (DAT and TH).
Evidence of the dose dependence of the Vmat2 phenotype demonstrates its role as an
important modulator of monoaminergic function, in contrast to vesicular glutamate
transport in which compensatory decreases in synaptic vesicle release result in no net
increase in neurotransmission (Daniels et al., 2004). Enhancement of Vmat2 function
may therefore be an attractive therapeutic target for disorders resulting from reduced
monoaminergic function, including depression and Parkinson’s disease.
2.4.4 Selective Deletion of Vmat2 in Serotonergic Neurons
Two models of the selective deletion of Vmat2 in serotonergic neurons have been
developed to address the developmental role of serotonin. The Slc18a2 transcript is
expressed transiently in several regions of the brain during early postnatal development in
mice, in spatial and temporal co-expression with the serotonin plasma membrane
transporter (SERT) (Lebrand et al., 1998). In Vmat2sert-cr e mice, the Slc18a2 gene is
selectively deleted in all SERT-expressing cells, causing a profound depletion of central
serotonin stores (~95%), a partial depletion of serotonin in the blood, and normal
serotonin levels in the gut (Narboux-Neme et al., 2011). In Vmat2pet1-cre mice, the
deletion of Slc18a2 is specifically targeted to raphe neurons, producing a partial (~75%)
reduction in brain serotonin levels (Narboux-Neme et al., 2013). In both models, an
overall somatic growth defect was observed, consistent with the phenotype of serotonin
32
depletion observed in Tph2-/- mice. Additionally, severe serotonin depletion was shown
to cause a mild delay in the development of the upper layers of the cerebral cortex, but no
impairment in barrel cortex development. Interestingly, the barrel cortex development
defects observed in Vmat2-/- mice are not reproduced in these mouse models (Stankovski
et al., 2007), and therefore likely do not derive from serotonin depletion. Generally, the
delay in cortical growth was much more mild than the overall growth defect in these
mice.
Behavioral testing of Vmat2sert-cre mice revealed decreased locomotor activity, decreased
anxiety, and decreased immobility in the tail suspension test; interestingly, the latter
result differs from observations in Vmat2+/- mice in which all monoamines are affected.
These changes were reversed by treatment with a monoamine oxidase inhibitor.
Behavioral correlates of Vmat2 depletion specifically in raphe neurons have not yet been
reported.
2.4.5 Selective Expression of Vmat2 in Noradrenergic Neurons
A Vmat2 transgene was selectively expressed in noradrenergic neurons of Vmat2-/- mice
(Vmat2-/-NE+) to investigate the role of noradrenergic defects in early postnatal lethality of
Vmat2 deletion (Ohara et al., 2013). These mice had a slightly extended lifespan
compared with Vmat2-/- mice (2–3 weeks), but a body weight of approximately half that
of wild-type littermates. Behavioral testing revealed pronounced akinesia and almost
complete elimination of the normal open field exploratory pattern. The data suggest that
noradrenergic function is critical to early neonatal survival, but survival beyond this point
is dependent on other monoaminergic systems.
33
2.5 Role of VMAT2 in Disease Processes The relationship between VMAT2, affective disorders, and Parkinson’s disease stems
from the early discovery that chronic administration of reserpine—initially introduced for
the treatment of hypertension—causes central nervous system effects including aspects of
depression and Parkinsonism. This observation lead to the monoamine hypothesis of
affective disorders (Freis, 1954), and the recognition of the role of dopamine depletion in
Parkinson’s disease.
2.5.1 VMAT2 and Parkinson’s Disease
Dopamine toxicity is a controversial topic suffering a lack of direct evidence in vivo.
Importantly, toxic dopamine is that displaced from vesicles and subject to oxidation;
greater than 90% of intracellular dopamine is normally sequestered in the reducing
environment of vesicles (Eisenhofer et al., 2004). Dopamine is highly reactive, and can
yield reactive species including hydroxyl radicals, superoxide, hydrogen peroxide, and
dopamine quinones (Graham, 1978, Hastings et al., 1996). Reaction of quinones with
proteins can create cysteinyl adducts; L-DOPA and DOPAC cysteinyl adducts are also
formed. Superoxide further reacts with nitric oxide to produce reactive nitrogen species.
All of these species may contribute to the oxidative stress underlying the multifactorial
pathogenesis of Parkinson’s disease, and the unique vulnerability of nigrostriatal
dopamine neurons (Jenner, 2007). Therefore, VMAT2 function and regulation may be
critical to neuroprotection through its role in maintaining dopamine homeostasis.
Several lines of indirect evidence suggest a role for VMAT2 in Parkinson’s disease
pathogenesis. These include the parkinsonian side effects of the specific VMAT
34
inhibitor, reserpine; its role in the sequestration of MPP+ conferring protection against
MPTP-induced parkinsonism; evidence of a direct interaction between VMAT2 and
α-synuclein and an increase in cytosolic dopamine and reactive species with α-synuclein
overexpression (Lotharius and Brundin, 2002, Mosharov et al., 2006, Guo et al., 2008);
and the mechanism of methamphetamine toxicity disrupting vesicular dopamine
sequestration (Cubells et al., 1994, Sulzer and Rayport, 1990). Furthermore, a
hypomorphic Vmat2 mouse model appears to recapitulate relevant molecular and
pathological hallmarks of PD.
2.5.1.1 Vmat2 Hypomorphic Mice
In addition to the previously described models of Vmat2 deletion, hypomorphic mice
were studied to assess the effects of significant Vmat2 deficiency in animals that are able
to overcome the barrier of early postnatal lethality exhibited by Vmat2-/- mice. These
mice were created accidentally during the process of generating complete knockout mice,
in which the knockout cassette was stably inserted into intron 3 (Mooslehner et al., 2001).
Termed the KA1 strain, these mice also unintentionally possessed a spontaneous deletion
encompassing the D-synuclein locus (not recognized in the initial reports).
KA1 mice exhibit 95% reduction in Vmat2 protein levels, and corresponding reductions
in tissue monoamines of 92% (dopamine), 87% (norepinephrine), and 82% (serotonin).
Interestingly, in vitro assay of electrically stimulated dopamine release measured only a
70% reduction compared with age-matched wild-type animals (Patel et al., 2003).
Compared with a 95% reduction in protein levels, this result demonstrates the presence of
compensatory mechanisms for the reduction in Vmat2—likely a redistribution of Vmat2
from reserve pools to actively cycling vesicles.
35
In KA1 mice, no differences were observed in D1/D2 receptor sensitization, but there
was supersensitization of D2/D3 autoreceptors (Colebrooke et al., 2007). In addition,
downregulation of TH phosphorylation was observed at residues involved in feedback
inhibition. No differences in DAT expression or activity were observed (Colebrooke et
al., 2006). Importantly, these mice were found to downregulate substance P expression,
and upregulate enkephalin in the striatum, demonstrating differences in underlying
architecture of the striatum (Mooslehner et al., 2001).
Behaviorally, the KA1 mice exhibit decreased locomotor activity and impairment in
motor coordination (measured by beam walking and rotarod) that worsens with age, but
normal reactivity in the novelty place preference task (Mooslehner et al., 2001). The
motor deficits were responsive to L-DOPA administration. KA1 animals were also
predictably more sensitive to MPTP toxicity and amphetamine.
No evidence of neurodegeneration was evident in Vmat2-deficient KA1 mice at any age,
but this was later attributed to the lack of D-synuclein in these mice. A strain of Vmat2-
deficient mice was therefore created in which wild-type D-synuclein was reintroduced
through selective breeding. These Vmat2-deficient mice exhibited an 85% reduction in
striatal dopamine with associated reductions in DOPAC and HVA (Caudle et al., 2007).
Importantly, age-related declines in striatal dopamine were observed in these mice, and
were associated with several hallmarks of PD (Caudle et al., 2007). These mice exhibited
molecular markers of oxidative stress and damage, including cysteinyl-DOPA and
DOPAC adducts at 2 and 12 months of age, and protein carbonyls and 3-nitrotyrosine at
12 months of age. Progressive cell death and loss of TH-positive neurons was observed
36
within the SNpc in older animals. More dramatic progressive cell loss was observed in
the locus coerulus (LC), preceding that in the SNpc, consistent with observations in
human patients with PD (Braak et al., 2003). Additional progressive motor deficits were
also observed at 28 months of age, including a shorted forepaw stride length that may
mimic shuffling gait. These motor deficits were responsive to L-DOPA.
Although age-dependent alterations in the nigrostriatal system were observed in both
Vmat2-deficient mouse strains, progressive cell loss was only observed in the Vmat2-
deficient mice with intact D-synuclein, implicating the latter in the severity of the
phenotype and the mechanism of cell death.
Nonmotor deficits analogous to the nonmotor symptoms of PD were also assessed in
these Vmat2-deficient animals. These mice were observed to have progressive deficits in
olfactory discrimination, altered sleep latency, delayed gastric emptying, and anxiety-like
and depressive phenotypes (Taylor et al., 2009). As in PD, these nonmotor deficits
preceded the severe motor deficits, and the pattern of L-DOPA responsiveness was
similar (with the exception of sleep latency, which was L-DOPA–responsive in Vmat2-
deficient mice and is not responsive in human patients with PD) (Taylor et al., 2009,
Taylor et al., 2011).
2.5.2 VMAT2, Neuropsychiatric Phenotypes, and Drugs of Abuse
That the monoaminergic systems play a role in neuropsychiatric phenotypes is well
established, although the precise mechanisms are poorly understood. The particular role
of VMAT2 in this respect, however, has not been clearly investigated. As discussed
above, mouse models of VMAT2 deficiency (Vmat2+/- and hypomorphic Vmat2 mice)
37
exhibit phenotypes related to depression and anxiety. In addition, SLC18A2 was
identified in multiple genome-wide association studies of schizophrenia, as well as
alcohol dependency and post-traumatic stress disorder (see Section 2.5.3). Nonetheless,
the critical role of VMAT2 in determining quantal size makes it an attractive target for
further study into the perturbations of monoaminergic function that underlie these
phenotypes.
More evidence exists for a role of VMAT2 in the mechanism of action of drugs of abuse.
Amphetamines have multiple effects within the synapse that combine to produce
non-exocytic release of dopamine. Efflux occurs through reverse transport of dopamine
through DAT. The amphetamine- and methamphetamine-induced efflux of dopamine out
of cells, however, first requires the desequestration of dopamine from vesicles.
Amphetamine prompts this by one or both of two proposed mechanisms: VMAT2
inhibition or eliciting DA reverse transport as a VMAT2 substrate, and by collapsing the
synaptic vesicle pH gradient through competition for intravesicular protons (Fleckenstein
et al., 2007, Sulzer et al., 2005). In the case of methamphetamine, desequestration was
shown to be tetrabenazine-sensitive (Volz et al., 2006).
Some evidence has been provided that drug treatments may cause differential
redistribution of VMAT2 within striatal synaptic terminals. Administration of
methamphetamine was shown to redistribute VMAT2 in striatal synaptosome
preparations out of an enriched vesicular fraction in conjunction with a decrease in
vesicular DA content (Riddle et al., 2002, Sandoval et al., 2003, Eyerman and
Yamamoto, 2005). Cocaine and methylphenidate primarily act as inhibitors of dopamine
reuptake. However, both have been shown to redistribute VMAT2 from a membrane-
38
associated vesicle fraction to a vesicle-enriched, non–membrane-associated fraction
(Sandoval et al., 2002, Riddle et al., 2002).
Importantly, differential sensitivity to the locomotor effects of amphetamine, cocaine,
and ethanol (Takahashi et al., 1997, Wang et al., 1997), and methamphetamine toxicity
(Fumagalli et al., 1999), as well as altered ethanol-associated behaviors (Hall et al., 2003,
Savelieva et al., 2006), have been demonstrated in Vmat2+/- mice. Much work remains
be done to truly understand the role of VMAT2 in the mechanism of action of drugs of
abuse.
2.5.3 Genetic Variation in VMAT2
Although no direct disease-associated VMAT2 mutations have been identified prior to
that reported in this thesis, some associations have been discovered between single
nucleotide polymorphism (SNP) alleles in SLC18A2 introns or promoter and Parkinson’s
disease (Brighina et al., 2013), schizophrenia (Talkowski et al., 2008, Chu and Liu, 2010,
Gutierrez et al., 2007, Simons and van Winkel, 2013), post-traumatic stress disorder
(Solovieff et al., 2014), and liability to or protection against alcohol dependency (Lin et
al., 2005, Fehr et al., 2013).
Gain-of-function promoter haplotypes may also be associated with a protective effect
against Parkinson’s disease in women (Glatt et al., 2006). As expected for traits that are
highly polygenic, the magnitudes of effect in each case are not conclusive, and functional
validation of mechanistic involvement of VMAT2 would be required to draw further
conclusions.
39
3 Thesis Overview A new pediatric neurotransmitter disorder was discovered in eight cousins of a
consanguineous Bedouin family in Saudi Arabia, and a causative mutation in VMAT2
(SLC18A2) was discovered in the present work. Understanding the underlying
mechanism of this disorder extends the spectrum of known pediatric neurotransmitter
diseases, serves as the first demonstration of mutation in VMAT2 causing a human
phenotype, and thereby provides new insight into the role of VMAT2 in monoamine
homeostasis.
This thesis additionally demonstrates the utility of implementing genomic diagnosis in
the clinic, with respect to providing simple and effective treatments in a timely manner to
improve outcomes for patients with rare inborn errors of metabolism.
My thesis objectives were the following:
(1) To characterize this new pediatric neurotransmitter disorder.
The disease was hypothesized to involve a defect in monoamine neurotransmitter
pathophysiology. To provide evidence of the underlying pathophysiology of the
disease, careful clinical investigations, including genetic analyses of candidate
genes, were undertaken to exclude known diagnoses and produce a
comprehensive clinical description of the disorder and its progression. In
particular, neurotransmitter metabolite profiles were assessed in both CSF and
urine. This data informs the differential diagnosis of this particular disease and
related disorders; however, genetic and treatment evidence were further required
to confirm this hypothesis.
40
(2) To identify the genetic mutation causing this disease and characterize the effect of
the mutation
I hypothesized that a homozygous genetic mutation was causing this autosomal
recessive disorder in affected individuals of a consanguineous pedigree. To
investigate this hypothesis, whole-genome SNP analysis was performed on 5
patients and 3 unaffected siblings, in combination with linkage and homozygosity
analyses, to identify the region containing the disease-causing mutation. Sanger
sequencing of candidate genes within the locus identified a nonsynonymous
variant in the SLC18A2 gene encoding the vesicular monoamine transporter 2
(VMAT2) protein. The possibility of other variants in the region and in other
candidate genes in the genome was excluded by whole-exome sequencing of the
proband.
(3) To infer treatment targets on the basis of the biochemical and physiological role
of the identified disease-causing mutation
Patients exhibited an initial positive response to treatment with
levodopa/carbidopa followed by a rapid decline and worsening of the phenotype.
This poor response to dopamine replacement is consistent with the presumed
normal dopamine biosynthetic capacity in these patients combined with aberrant
dopamine storage and release. It was then hypothesized on the basis of the known
defect in VMAT2 function that treatment with a dopamine agonist would
stimulate dopamine receptors while bypassing the requirement for vesicular
storage and release. Subsequent treatment with a dopamine agonist, pramipexole,
41
resulted in a remarkable response in all patients, with the most substantial
recovery occurring in younger patients.
Much of the work contained in this thesis and the major conclusions were published in:
RILSTONE, J. J., ALKHATER, R. A. & MINASSIAN, B. A. 2013. Brain
dopamine-serotonin vesicular transport disease and its treatment. N Engl J Med,
368, 543-50.
42
Chapter 2 Clinical Characterization of a New Pediatric Neurotransmitter
Disease
1 Introduction The pediatric neurotransmitter diseases (PNDs) mimic the phenotype of other
neurological disorders, and are therefore often misdiagnosed or remain without diagnosis
for years. Common misdiagnoses include cerebral palsy, hypoxic ischemic
encephalopathy, paroxysmal disorders, inherited metabolic diseases, and genetic dystonic
or parkinsonian syndromes. Further hindering accurate and timely diagnosis, several of
the pediatric neurotransmitter diseases are exceedingly rare. The most similar syndromes
to that presented in this thesis (TH deficiency and AADC deficiency) reflect worldwide
diagnoses of just 50 and 100 patients, respectively. Increasing awareness in the clinical
community of this group of disorders and the clinical spectrum of monoamine deficiency
will aid the recognition of these disorders. In this chapter, the clinical investigation and
diagnosis of a new autosomal recessive pediatric neurotransmitter disease is described.
The diagnosis of monoamine neurotransmitter disorders is based on clinical history,
neurological examination, biochemical investigations (including specific cerebrospinal
fluid investigations), enzyme analysis, and genetic analysis. Conclusive diagnosis of
biogenic amine deficiency in the central nervous system is often demonstrated by clinical
measurements of their primary metabolites in cerebrospinal fluid. In the present case,
several lines of evidence coincided—most importantly, the genetic results described in
Chapter 3—to conclusively diagnose this new disease. The relevant investigations are
presented in this chapter to characterize this new syndrome, demonstrate the involvement
43
of biogenic amines in its etiology, and present a successful treatment strategy.
Additionally, difficulties in diagnostic strategy are discussed, including the pitfalls of
cerebrospinal fluid metabolite measurements as a conclusive diagnostic method.
2 Methods
2.1 Patients The patients in this study were identified in the Khobar province of Saudi Arabia by Dr.
Reem Alkhater, who undertook neurological examinations and relevant investigations.
The study was approved by the Hospital for Sick Children’s research ethics board.
Written informed consent was provided by the parents, with the patients providing their
assent for participation in the study.
2.2 General Investigations Investigations presented here were performed as part of regular clinical care for these
patients, and included neurological examination, bloodwork and metabolic screens,
video-EEG, magnetic resonance imaging (MRI), and MR spectroscopy.
2.3 Clinical Measurement of Neurotransmitter Metabolites Lumbar puncture was performed in a younger affected sibling of the proband at the age
of two to allow measurement of monoamine neurotransmitter metabolites, intermediates,
and precursors. Samples were drawn sequentially into numbered tubes to ensure
collection of the appropriate fraction. The tube for analysis contained 1.0 mL of CSF
collected directly from the tap needle after previously dispensing 1.0 mL of CSF. This
tube contained appropriate antioxidants for preservation of the analytes. The tube was
44
immediately frozen at –80°C and shipped on dry ice for analysis. The sample contained
no visible blood contamination.
Metabolites were measured at the Service de Génétique Médicale of the Centre
Hospitalier Universitaire de Sherbrooke Fleurimont by liquid chromatography tandem
mass spectrometry (LC-MS/MS) according to established methods.
A urine sample was also collected from the same patient. This sample was analyzed for
relevant neurotransmitter metabolites at the Mayo Clinic in Rochester, MN.
2.4 AADC Enzyme Test The activity of aromatic L-amino acid decarboxylase (AADC) was assayed commercially
by plasma enzymology at Medical Neurogenetics. Plasma was separated immediately
upon collection of a blood sample in an EDTA tube and stored at –80°C before shipping
on dry ice for analysis.
2.5 Candidate Gene Sequencing A total of 5 mL of whole blood was collected in EDTA tubes. Genomic DNA was
isolated at the Hospital for Sick Children’s DNA lab. Commercial sequencing of a panel
of genes associated with spinocerebellar ataxia (SCA) and those associated with
mitochondrial DNA disorders was performed.
Primers were designed to amplify the exons of each gene and approximately 50
bp of flanking intronic sequence (Table 3). The reaction was performed using Picomaxx
DNA polymerase (Stratagene) according to the manufacturer’s specifications. Cycling
conditions incorporated an initial denaturation at 95°C for 5 minutes, followed by 30
cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension step at
45
72°C for 10 minutes. Amplification products were visualized by gel electrophoresis, and
purified from bands of the appropriate size using a Qiagen Gel Purification Kit according
to manufacturer’s specifications. Purified PCR products were sequenced using both
forward and reverse primers in separate reactions.
Sequences were compared to reference genome sequence using the BLAT tool in
the UCSC Genome Browser (Meyer et al., 2013). Putative variants were verified by
manual inspection of electropherograms.
Table 3 Primer sequences for the amplification of exons of candidate genes associated with known pediatric neurotransmitter diseases Gene Exon Sequence Size PTS 1 AGC GGA GAC GCA CTT CCT A 389 bp
GAC ACT CCA GCC CCC ATC 2 TTG GTG AGC TAA AGT AAT AAA TTG
G 300 bp
TCC GTA AGT TTT CCC ATT CTT 3 AGC TTT TGG GGA CAG ATC TAA 250 bp
AAG CAA TAC TGA CTG GAA CAG TTT 4 GGA TGA AGG CAA ATG TGC AA 298 bp
CCA GTT CTA TTC ACA AAG TCA TGG 5 GAC AGC TGG GCC TGA CTT TA 380 bp
GAA ATT CTA GTT TCG AAA GAT TTC AT
6 TTT GAT TGT TGT GTG ATT TCT GA 392 bp AAT AGG CAC TCC AGA GCA CAA
SPR 1 CAG ATC CCA AGG GAA CCA G 600 bp GCT GGA ACA ACT AGG GCT TTA
2 TAG GTT CCC CAG CTG TGT CT 575 bp ATC CTG TGG GTT GTT TCC TG
3 AAG ATG ACG TGT TTC CTC TGG 400 bp CCC TAT GGC AGG GTG TGG
QDPR 1 GCC TGG CCG AAG TTA CAG T 428 bp GTG CAA GCA ACA CGA GTC AG
2 TCC CAA AGT GCT GGC ATT AC 298 bp GCC AAA GGA AGA ACA TAC AGC
3 CAA AGC ATT AAT TGC CAG GT 300 bp AGT GCA AAC CCA ATC CTT GT
4 GCC CTG TGC TGT TTG TGT TA 371 bp
46
TCC TCA TCC CAT GAA AGT GC 5 CGT CTG ACC TGA AGG AGG AG 363 bp
CAG AAC CAG AGG TGA GAG CA 6 TAG CAC CCT CAG TGC CAG A 300 bp
GGG AAC ACA GAC TTG TCC TCT 7 TTA AAC AGT CGC TGC TGT GC 377 bp
GGA GAG CAA ATG CAT ATT ATG TGA GTCPH1 1 GCG TAC CTT CCT CAG GTG AC 561 bp
AGT GAG GCA ACT CCG GAA AC 2 CAG AAA AGG ACT TTG CTA CTT TGA 476 bp
GCC TTC TGC TAC TTT GGT TTT G 3 AAC AGT TCC AGA TGT TTT CAA GG 379 bp
GTA GGG GAC GAG AAG GAA GG 4 GTC CTT TTT GTT TTA TGA GGA AGG C 288 bp
GGT GAT GCA CTC TTA TAA TCT CAG C 5 TGG TGT GTC TTG GCT CTT AAA 300 bp
CCT GGT GCT ACA AAA TAT GAG AA 6 CCG CAG TTA CTT TTG CAT GA 396 bp
CAC ATC TGT AAC AAT TGA AAA TGG A
SLC6A3 1 TGG CTG AAG ACC AAG AGG G 425 bp CTC GTT TCC GTA CGT GCC
2 ATG GAT GGT TGA CTG GGG TA 335 bp TAG CAA AGC AGG GCT GGA T
3 TGG GCT CAG GGT AAT GTC TC 490 bp CCA GAG CAC TAA AGG GAT GG
4 AGT TCC AGG TGG GTT GAC AG 304 bp AGC ACA AAA CCC AAC TGA GG
5 CTC CCA ATC AGA GGA CAA GC 354 bp CCT GGT GTC TGC AAC TCT GA
6 CAC TTC CTG AGG CTG CAT CT 361 bp TCT CAT CCA GGG ACA CCC TA
7 AAA AGT AGC CCC TCC GAA GA 383 bp TGA GGC AGC AAC TCT CAC TG
8 CGC ACC AGC CCT AGT CTC TA 424 bp TCC AGT CAC CAC TCA CTC CA
9 AAA CCC CCT ACC GTG GAT AC 561 bp TGA CTC TGG GAC CAA GCT CT
10 TAC ACG TGG CCC TAA GAA GC 442 bp AGC ACC TCA CTG ATC CCA TC
11 CTT GGG AGT CAG CGA GGA 314 bp AGT CTT GAG GCC CCT GAC TC
12 GAG ACG CTC TGC CAT GAA GT 311 bp CTT TCT GGT GGC CTC ACA CT
13 CTG GCA GTG GGT ACT GGT CT 200 bp
47
CTG GGG GCT AAG AAC ACT GA 14 CAG GAG GCT GCA AAC TGT CT 2.2 kb
AGG CCG GAA GGA GAG ATG TH 1 GCC TCC CTC CTT CCT CAC 379 bp
GGT TTG CAT GGA CCC TGA 2 AAA TGG GTT TTT ATT TAT GGA CCT T 400 bp
GGG ACT TGG CAG ACA CCT G 3 CTC AAA AAC GTG CTC TCA TCC 500 bp
AGC TGA GGC CTG AGA CTC C 4 AAG AAC GGG ATC TGT GTG CT 479 bp
CAC GGA TGT GTA GCA AAA CG 5, 6 CCC CCG GAA GTC TTG TAG G 500 bp
GGT CCT CCC CTT TGT CCT T 7 CAC CCT CCT GTC CAT CCT C 293 bp
CTC TCC TGT TGT GCC AAG GT 8 ACT GGG GTG GGG CAT TAG 396 bp
CAC TGG AAC ACG CGG AAG 9 GGG GAT GGT CAG CCA AGC 297 bp
CGC GTA GGA GGG AGA AGG 10 ACT CCC CTG AGC CGT GAG 268 bp
AGC AGG CAG CAC ACT TCA C 11 AGG GAA GTG TCC CAG AGA CC 235 bp
AGA GGG TGA GGC CTG GAT T 12 TGT CTC TGG GCT GAT GCT G 250 bp
AGA GCC TGA GTC CTG GAG GT 13 TGG AGT CAG TGA TGC CAT TG 242 bp
CTC AAG GCC AGA AGG AAG G 14 TCT GAG CCA CTG TGA AGG TG 296 bp
GTT GGG AAG GGC CCT CAG DDC 1 GGG GAG GCA GAC ACT CTG T 228 bp
GGA GGA GAA TTC AGC ACA GC 2 TCC TAC AGA CAT GGA GGG AAA 494 bp
TGC CAT AGG GAT TCC TTG AA 3 ACA TTT GGG GAA CTG CAC TC 340 bp
CAG GTC CCT TGT GCA TAG GT 4 TCT GGG CTT TAG TGG AAG GTT 300 bp
GTG CCT CTT TCC CCA CCT 5 GGA CAC AAA ACA ATA TGT CTT CCA 300 bp
TGG TTT GGT TTG AAT TTG ACA 6 CCT TGT GTT TGC AAT GTT GG 300 bp
CCC GAG TAG CTG GGA TTA CA 7 CAT TGG GAT CTC GGC TCA T 300 bp
GGC AAA CCA TCA CAA TAT GAA 8 CAA GAA GGT GCT CAG ACA GG 299 bp
TTG GCT GAA ACA AAC CTC AA
48
9 CAC TGT GGA TTA GTT GTG CCA TA 296 bp GCA GCA AGC AGT GAG CTA AG
10 CCC AGG TAC TTG GAG CAG AG 390 bp TAC AAG GGC AAA TCC AGG AA
11 GCC TTT GGG CAG TTT TAT TTC 249 bp CGT GCT GAT CAT GAG AGT GG
12 GTT GGC CAC CAG GGA ATC 300 bp GCA GTG AGC TGA GAT TGT GC
13 TGC CAA GAG CGT CTA AAT GA 392 bp CGT GGA AAC AAG GCT GTG TA
14 GTA GGG TTG CCA AGC ACT GT 395 bp CCT GTA GCT GGG TCT GGA CT
15-1 ACC GTG GAA AGA GAG GGA GA 475 bp GTT GCG TGA ACA TTG ATT GC
15-2 GAG GGT TGT GAT TTT GTC TGC 400 bp TGC CGT TTA AAA ACA TCC AA
3 Results
3.1 Patient History and Neurological Examination The index case was a 16-year-old female with global developmental delay and abnormal
movements. She had initially been brought to medical attention at 4 months of age with
hypotonia, loss of acquired head control, and paroxysmal stereotyped episodes of
persistent eye deviation, and crying lasting hours. Video-EEG monitoring had excluded
seizures, and a symptom diagnosis of oculogyric crisis had been made. Development had
been normal initially, but had slowed after presentation. She had developed the ability to
sit at 30 months, crawled at 4 years, and walked at age 13.
Upon presentation at the age of 16, she was experiencing fatigue, excessive diaphoresis,
profuse nasal and oropharyngeal secretions, noisy breathing, hypernasal speech, poor
distal perfusion, cold extremities, disrupted sleep, hypotonia, ataxia, dysarthria, and
incoordination. There was no diurnal variation and no improvement with vitamin B6 or
folinic acid.
49
Neurological examination of the proband at age 16 revealed ptosis, hypomimia, facial
dyskinesia, and limited upward gaze. She had axial hypotonia and appendicular
hypertonia specifically involving the extensor muscles of the upper and lower
extremities, and her power was 4+/5 in all muscle groups. Deep tendon reflexes were
2+/4 and symmetric. Plantar reflexes were flexor, and there was no clonus. Coordination
testing revealed a fine tremor, and dysdiadochokinesia in the upper and lower extremities.
Her gait was parkinsonian with typical shuffling, her posture was stooped, and her
postural reflexes were diminished. She walked with bilateral alternating dystonia of
hands and feet with intermittent toe walking and foot inversion, and was unable to
tandem walk.
3.2 Investigations The patient’s basic bloodwork and metabolic screens were normal (Table 4). Repeat
overnight video-EEG revealed neither seizures nor interictal abnormalities, and MRI and
MR spectroscopy were normal (Figure 5).
Table 4 Serum metabolic screen revealed no abnormalities
Amino acids Vitamin B12 Biotinidase Copper Ceruloplasmin Lead Very long chain fatty acids Carnitine Ammonia Lactate Pyruvate
50
Figure 5 T2-weighted magnetic resonance images of proband at age 14 revealed no abnormalities.
51
Lumbar puncture was performed in a younger affected sibling at the age of 2 to measure
monoamine neurotransmitter metabolites, intermediates, and precursors—all of which
were normal (Table 5). The urine neurotransmitter profile, however, revealed increased
monoamine metabolites [5-HIAA = 17.6 µg/dL (reference range: 0–6 µg/dL); HVA =
14.1 µg/mg Cr (0–13.4 µg/mg Cr)] and a decrease in measurable urine monoamines
[norepinephrine = 1.1 µg/dL (4–29 µg/dL); dopamine = 19 µg/dL (40–260 µg/dL)]
(Table 5). Plasma was tested commercially for AADC enzyme activity; this result was
also normal.
52
Table 5 Cerebrospinal fluid and urine neurotransmitters and their metabolites measured in a younger affected sibling of the proband reveal decreased monoamines and elevated metabolites in urine, but not in cerebrospinal fluid. Values outside reference ranges are presented in bold.
Cerebrospinal Fluid 5-HIAA HVA HVA/5-HIAA 3-OMD 5-OHTrp 5-HT Normal Range 74–345 nM 233–928 nM 1.5–4.1 <150 nM <25 nM no reference Patient 169 nM 314 nM 1.9 20 nM 12 nM <5 nM Urine 5-HIAA Norepinephrine Epinephrine VMA HVA Dopamine Normal Range 0–6 µg/dL 4–29 µg/dL 0.0–6.0 µg/dL 0–12.9 µg/mg Cr 0–13.4 µg/mg Cr 40–260 µg/dL Patient 17.6 µg/dL 1.1 µg/dL 0.5 µg/dL 5.5 µg/mg Cr 14.1 µg/mg Cr 19 µg/dL HIAA, hydroxyindoleacetic acid; HVA, homovanillic acid; OMD, O-methyldopa; OHTrp, hydroxytryptophan; HT, hydroxytyramine; Cr, creatinine; VMA, vanillylmandelic acid.
53
3.3 Family Structure The proband is a member of a consanguineous Saudi family that includes 6 children
sharing an identical clinical picture of complex movement disorder (Figure 6). The
proband (V:6) was the eldest of 6 siblings, three of whom also presented with the disease
(V:9, V:10, and V:11). Two siblings were unaffected (V:7 and V:8). The parents had
also experienced two spontaneous abortions. An additional affected individual (V:3) is a
second cousin of the proband. A sixth affected individual (VI:2) was a first cousin once
removed of the affected children.
The parents of the affected individuals exhibited no movement disorder. The ratio of
male to female affected individuals is approximately 1:1, and there is no evidence of
maternal inheritance. The mode of inheritance is therefore autosomal recessive.
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Figure 6 Family structure of kindred demonstrates autosomal recessive mode of inheritance and consanguineous pedigree structure. Proband is labelled V:6. Cerebrospinal fluid and urine neurotransmitter analyses were performed on individual V:10. Black, affected; white, unaffected; square, male; circle, female; diamond, spontaneous abortion. Numbers in squares or circles indicate number of male or female offspring, respectively. From New England Journal of Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission.
55
Two additional affected cousins were identified. These two affected individuals were full
siblings of each other (VI:3 and VI:4) and first cousins of one of the affected individuals
(VI:2). At the time of analysis, the eldest was a 31-month-old boy. In addition to the
phenotype of hypotonia, these siblings exhibited bilateral anopthalmia. There is no clear
theoretical relationship between the neutrotransmitter phenotype and this developmental
phenotype. There is also no documented anopthalmia in other pediatric neurotransmitter
diseases.(Kurian et al., 2011a, Pons, 2009) Given the consanguineous background of
these affected individuals, they may simultaneously express an unrelated autosomal
recessive trait caused by an independent genetic mutation. A karyotype should be
performed for these siblings to identify any chromosomal abnormality, and these patients
may further opt to undergo whole-exome sequencing to identify additional homozygous
nonsynonymous variants.
3.4 Genetic Screening of Candidate Genes The clear autosomal recessive mode of inheritance in this consanguineous family belies a
single causative genetic mutation. Initial screening of some candidate genes was
performed in the clinical laboratory setting. Clinical genetic screening of candidate genes
revealed no mutations associated with SCA or mitochondrial disorders.
After the suggestion was made of biogenic amine involvement, the causative
genes for all known dopamine deficiencies were screened. Genes known to be associated
with pediatric neurotransmitter diseases were screened by sequencing of exons and
flanking intronic sequences (Table 6). The causative gene for pterin-4α-carbinolamine
dehydratase deficiency (PCD) was not sequenced because PCD does not present with
56
movement disorder features, and the causative gene for PLP was not sequenced because
seizures were not a component of the patients’ histories. No coding mutations or putative
splice variants were identified in any of the sequenced candidate genes.
Whole-genome investigations were undertaken to identify the causative mutation,
and a mutation was discovered in the SLC18A2 gene encoding vesicular monoamine
transporter 2 (VMAT2). These genetic studies are described in Chapter 3.
Table 6 Pediatric neurotransmitter disease–associated genes screened in the proband revealing no putative mutations Gene Symbol
Number of Coding Exons
Protein Name Associated Disorder
DDC 9 Aromatic L-amino acid decarboxylase
AADC deficiency
SLC6A3 14 Dopamine transporter (DAT) Dopamine transporter deficiency syndrome (DTDS)
QDPR 7 Dihydropyridine Receptor (DHPR) DHPR deficiency GCH1 6 GTP cyclohydrolase I GTPCH1 deficiency
Segawa disease Dopamine-responsive dystonia (DRD)
PTS 7 6-Pyruvoyltetrahydropterin synthase (PTPS)
PTPS deficiency
SPR 3 Sepiapterin reductase (SR) SR deficiency TH 13 Tyrosine hydroxylase (TH) TH deficiency
3.5 Drug Response On the basis of the parkinsonism and the diminished urine dopamine, the proband and
three of her younger affected siblings were initially treated with L-DOPA/carbidopa
(approximately 1 mg/kg/day), which resulted within 1 week in major deterioration, with
57
the appearance of intense chorea and worsening of the dystonia. Discontinuation of the
medication led to rapid return to baseline in all four children.
On the basis of the identification of the underlying VMAT2 mutation (described in
Chapter 3), the decision was made to attempt treatment with a direct dopamine receptor
agonist (pramipexole; 0.02 mg/kg/day). This treatment resulted within 1 week in
dramatic and sustained disappearance of parkinsonism and dystonic attacks, and
improvement of other symptoms.
The younger affected siblings were then treated. They exhibited even more remarkable
recoveries; the younger the particular child at the time of initiation of treatment, the
greater the number of symptoms that were corrected and the greater the extent to which
symptoms improved. The effect of treatment at each age is presented in Table 7.
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Table 7 Age at initation of dopamine agonist affects disease course
Feature Age at treatment
18 years 11 years 7 years 3 years Cognition and ability to learn
Mildly improved Mildly improved
Moderately improved
Greatly improved. Able to make stories from pictures.
Occulogyric crises
No further events; on a dose higher than her siblings
No further events
No further events
No further events
Dystonia Gait dystonia persists Gait dystonia persists
Gait dystonia persists
Gait dystonia improved
Parkinsonism Improved Improved Improved Improved
Fine motor skills Improved coordination, able to feed self, drink from cup, and hold a pen. Improved handwriting
Improved coordination, learning to hold a pen. Unable to write or read
Learning to hold a pen and drink from a cup independently. Unable to write or read
Able to write, learning to read
Language and speech
Dysarthric No language development
Mama and Papa Normal language development and mild dysarthria
Gait Improved posture and fatigue (had started walking at age 13)
Started walking within days of treatment
Started walking within days of treatment
Started walking within days of treatment
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3.6 Summary of Clinical Features and Differential Diagnosis
The salient features of the disorder are summarized in Table 8. The syndrome is onset in
early infancy (approximately 4 months of age) with the appearance of hypotonia and the
loss of motor milestones. As the affected children age, they develop features of
movement disorder, cerebellar symptoms, and autonomic symptoms. As initially
observed in the proband (the eldest patient), the movement disorder phenotype exhibited
in early childhood (dystonia, oculogyric crises, facial dyskinesia, and chorea) evolves by
the age of 11 into parkinsonism characterized by bradykinesia, rigidity, stooped posture,
and shuffling gait.
Table 8 Clinical features common to all affected individuals in the pedigree Features Onset at age 4 months Hypotonia Hypomimia Paucity of movements Oculogyric crises Attacks of dystonia Dysarthria Ataxia and incoordination Excessive diaphoresis Profuse nasal and oropharyngeal secretions Poor distal profusion and cold extremities Disrupted sleep Mild cognitive impairment No diurnal variation Evolution of the movement disorder by age 11 years into a picture closely resembling Parkinson’s disease, with dyskinesias
The composite clinical picture in this family includes features that can be attributed to
deficiencies of particular biogenic amines. A categorization of the clinical symptoms is
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presented in Table 9. The movement disorder symptoms, as well as ptosis and
hypersalivation, are considered signs of dopamine deficiency. The manifestations of
serotonin deficiency include temperature instability, sleep disruption, sweating, and
possibly dystonia. Many of the autonomic disturbances experienced by these patients
may be attributable to the deficiency in norepinephrine. Although this model is useful for
understanding, it should be recognized that significant interaction among the biogenic
amine pathways clouds these distinctions. Additionally, some features are difficult to
directly attribute to the actions of the biogenic amines, such as mild cognitive
impairment.
Table 9 Clinical features of the disease organized by category Onset 4–7 months
hypotonia and developmental regression Movement Disorder oculogyric crises, dystonia, facial
dyskinesia, chorea Parkinsonism bradykinesia, rigidity, shuffling gait,
stooped posture Cerebellar symptoms ataxia, dysarthria, tremor Autonomic symptoms ptosis, fatigue, diaphoresis, excessive nasal
and oropharyngeal secretions, noisy breathing, hypernasal speech, disrupted sleep, poor distal perfusion
Medication responsiveness L-DOPA/carbidopa: temporary improvement, rapid decline and worsening of initial phenotype Dopamine agonist (pramipexole): Responsive
The multisystemic constellation of symptoms of this disorder fall within the spectrum of
pediatric monoamine deficiency. A comparison with the most closely related pediatric
monoamine deficiencies is presented in Table 10; as illustrated, a normal CSF
neurotransmitter metabolite profile is the major distinguishing feature of VMAT2
61
deficiency among these syndromes. As with other PNDs, the motor manifestations
dominate the clinical picture of VMAT2 deficiency, and the autonomic features of the
syndrome may be overlooked. As such, the underlying monoamine pathophysiology may
not be recognized by many physicians, and the most common misdiagnosis would be
cerebral palsy. Furthermore, physicians who suspect biogenic amine involvement may
opt for confirmatory CSF neurotransmitter analysis, which would produce a negative
result, further impeding proper diagnosis. The sole conclusive diagnostic for VMAT2
deficiency at the present time is genetic analysis.
These caveats underscore the importance of including genomic investigations in the
diagnostic protocol for pediatric movement disorder. In addition to being the sole
diagnostic for VMAT2 deficiency, the rate of misdiagnosis (or lack of diagnosis) is high
for the majority of pediatric neurotransmitter diseases, and more broadly, all inborn errors
of metabolism presenting with motor features.
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Table 10 Comparison of clinical features of the present disease with those of closely related monoamine deficiency syndromes
Our Family AADC Deficiency Tyrosine Hydroxylase
Deficiency DTDS Age at onset Infancy Infancy Infancy Infancy Muscular hypotonia + + + + Dystonic spasms + + + + Occulogyric crises + + ++ + Parkinsonism + + infantile onset + Delayed motor milestones + + ++ +
Autonomic symptoms + + + + Depression + + – –
CSF neurotransmitters a Normal b
Abnormal (Decreased HVA,
5-HIAA, and increased 3-OMD)
Abnormal (Decreased HVA)
Abnormal (Increased
HVA/5-HIAA)
Treatment dopamine agonist dopamine agonist L-DOPA
dopamine agonist (mild success in some patients)
AADC = aromatic amino acid decarboxylase; CSF = cerebrospinal fluid; DTDS = dopamine transporter deficiency syndrome; 5-HIAA = 5-hydroxyindoleacetic acid; HVA = homovanillic acid; 3-OMD = 3-O-methyldopa; a Complete CSF neurotransmitter metabolite profiles are presented in Table 11. b Based on a single case.
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4 Discussion Until the identification of dopamine transporter deficiency syndrome (DTDS) in 2009
(Kurian et al., 2009), the spectrum of PNDs included multiple defects in the biosynthesis
of one or more of the biogenic amines. The gold standard diagnostic test in patients with
suspected diseases of monoamine metabolism is the measurement of monoamine
metabolites in the CSF. Because each specific defect results in a particular metabolite
profile, this single test specifies the disease (Hyland, 2008, Kurian et al., 2011a, Pons,
2009). The particular metabolite profiles of each disease are presented in Table 11.
DTDS was the first PND discovered to result from a defect in biogenic amine transport
(specifically, dopamine uptake at the plasma membrane). This syndrome depicted a
normal serotonergic profile (normal 5-HIAA), but elevated HVA concentrations. This is
also reflected as an increased HVA/5-HIAA ratio, as recently observed in 11 unrelated
children with DTDS (range, 5.0–13.2; normal range, 1.3–4.0) (Kurian et al., 2011b). The
elevation of the primary metabolite of dopamine indicates the production of dopamine in
relevant quantities, as well as an increased rate of dopamine turnover. The latter is
expected to result from increased exposure of dopamine to degradative processes in the
extraneuronal space.
The measurement of CSF neurotransmitters in 1 patient in the present study revealed
metabolite concentrations within the normal ranges (Table 5), including a normal HVA
concentration and HVA/5-HIAA ratio (314 nM and 1.9, respectively). Acknowledging
the limitation in sample size, this result is similarly distinct to that observed for patients
with biosynthetic monoamine deficiencies.
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Defects in monoamine transport processes within the central nervous system would be
expected to produce an abnormal distribution of the affected transmitters with respect to
the relative quantities present within the vesicles, the cytosol, and the extraneuronal
space. Vesicular monoamine transport is driven by the pH gradient across the vesicular
membrane; namely, the acidification of the vesicles. In the acidic, reducing environment
of the vesicles, the monoamines are protected from oxidative processes. Additionally,
they are sequestered from degradative enzymes (MAO and COMT). Aberrant
redistribution of the biogenic amines would therefore be expected to increase the rate of
turnover, and subsequently increase the ratio of metabolite to transmitter. Importantly,
Vmat2-/- mice exhibit normal levels of DOPAC, HVA, and 5-HIAA despite dramatically
reduced levels of brain monoamines (Wang et al., 1997, Fon et al., 1997), consistent with
observations in this patient.
The biogenic amines are synthesized in the cytosol (with the exception of norepinephrine,
which is synthesized intravesicularly from dopamine, which is in turn synthesized in the
cytosol). In the case of VMAT2 deficiency, it is possible that the increased cytosolic
concentrations of dopamine and serotonin may provide some feedback inhibition on their
biosynthesis. This presumed difference in distribution relative to DTDS—in which the
neuronal reuptake of monoamines is defective—may underlie the difference in measured
HVA concentrations between DTDS and this case of VMAT2 deficiency (increase in
HVA versus normal HVA, respectively).
It will be necessary to perform metabolite measurements in the CSF of a larger cohort of
patients to conclusively characterize the particular metabolite profile of VMAT2
deficiency. This opportunity may arise as unrelated patients with suspected
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neurotransmitter deficiency phenotypes are screened genetically for VMAT2 mutation.
Many of these patients may have previously undergone CSF metabolite profiling, but
remain undiagnosed given normal results. CSF neurotransmitter metabolite analysis has
been instrumental in the discoveries of the monoamine disorders (Hyland, 2008, Kurian
et al., 2011a, Pons, 2009), but not may have captured VMAT2 deficiency because of its
normal CSF profile.
Table 11 Comparison of metabolite profiles in cerebrospinal fluid for pediatric neurotransmitter diseases, including VMAT2 deficiency Affected Enzyme
HVA 5-HIAA MHPG 3-OMD
Disorders of BH4 synthesis (recessive)
decrease decrease decrease normal
GTP cyclohydrolase (dominant)
decrease normal normal normal
Tyrosine hydroxylase
decrease normal decrease normal
AADC decrease decrease decrease increase PNPO decrease decrease decrease increase Dopamine β-hydroxylase
increase normal decrease normal
Dopamine transporter (Kurian et al., 2009, Kurian et al., 2011b)
increase normal NR NR
VMAT2 (single case)
normal normal NR normal
AADC = L-aromatic amino acid decarboxylase; BH4 = tetrahydrobiopterin; GTP = ; NR = not reported; PNPO = ; VMAT2 = vesicular monoamine transporter 2 Note: Data in table derived from Hyland 2008, except for the present case and where otherwise noted.
66
The specificity of HVA abnormalities in diagnosing primary neurotransmitter disorders
has also recently been called into question by the demonstration of decreased HVA in
15.4% of cases and increased HVA in 4.6% of cases in a cohort of 1388 pediatric patients
with neurological disorder, whereas genetic dopaminergic deficiency was only
discovered in 21 (1.5%) patients by genetic screening (Molero-Luis et al., 2013). The
magnitude of the HVA decrease was greater in this study for patients with genetic
dopaminergic deficiency compared with patients with other neurological disorders, but
the ranges overlapped significantly.
The analysis of monoamines or their metabolites in urine is not reliable in the diagnosis
of monoamine neurotransmitter diseases (Hyland, 2008, Kurian et al., 2011a, Pons,
2009), except in one—AADC deficiency—in which increased 3-O-methyldopa (3-OMD)
with decreased vanillylmandelic acid (VMA) in the proper clinical context is highly
suggestive and generally confirmed by mutation analysis (Brun et al., 2010, Lee et al.,
2012, Pons et al., 2004, Swoboda et al., 1999). In the present condition, urine shows
abnormalities because VMAT2 also functions outside the central nervous system,
including in the peripheral nervous system, adrenal medulla, and platelets (Eiden and
Weihe, 2011). The reason abnormalities are detected in urine and not CSF may pertain to
differences in monoamine and metabolite stabilities, processing, and ranges of normal
values between brain and periphery. In any case, it appears that the pair of metabolically
and clinically similar diseases, the AADC and VMAT2 deficiencies, could be screened
for by urine testing, and then confirmed by corresponding gene sequencing, obviating
lumbar puncturing.
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The initial selection of treatment of the affected children on the basis of clinical
phenotype alone (parkinsonism) led to severe, immediate worsening of the movement
disorder. Subsequent identification of the underlying pathophysiology allowed the
rational selection of an appropriate treatment.
It has been suggested that the immediate worsening of the movement disorder observed
in these patients upon treatment with L-DOPA may reflect a severe dyskinesia resulting
from the overactivation of sensitized receptors. In this hypothesis, the supplementation
with exogenous L-DOPA at a high dose would have been sufficient to overcome the
vesicle-loading deficiency, resulting in excessive release of dopamine at the synapse. L-
DOPA responsiveness has been observed in a pair of AADC-deficient siblings
harbouring a G387A mutation (Chang et al., 2004). The mutation affected the binding
site of the AADC protein, causing an approximately 60-fold decrease in affinity for the
L-DOPA substrate. However, saturation of the enzyme by treatment with exogenous L-
DOPA allowed sufficient enzyme activity to improve motor functioning in these patients.
Given the partial functioning of VMAT2-P387L observed in vitro (see Chapter 4,
Section 3.3), it is plausible that the supplementation of cytosolic dopamine would allow
the transport mechanism to come to equilibrium with a sufficient quantal size to allow
neurotransmission. The plausibility of this suggestion is supported by the L-DOPA
responsiveness of the motor phenotype in VMAT2-LO mice (Caudle et al., 2007, Taylor
et al., 2011). Another possibility that may underlie neurotransmission in these patients
under conditions of an extreme excess of cytosolic dopamine include reverse efflux
through the plasma membrane transporter.
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Willemsen et al. suggested that, as in patients with TH deficiency (Willemsen et al.,
2010), careful selection of minimal L-DOPA doses may rescue the dopamine-deficient
motor phenotype and avoid extreme dyskinesia. This would have the additional benefit of
restoring norepinephrine neurotransmission and ameliorating the autonomic features of
the syndrome.
As a treatment strategy, smaller doses of L-DOPA may indeed be effective. However,
treatment with L-DOPA for an extended period of time in a setting of disrupted vesicular
storage confers a potential risk of oxidative damage leading to neurodegeneration, and
hypomorphic Vmat2 mice do exhibit age-related neurodegeneration (Caudle et al., 2007,
Taylor et al., 2011).
An additional clinical feature of note is the very high rate of major depression observed
in the patients’ parents. Heterozygous mice possessing a single Vmat2 allele exhibit no
overt motor phenotype, but do express a depressive behavioral phenotype (Fukui et al.,
2007). Major depression is also observed in parents of patients with AADC deficiency
and thought to be caused by clinically significant reductions in serotonin in these
individuals with hemizygous defects in the serotonin pathway (Brun et al., 2010, Lee et
al., 2012, Pons et al., 2004, Swoboda et al., 1999). To what extent mutations in the AADC
and VMAT2 genes contribute to common depression and its heritability remains to be
investigated.
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Chapter 3 Identification of Disease-Causing Genetic Variant
1 Introduction In Chapter 2, the clinical features of a new autosomal recessive pediatric neurotransmitter
disease are described, and the difficulties are discussed of determining a precise diagnosis
by clinical characterization alone, comprising clinical history, neurological examination,
biochemical investigations (including specific urine and cerebrospinal fluid
investigations), enzyme analysis, and genetic sequencing of candidate genes.
In this chapter, the discovery of the disease-causing variant is presented—initially by
genome-wide single nucleotide polymorphism (SNP) genotyping, homozygosity analysis,
linkage analysis, and mutation screening. Additionally, the advent of whole-exome
sequencing allowed the exclusion of the possibility mutations in other genes in the locus
by whole-exome sequencing of the proband, as well as demonstrating the possibilities of
genomic diagnosis of rare diseases in a clinically relevant timeframe. This is particularly
relevant in the present case, in which a group of related diseases has an overlapping
phenotypic spectrum that is difficult to discern by clinical investigations alone, but the
determination of the underlying genetic etiology precisely determines the successful
course of treatment.
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2 Methods
2.1 Patient Samples The study was approved by the Hospital for Sick Children’s research ethics board and
parents provided informed consent. A total of 5 mL of whole blood was collected in
EDTA tubes. Blood samples were stored at 4°C for up to 1 week.
Genomic DNA was isolated from each sample using a QIAGEN FlexiGene DNA Kit
according to manufacturer’s specifications. Briefly, blood samples were diluted in buffer
and centrifuged to obtain a cell pellet. The cell pellet was resuspended and incubated
with a protease mixture at 65°C for 10 minutes to release genomic DNA into solution.
This genomic DNA was then precipitated by the addition of isopropanol to a final
concentration of 50% and centrifugation. The resulting genomic DNA pellet was then
washed with 70% isopropanol and re-pelleted. After air drying, the genomic DNA was
resuspended in water or Buffer FG3.
2.2 Genotyping of Single Nucleotide Polymorphisms The genotyping of SNPs was performed by the Finnish Genome Center at the University
of Helsinki. A 300K Illumina SNP microarray was used to genotype >300,000 SNPs in
the genomic DNA of eight family members (V:2,3,6,7,8,9;VI:2; see Figure 6).
2.3 Homozygosity Analysis Regions of homozygosity across the genome were identified using PLINK v0.99s
(Purcell et al., 2007). Raw data from the SNP genotyping array was parsed into the input
file format for PLINK using custom PERL scripts. One set of input files was created for
71
data from each chromosome (i.e., data was divided among 22 sets of input files), and
analysis was performed by chromosome.
The parameters specified for homozygosity analysis were a minimum size of 100 kb for
each homozygous region. Additionally, a SNP density of at least 1 SNP per 50 kb was
mandated.
Regions of homozygosity were identified separately in all eight genotyped individuals,
and overlap among homozygous regions was then identified between family members.
These shared regions of homozygosity were then further classified by the disease
affection status of the individuals.
2.4 Linkage Analysis The complexity of this consanguineous family structure prohibited successful power
simulation. To perform linkage analysis, it was necessary to alter the analyzed family
structure to remove consanguineous connections. A modified family structure lacking
consanguineous marriages was therefore used in this analysis.
A subset of 2500 SNPs was selected for parametric linkage analysis. To ensure a
maximally informative SNP subset, the selected SNPs had a minimal allele frequency of
at least 0.4 in the available HapMap dataset (International HapMap Consortium 2003).
An average spacing of approximately 1.0 Mb was selected to provide sufficient
resolution across the genome. This subset was determined manually, and PERL scripts
were designed to parse the data appropriately.
72
Parametric linkage analysis was performed using Merlin software (Abecasis et al., 2002).
A fully penetrant autosomal recessive mode of inheritance was assumed. Marker allele
frequencies were derived from marker allele frequencies in the HapMap dataset, and
disease allele frequency was estimated at 1%. Analysis was performed independently for
each chromosome.
2.5 Mutation Screening Sanger sequencing of candidate gene exons was performed to identify sequence variants
within the homozygous region.
Eight genes in the region were selected as candidates for mutation screening on the basis
of neuronal function or localization: GRK5, EMX2, KCNK18, PRLHR, SLC18A2,
GFRA1, VAX1, and NANOS1. Primers were designed to amplify the exons of each gene
and approximately 50 bp of flanking intronic sequence (Table 12 and Table 13). The
reaction was performed using Taq DNA polymerase (Stratagene) according to
manufacturer’s specifications. Cycling conditions incorporated an initial denaturation at
95°C for 5 minutes, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for
30 s, and a final extension step at 72°C for 10 minutes. Amplification products were
visualized by gel electrophoresis, and purified from bands of the appropriate size using a
Qiagen Gel Purification Kit according to manufacturer’s specifications. Purified PCR
products were sequenced using both forward and reverse primers in separate reactions.
Sequences were compared to reference genome sequence using the BLAT tool in the
UCSC Genome Browser. Putative variants were verified by manual inspection of
electropherograms.
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Table 12 Primer sequences for amplification of exons of candidate genes located at 10q26 Gene Exon Primer sequences Size GRFA1 1 TCA CTG GAT GGA GCT GAA CTT 300 bp
CTT CCT TCC ACA TCC ACC AC 2 CGA AGG CTG GTC AAG CAT C 497 bp
AAG TGA GTG GAG GAT GAG ATC AG 3 GGG GAG AAC AAG GAC AAC AA 398 bp
CCA GAA ACA CTG TGC CAT TC VAX1 1 GCG GGG ACA TTC ATT CTT 534 bp
GCC AAC AAC TTT CTC CCA AG 2 GCC CTC CAC ACA GTG TCT TT 463 bp
CCC AAG GGT AGT TCT GTC CA 3/4 TCT GAA GCA AGC GAA AAA CA 496 bp
TCG AGA GCG AAA CAC TCA AG NANOS1 1 AGT GGG CCC GAT AAA AGG 456 bp
GTC GTC GTC CTC GTC GTA GT 2 CGC ACA CCA TCA AGT ACT GC 393 bp
AGC GCC TCT AAG TTG CCA TA EMX2 1-1 ACC CCA AAC AAA CGA GTC C 490 bp
ATG AGC CAG GGG TAG AAG GT 1-2 GGT AGG GGC GTC TAC TCC A 375 bp
CCG CCT AGT TTC CCA ACA G 2 GTG AGC CCT TGG GAG GAC 474 bp
CAG GCG TGG AAC CAG CTA C 3 GGA GGC TGG ACC TTA GGA CT 422 bp
GTG AAC GTG TAT GCG GTT TG KCNK18 1 CAC ACG CAC CAT CCA CTT AG 486 bp
CTT AAA GTG CCC AGG CAT GA 2 CAC CGA GCT TTG GTG TTG AT 392 bp
GGA AGG GAG GGA AGA AGA GA 3-1 GTA TTT TCA AAA ACA ATG TGT AAA
ATG 500 bp
GTT GCA GTG TGT TCT GTT TCT C 3-2 GCA GAT GAA GCT GTC CCT CA 598 bp
TGT CAC CTG AGA GAT AAT GAA ACC PRLHR 1-1 GTT GTT CTG TGG CCG GTT AT 418 bp
AAG TTC GTC ACG TTG TGC AG 1-2 GGC TGA TCG TGC TGC TCT A 427 bp
AGC TCC ACG TGA TAG GTG TG 1-3 CGC TAC GTC GTG CTG GTG 495 bp
GAG CCA GTG GCA GAG CAG 1-4 GGT GAT CGT GGT GGT GTT C 489 bp
CCC AAG CAA AGA GCA AGA CT
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GRK5 1 GGA ATA ATG CGG TAG GCA AG 337 bp CTC TCC GAA GTG TCC TGC TC
2 TGG GAG GAA GAG TGT GTG TG 400 bp TGT GTC CAC TGA TTG GCA TT
3 GAG TTT GCC AGT CAC CTT CC 398 bp CCT CAG ACA ATT TTG CAT TCC
4 CAG GAG AAG GGG GTT GGT C 300 bp CAC TTA ACA GCT CCC CAT GT
5 GCT GCC AGA TGT ACC AGC A 350 bp TGG TAT AAC TTG GCT GAA GCT G
6 GCA TCT GCA TGG GTT GAG A 300 bp CGG GAG GTA ACT GAT TTT TAT ATG
7 CTC GAA GGT CCA GTC TCC AG 391 bp CAA GGC CAC TGA CTC TCT CC
8 AGG CAT CAC TGG GTC CTG 391 bp ATT AGA GGA CCA CGC CCT TC
9 ACT GAA AGG GAG GAG CAG GT 498 bp AGG GCT GAT TCC CAG AGC
10 AGG AGG TGG GAA GGA AAC AC 299 bp CAG GTA CCC AGC ACT GAG C
11 GAG TGG GCA TTT TCC TGT GT 399 bp AGT GCA TGA AGA GGC GAG AG
12 GGG CAC AGA GGA GAG TCA TC 484 bp CTC AGA CCC CTG TCC CTT C
13 AGC CCT GAA GCA AGA CCT TT 369 bp CAC ACT CTG CAC CAG CTC AC
14 CAT AGC AGT TCT GGG GGT GT 353 bp CTG CCG CCA AAC TCA TAT TC
15 CCA GTG GCT TTG CTG CTG 299 bp CAA GGG TCT CTA CTG TGG GTC T
16 CTG AGG GGA GAC TGC AAA AG 377 bp GTT CTA CGT CGA CGG GAT G
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Table 13 Primer sequences for amplification of SLC18A2 exons SLC18A2 Primer sequences Size 0 GAC TGA CGG AGC CCA CTG 266 bp
ACT GTG CCA CCT CCC AAA CT 1 GCG CTC GAG GCA GGT GAG 438 bp
CCT GGA GGG GAT GTC TTT TT 2 CTC AGC TTC CCA AAG TCC TG 300 bp
CCA CCA TGG ATT TTC CAG AC 3 TTT CAG AAA AGT CCA CCA AAC A 279 bp
CGC CGT CCT TAG GTT GTA TT 4 AAG CAC AGG GTG GCT AAC AT 298 bp
CAA CCA CCA CCA ATA CCT GA 5 AAG GCA CCC ACT TTC CTC TT 365 bp
GGA CCT CTG TGT CAG TGC AA 6-8 CCC AAA GCC TTA TTG GAA CA 595 bp
GCA GCA AAA CGA AAA TCA GC 9 AGG AGG CAG AAG CCA CTA CA 182 bp
GGG GAC AAA TGC TCT TGA AT 10/11 GGG GCT TCG TTT TAT CTG CT 497 bp
AAA TGT TTC TTG GTG ATT CTT GC 12 CCA CCC TTC TTC CTC CTG TT 324 bp
CCT GCA GCC TCC TTC TAA GAT 13/14 CTG GCA GGG TGG TGA GTT TA 599 bp
TTC TCA CCA TTT ATG TTT GAA GG 14 TTG CAT CTT TCA GTC TAC AAG ACA 269 bp
CCA TGA GGA AGC TAT CAG GAA 15 CGA TTG CTC CAA ATG ACT GG 398 bp
CAA TCG ACC ATA ACC ATG GAA
2.6 Whole-Exome Sequencing Whole-exome sequencing was performed to exclude the possibility of additional
mutations present in genes within the homozygous disease-associated region at 10q26.
Exon sequences were first enriched using an Agilent SureSelect V4 50-Mb capture kit
that uses a biotinylated library of RNA baits and streptavidin-coated magnetic beads to
76
specifically capture exon sequences. The exon-enriched samples were sequenced using
the Illumina Hiseq 2000 next-generation sequencing system. These protocols were
performed by The Centre for Advanced Genomics (TCAG). The resulting sequence data
was filtered for coding variants.
2.7 TaqMan SNP Genotyping Assay Genotyping was performed to confirm the absence of the identified mutation in controls.
Differentially fluorescently labelled TaqMan probes were synthesized to represent each
of the SLC18A2 c.1160C and c.1160T alleles. Within an amplification reaction,
fluorescence is released from probes that hybridize to the template DNA through the
5' nuclease activity of DNA polymerase. The ratio of the detected fluorescent labels
thereby determines the genotype of the individual at that locus. The samples were
processed in 96-well format. This assay was performed by TCAG.
3 Results
3.1 Homozygosity Analysis Homozygosity mapping of the microarray results identified a single homozygous 3.0-Mb
interval in 10q25.3–26.11 that was uniquely shared by the affected family members
(chr10:117,937,133–120,972,346), and not homozygous in the unaffected family
members who were genotyped (Figure 7).
Because of the consanguinity of the family structure, a large number of homozygous
regions were expected in individual genomes. Indeed, an average of 30 homozygous
regions of >500 kb were discovered in each genome. These regions of homozygosity in
individual genomes ranged from 780 kb to 68 Mb. Several of these regions were
77
inherited in their entirety by multiple siblings, and overlapped with homozygous regions
inherited by cousins.
A limitation of this homozygosity analysis was related to the density of SNPs. Whereas
the region at 10q25.3–26.11 was the only homozygous region shared by the affected
individuals, the parameters for the analysis specified a minimum size of 100 kb, and it is
possible that smaller homozygous regions would not have been identified. The average
size of a gene in the human genome is 27 kb (Lander et al., 2001), smaller than the
minimum identifiable region in this analysis. More dense SNP coverage would allow
higher confidence assessment of smaller homozygous regions.
78
Figure 7 Single nucleotide polymorphism alleles for all genotyped family members in the homozygous region uniquely shared by affected family members. Black, affected; white, unaffected; square, male; circle, female. From New England Journal of Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission.
79
3.2 Linkage Analysis Parametric linkage analysis produced a significant logarithm of odds (LOD) score of 4.1
within the region of 10q25–26 (Figure 8). One additional significant LOD score of 3.1 on
chromosome 3 did not correspond to a region of homozygosity shared by all affected
individuals. A small positive LOD score was also obtained on chromosome 16, also not
representing a region of homozygosity shared by all affected individuals (Figure 9).
Methodological limitations, however, were several. These included the difficulty in
calculating a logarithm of odds (LOD) score for each putative locus in a highly
consanguineous family. For the purposes of calculation, the consanguineous connections
in the pedigree had to be removed, thereby not accounting for all of the available
inheritance information and reducing the power of the analysis.
80
Figure 8 Multipoint LOD scores estimated across chromosome 10
81
Figure 9 Multipoint LOD scores estimated across chromosomes 3 and 16
82
3.3 Characterization of Genes in the Locus The homozygous region at 10q25.3–26.11 contained 26 known protein-coding genes
(Figure 10), of which eight genes had known neuronal localization or function: EMX2,
GFRA1, GRK5, KCNK18, NANOS1, PRLHR, SLC18A2, and VAX1. The known function
of these eight genes is summarized in Table 14. The exons and exon–intron boundaries of
each of these genes were sequenced in one affected patient, and all identified sequence
variants are listed in Table 15. Additionally, KIAA1598 was not initially identified as a
candidate (and therefore not initially sequenced), but has since been annotated to have
neuronal localization and is included in Table 14. Sequencing revealed two
nonsynonymous variants among the eight sequenced genes: c.847A→G in PRLHR and
c.1160C→T in SLC18A2. The variant in PRLHR was previously identified in the dbSNP
database with a global minor allele frequency of 0.17; this variant is therefore unlikely to
be causative of this disease. The c.1160C→T variant in SLC18A2, however, appears to
be novel and predicts a substitution of proline with leucine at position 387 (p.P387L)
in the VMAT2 protein.
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Table 14 Genes of known neuronal function or localization present in the disease-associated bomozygous region Gene Functional Information (Meyer et al., 2013) EMX2 Homeobox-containing transcription factor homologous to ‘empty spiracles’
gene in Drosophila melanogaster. Expressed in the dorsal telencephalon during development and is proposed to pattern the neocortex into defined functional areas.
GFRA1 A receptor for glial cell line–derived neurotrophic factor (GDNF) and neurturin (NTN). Candidate gene for Hirschsprung disease.
GRK5 G protein–coupled receptor kinase 5. Accumulates in Lewy bodies, a histological hallmark of Parkinson’s disease (Arawaka et al., 2006).
KCNK18 Outward-rectifying potassium channel associated with migraine with aura. KIAA1598 Involved in neuronal polarization NANOS1 Zinc-finger RNA-binding protein expressed in the developing nervous
system and adult brain. PRLHR Prolactin-releasing hormone receptor expressed in the brain and pituitary
gland. Transcription of this gene was shown to be regulated by dopamine D2 receptor agonist bromocriptine (Ozawa et al., 2002).
SLC18A2 Vesicular monoamine transporter 2 responsible for packing monoamines into synaptic vesicles.
VAX1 Homeodomain-containing transcription factor that may play a role in development of the anterior ventral forebrain and visual system.
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Figure 10 Genes present within the homozygous region shared by all affected individuals in the family. C10orf82, chromosome 10 open reading frame 82; CACUL 1, CDK2-assocaited cullin domain 1; CASC2, cancer susceptibility candidate 2 (non-protein coding); CCDC172, coiled-coil domain containing 172; EIF3A, eukaryotic translation factor 3 subunit A; EMX2, empty spiracles homeobox 2; ENO4, enolase family member 4; FAM204A, family with sequence similarity 204 member A; GFRA1, GDNF family receptor alpha 1; GRK5, G protein–coupled kinase 5; HSPA12A, heat shock 70 kDa protein 12A; KCNK18, potassium channel subfamily K member 18; KIAA1598, Shootin1; MIR, microRNA; PDZD8, PDZ domain containing 8; PNLIP, pancreatic lipase; PNLIPRP, pancreatic lipase–related protein; PRDX3, peroxiredoxin 3; PRLHR, prolactin-releasing hormone receptor; RAB11FIP1, RAB11 family interacting protein 2 (class I); SFXN4, sideroflexin 4; SLC18A2, solute carrier family 18 member 2; SNORA19, small nucleolar RNA H/ACA box 19; VAX1, ventral anterior homeobox 1. Image from UCSC Genome Browser [http://genome.ucsc.edu]
chr10 (q25.3-q26.11) 10p14 10p13 p12.1 10q21.1 21.2 10q21.3 q22.1 q22.3 10q23.1 10q25.1 q25.3 26.13 q26.3
Scalechr10:
1 Mb hg19118,500,000 119,000,000 119,500,000 120,000,000 120,500,000 121,000,000
UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics)GFRA1GFRA1GFRA1
CCDC172PNLIPRP3
JA611286PNLIP
PNLIPRP1PNLIPRP1
PNLIPRP1PNLIPRP1
PNLIPRP2PNLIPRP2PNLIPRP2
C10orf82
C10orf82
HSPA12AHSPA12A
DQ596646ENO4
ENO4KIAA1598
KIAA1598KIAA1598KIAA1598KIAA1598AK092331
KIAA1598
KIAA1598
VAX1VAX1
BC039338MIR3663
KCNK18SLC18A2SLC18A2
PDZD8EMX2OSEMX2OS
EMX2
EMX2
RAB11FIP2RAB11FIP2
CASC2CASC2CASC2CASC2CASC2
FAM204AFAM204A
PRLHRPRLHR
Mir_584CACUL1CACUL1
NANOS1EIF3AEIF3AEIF3A
SNORA19SNORA19
FAM45BFAM45BFAM45BFAM45BFAM45B
BC042590SFXN4SFXN4SFXN4
PRDX3
PRDX3
GRK5GRK5
GRK5
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Table 15 Sequence variants identified among eight candidate genes in the disease-associated locus by direct sequencing of exons Gene Segment Sequence Variation Present in
dbSNP EMX2 Exon 1 none –
Exon 2 none – Exon 3 none –
GRK5 Exon 1 none – Exon 2 none – Exon 3 c.149-6G→A rs2275036
KCNK18 Exon 1 none – Exon 2 none – Exon 3 none –
PRLHR Exon 1 c.847A→G (I283V) rs1613488 SLC18A2 Exon 1 none –
Exon 2 none – Exon 3 none – Exon 4 none – Exon 5 none – Exon 6 c.700+15C→T rs2072362 Exon 7 c.790+54C→T rs363420 Exon 8 c.791-42C→A rs363343 Exon 9 none – Exon 10 none – Exon 11 none – Exon 12 c.1122+20T→C rs363227 Exon 13 c.1160C→T (P387L) – Exon 14 c.1306+43A→G rs363272 Exon 15 none – Exon 16 c.1441-48T→C rs363279
VAX1 Exon 1 none – Exon 2 none – Exon 3 none – Exon 4 none –
GRFA1 Exon 1 none – Exon 2 none – Exon 3 none –
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3.4 Whole-Exome Sequencing of the Proband We also performed whole-exome sequencing in the proband, and the data were filtered to
reveal 9,821 coding variants in the genome. Of these, 3,900 coding variants were present
in the homozygous state. This large number reflects the extent of homozygosity in the
genome of this individual resulting from her consanguineous background. A total of 70
homozygous, non-coding variants were not previously represented in the NCBI dbSNP
database. As a member of an isolated, Bedouin population, there may be poor
representation of this patient’s particular ethnic background among the ethnically diverse
samples genotyped to date. This presents a current limitation of the ability to exclude
variants on the basis of their presence in the general population. In the absence of a clear
functional candidate, it would be warranted to perform whole-exome sequencing on
additional affected and unaffected family members to narrow the list of shared
homozygous sequence variants, and identify those that are present homozygously more
broadly within the pedigree.
However, this analysis independently identified the homozygous variant in SLC18A2
(VMAT2-P387L) and the aforementioned c.847A→G variant in PRLHR, and revealed no
other novel non-synonymous variant in the linked region of shared homozygosity at
10q25.3–26.11
3.5 Identification of VMAT2 Variant as a Causative Candidate
The identified novel variant in SLC18A2 (c.1160C→T) was present in exon 13 (Table 15)
and predicts a substitution of proline with leucine at position 387 (p.P387L) in the
VMAT2 protein. To confirm that the variant segregated with the disease, the region was
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sequenced in all available affected and unaffected family members, including siblings
and parents. The mutation was present in homozygous form in all affected family
members and was not present, or was present in heterozygous form, in unaffected
siblings (Figure 11). All genotyped parents carried the variant in heterozygous form.
The SLC18A2 c.1160C→T variant has not previously been identified in the literature, and
is not listed in dbSNP or the 1000 Genomes Database.
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Figure 11 Electropherogram depicting sequence variation in unaffected and affected family members. Top panel, unaffected sibling with wild-type genomic sequence; middle panel, asymptomatic parent (IV:3) possesses the SLC18A2 c.1160C→T variant in heterozygous form;; lower panel, proband (V:6) is
homozygous for SLC18A2 c.1160C→T. From New England Journal of Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission.
89
3.6 Controls To confirm that the SLC18A2 c.1160C→T variant is not present in unaffected members
of the extended kindred, TaqMan genotyping was performed on DNA samples isolated
from direct relatives of the patients. The SLC18A2 c.1160C→T variant is not present in
homozygous form in 78 unaffected members of the extended family, 26 of whom do
carry it in the heterozygous state.
In addition, as one of the most studied candidate genes for involvement in Parkinson’s
disease, SLC18A2 was previously screened in 704 healthy individuals of diverse ethnic
backgrounds and 452 Parkinson’s disease patients (Burman et al., 2004, Glatt et al., 2001,
Iwasa et al., 2001), none of whom had the c.1160C→T change. The 1000 Genomes
Database (Abecasis et al., 2010) identifies 44 missense variants in SLC18A2 that are not
associated with an overt phenotype, but none within the same codon.
4 Discussion Collectively, the results presented in this chapter identify and confirm SLC18A2
c.1160C→T as the disease-causing mutation in this family. Consistent with the observed
autosomal recessive mode of inheritance, the mutation is present in homozygous form in
all affected individuals, and those who possess the mutation in heterozygous form do not
exhibit the movement disorder phenotype. All affected individuals are direct descendants
of individuals I:1 and I:2 (see Figure 6). The expression of the movement disorder
phenotype begins in the fifth generation of this consanguineous kindred, consistent with a
founder mutation likely arising in one of these two individuals.
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No disease-associated mutations have been identified in SLC18A2 (VMAT2) to date.
There is no evidence of the SLC18A2 c.1160C→T variant in genomic databases of
healthy individuals. Additionally, the 1000 Genomes Database identifies no variants
predicted to cause a premature stop codon in VMAT2. Of 44 identified VMAT2
missense variants in this database, 12 are predicted to be deleterious (“probably
damaging”) by PolyPhen (a further 10 are “possibly damaging”). By the SIFT algorithm,
20 were considered deleterious. In the Exome Variant Server of the NHLBI GO Exome
Sequencing Project, a single frameshift deletion allele was identified, but this deletion
involved the coding sequence for the final amino acid of the protein and may therefore be
benign. Importantly, none of the 25 missense variants identified in this database (a subset
of the 44 missense variants catalogued in the 1000 Genomes Database) were present in
homozygous form.
In addition, the screening of SLC18A2 in 704 healthy individuals of diverse ethnic
backgrounds and 452 Parkinson’s disease patients identified only 5 missense variants
(only two of which are not missense variants represented in the 1000 Genomes Database)
(Burman et al., 2004, Glatt et al., 2001, Iwasa et al., 2001). Four of these missense
variants were functionally screened, and determined to have only modest effects on
protein function (Burman et al., 2004).
It has been suggested that VMAT2 exhibits a low rate of variation relative to genes of
related function, such as the dopamine and serotonin plasma membrane transporters
(DAT and SERT, respectively) (Glatt et al., 2001). This implies functional significance
of the protein, and therefore suggests that a dramatically deleterious mutation in VMAT2
would have correspondingly dramatic phenotypic effects. Indeed, the 1000 Genomes
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Database reports 151 missense mutations in DAT and 317 missense mutations in SERT,
both transmembrane proteins of comparable length to VMAT2 and related function in the
central nervous system. Importantly, 726 missense mutations are reported in the 1000
Genomes Database for the non-neuronal VMAT isoform, VMAT1. In comparison with
the respective 44 missense mutations currently identified in VMAT2 in the same sample
set, these figures therefore illustrate that a functionally deleterious mutation in VMAT2
would be predicted to have significant phenotypic effects—particularly if identified in
homozygous form, as observed here for the VMAT2 p.P387L variant.
Proline residues located adjacent to transmembrane domains have major structural
implications, and are overrepresented among disease-causing substitutions in
transmembrane domains (Partridge et al., 2004). The P387L substitution is immediately
adjacent to a transmembrane segment (Figure 12), and the constraints imposed by the
proline residue on the flexibility of the peptide backbone are likely important for
insertion of the transmembrane domain into the membrane to form the proper tertiary
configuration.
Sequence alignment shows that the P387 residue is highly conserved through evolution,
and is therefore of likely structural or functional significance. It is also conserved in the
VMAT1 paralog, as well as in C. elegans CAT-1—the single vesicular monoamine
transporter in nematode (Figure 13) (Duerr et al., 1999). Interestingly, the residue is not
conserved in the vesicular acetylcholine transporter (VAChT), which maintains 39%
identity to VMAT2, implying that P387 may have a specific implication in monoamine
transport.
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Furthermore, the P387L substitution is predicted to be “probably damaging” by the
PolyPhen-2 algorithm (Adzhubei et al., 2010) with a score of 1.000. This algorithm
considers eight sequence-based and three structure-based predictive features, and was
developed using large datasets of known damaging (Mendelian disease-causing or protein
destabilizing) and nondamaging alleles.
The evidence presented in this discussion—the segregation of the variant consistent with
an autosomal recessive inheritance pattern, the absence of the variant in healthy
individuals of ethnically diverse populations, evolutionary conservation of the residue, its
potential structural relevance, and the low rate of genetic variation in SLC18A2—together
predicts the likely functional relevance of the p.P387L substitution in VMAT2 (SLC18A2
c.1160C→T).
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Figure 12 Predicted structure of VMAT2 comprises 12 transmembrane domains, a large lumenal loop including four proposed glycosylation sites, and both N-terminal and C-terminal cytoplasmic regions. Proline residue 387 is located immediately adjacent to the insertion of transmembrane domain X. From New England Journal of Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission.
94
Figure 13 Multiple sequence alignment of VMAT2 in the region of the p.P387L variant. TM9 and TM10 represent sequences associated with transmembrane domains 9 and 10, respectively. Residues that differ from human VMAT2 sequence are indicated in gray. Amino acid position 387 is indicated by an asterisk. Homo, homo sapiens (human); Patient, proband; Pan, Pan troglodytes (chimpanzee); Macaca, Macaca mulatta (Rhesus macaque); Mus, Mus musculus (mouse); Rattus, Rattus norvegicus (rat); Canis, Canis familiaris (dog); Bos, Bos taurus (cow); Monodelphis, Monodelphis domestica (opossum); Gallus, Gallus gallus (chicken); Tetraodon, Tetraodon nigroviridis (pufferfish); Danio, Danio rario (zebrafish); Drosophila, Drosophila melanogaster (fruit fly); C. elegans, Caenorhabditis elegans (nematode); hVMAT1, human VMAT1 isoform. From New England Journal of Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission.
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Chapter 4 Functional Characterization of the Disease-Causing Variant
1 Introduction The identification of the putative disease-causing mutation is described in Chapter 3. As
presented in that chapter, the SLC18A2 c.1160C→T variant segregates in a manner
consistent with the observed autosomal recessive inheritance of the disease. The
corresponding VMAT2 p.P387L substitution is predicted to have a structural role within
the protein, and the proline residue at this position is conserved in all vesicular
monoamine transporters isoforms in all species.
The phenotype expressed by the patients harboring SLC18A2 c.1160C→T is similar to
that expressed by patients with biosynthetic deficiencies of the biogenic amine
neurotransmitters, predicting a loss of VMAT2 function that would interrupt vesicular
storage in the brain and expose biogenic amines to degradation. The patient CSF
neurotransmitter metabolite profile presented in Chapter 2 is consistent with this
hypothesis, and the hypothesis is tested in the present chapter.
2 Methods
2.1 Construct Design and Site-Directed Mutagenesis Human VMAT2 cDNA sequence was acquired from the Mammalian Gene Collection
and subcloned into pcDNA3.1A (Invitrogen). The primers used for amplification were
designed to incorporate restriction sites flanking the open reading frame (Table 16). The
P387L (c.1160C→T) mutation was introduced by site-directed mutagenesis using the
QuikChange Site-Directed Mutagenesis kit (Stratagene). The oligonucleotide sequences
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for site-directed mutagenesis are presented in Table 17. The final sequence of the vector
was confirmed by sequencing using standard M13 primers that are complementary to
vector sequences.
Table 16 Primers to amplify SLC18A2 cDNA sequence for subcloning into pcDNA3.1 Primer Sequence VMAT2-KpnI-F CAT GGT ACC AGT AGT ATG GCC CTG AGC GAG CTG G VMAT2-PmeI-R CAT GTT TAA ACA CTA CTT CAG TCA CTT TCA GAT TCT
TCA TC
Table 17 Oligonucleotides to introduce cytosine to thymine substitution at position 1160 of SLC18A2 using site-directed mutagenesis Primer Sequence VMAT2-P387L Quikchange-F
AAA CAT TTA TGG ACT CAT AGC TCT GAA CTT TGG AGT TGG TTT TGC
VMAT2-P387L Quikchange-R
GCA AAA CCA ACT CCA AAG TTC AGA GCT ATG AGT CCA TAA ATG TTT
2.2 Cell Culture and Transfection The wild type and mutant constructs were expressed in Cos7 cells, as there is no
endogenous VMAT1 or VMAT2 expressed in these cells. Cos7 cells were maintained in
Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) at
37°C and 5% CO2. Cells were passaged by trypsinization, except immediately prior to
transfection for the serotonin uptake assay; cells prior to the uptake assay were removed
from the surface of the plate by scraping. In both cases, cells were diluted 1:10 for
seeding onto fresh plates.
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Transient transfection of Cos7 cells was performed at 80% confluency using Fugene HD
transfection reagent (Roche) according to manufacturer specifications. After 48 hours,
cells were washed once in 1× phosphate-buffered saline (PBS), scraped, and pelleted for
further experiments. Cells from three 10-cm plates that had been transfected in parallel
were combined into a single pellet.
2.3 Serotonin Uptake Assay Vesicular serotonin uptake was assayed in a heterologous cell system. VMAT2 transport
activity was measured by incubating membrane preparations with tritiated serotonin,
followed by rapid washing and filtration to retain vesicles with trapped substrate.
Transiently transfected Cos7 cell pellets were immediately resuspended in 320 mM
sucrose-HEPES buffer (pH 7.4) and sonicated by 20 × 1 s pulses. Lysates were
centrifuged at 4,000g for 5 min, and supernatants were stored at –80°C until use.
Reaction buffer contained 150 mM choline gluconate, 10 mM HEPES-Tris (pH 7.4), 2
mM Mg-ATP, and 90 nM 3H-serotonin (New England Biolabs). Where indicated,
reaction buffer also contained 10 µM reserpine. To each tube was added 10 µL of
microsomal lysate, followed by incubation at 30°C for the specified time. Reactions were
stopped by rapid filtration, and retained serotonin was measured by scintillation counting.
Retained serotonin was also measured from samples incubated on ice and filtered
immediately upon addition of the lysate, as well as from reaction buffer alone.
2.4 Western Blotting Western blots were performed by equal loading of protein lysates onto 10% SDS-PAGE
gels. Proteins were transferred to PVDF membrane in a Bio-Rad semidry transfer cell at
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1 mA/cm2 for 45 min. Transfer buffer was 200 mM glycine, 25 mM Tris-HCl, pH 7.5,
and 20% methanol. The membrane was blocked for 1 h in 50 mM Tris-HCl, pH 7.5, 150
mM NaCl, and 0.05% Tween 20 (TBS-T) with 5% skim milk. Blots were incubated in
blocking solution with primary antibody for 1 h at 4°C, washed three times with TBS-T,
and then incubated with secondary antibody in blocking solution for 1 h at 4°C, followed
by three TBS-T washing steps and detection by chemiluminescence according to
manufacturer’s recommendation.
The specificity of a commercial antibody against a 20–amino acid C-terminal peptide of
VMAT2 (Abcam) was verified by western blot, showing no non-specific signal in
untransfected or vector-transfected cultures (see inset to Figure 19). Secondary antibody
was horseradish peroxidase–conjugated anti-rabbit antibody.
2.5 Sucrose Gradient Centrifugation To compare the subcellular localization of the wild-type and p.P387L VMAT2 proteins
in Cos7 cells used for serotonin uptake assays, cell lysates were fractionated by sucrose
gradient centrifugation, and the presence of the protein in each fraction was visualized by
western blot.
Transiently transfected Cos7 cell pellets were immediately resuspended in 320
mM sucrose-HEPES buffer (pH 7.4) and sonicated by 20 × 1 s pulses. Lysates were
centrifuged at 4000 g for 5 min, and protein concentrations were measured by Bradford
assay to ensure equal protein loading on gradients tested in parallel. Supernatants were
immediately applied to a linear sucrose gradient of 20%–55% sucrose (approximately
0.6–1.6 M) in HEPES (pH 7.4) and centrifuged at 150 000 g for 16 h at 4°C.
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Fractionation was performed using a Brandel Density Gradient Fractionation
system. Aliquots of equal volume were separated by SDS-PAGE on 4%–12% Bis-Tris
gels (Invitrogen). Blots were prepared as described in Section 2.4.
Linear sucrose gradients were generated by tilted tube rotation on a BioComp
Gradient Master. The linearity of select gradients was confirmed by the addition of ATP
in the heavy phase (50% sucrose) prior to gradient generation, and measurement of
absorbance at 260 nm during fractionation (Figure 14).
Figure 14 Visualization of ATP by absorbance at 260 nm to illustrate sucrose gradient linearity
The consistency of gradient fractionation between samples was demonstrated by
the measurement of absorbance at 280 nm during the fractionation process. An initial
peak in the first fraction, likely representing free cytosolic contents, is followed by a
trimodal peak present approximately between fractions 7 and 12. All fractionations
exhibited the same pattern of absorbance, and parallel fractionations produced
absorbances of equal intensity (Figure 15). This demonstrates the reproducibility of the
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gradient centrifugation process. Some minor shift in the absorbance peaks relative to
particular numbered fractions resulted from variation in the fractionation process.
Figure 15 Consistent fractionation demonstrated by absorbance at 280 nm of parallel sucrose gradient fractionations
Protein markers of cellular compartments were visualized by western blot. GAPDH was
used as a cytosolic marker, and was present only fractions 2 and 3, verifying a lack of
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cytosolic contamination in later fractions. Transferrin receptor (TfR) was used as a
marker for recycling endosomes, and was reproducibly visualized at highest
concentration in fractions 5–9, with faint signal detectable in later fractions. Calnexin
was used as a marker for endoplasmic reticulum, and was present primarily in fractions
9–13, but with signal detectable in all fractions. Overlapping signal demonstrates
incomplete separation of the microsomal components of the cell, but the relative order of
components is as expected.
Figure 16 Markers of cellular compartments in sucrose gradient fractions
3 Results
3.1 Steady-State Protein Levels of Transfected Protein Unaffected by P387L Variant in Heterologous System
To determine the effect of the p.P387L mutation on VMAT2 transport activity, we
transiently expressed wild-type and mutant human VMAT2 in Cos7 cells.
Immunoblotting of membrane preparations confirmed equivalent protein levels of
VMAT2 in parallel transfections, suggesting no major defect in protein processing
(Figure 17).
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Figure 17 Parallel transient transfections of wild-type and p.P387L VMAT2 in Cos7 cells, and vector-transfected control. GAPDH, protein loading control.
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The wild-type and p.P387L VMAT2 constructs exhibit the same intensity and banding
pattern on western blot, indicative of multiple glycosylation forms. This banding pattern
is better illustrated in Figure 18. In transiently transfected Cos7 cells, bands of
approximately 57 and 45 kDa are most prominent, with a lower molecular weight band of
lesser intensity at approximately 35 kDa. These bands are consistent with those observed
in previous studies by Tong et al., who examined the expression of VMAT2 in autopsied
human brain tissue, and reported four protein bands of approximately 75, 55, 45, and 35
kDa (Tong et al., 2011). A pair of higher molecular weight bands are also visible, but
have a molecular weight of approximately 90 kDa in this heterologous cell system. The
bands less distinct than is observed in brain tissue, and multiple bands of intermediate
size are also visible and appear as a smear—this was interpreted as an artifact of
overexpression in the heterologous system. Because glycosylation does not substantially
affect VMAT transport activity (Yelin et al., 1998), no further investigation of the
identity of the protein bands was performed.
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Figure 18 Comparison of banding pattern observed for wild-type and p.P387L VMAT2 expressed transiently in Cos7 cells. unt, untransfected; WT, wild-type.
3.2 Highly Reduced Transport Activity of VMAT2 p.P387L in Heterologous System
The p.P387L variant of VMAT2 was assayed for its ability to transport tritiated serotonin
in a crude vesicle lysate isolated from Cos7 cells transfected in parallel with either the
wild-type or p.P387L VMAT2 construct, or vector alone. After incubation with tritiated
serotonin, samples were rapidly filtered to retain microsomes, and the trapped substrate
retained on the filter was measured by scintillation counting.
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The p.P387L variant of VMAT2 showed dramatically decreased serotonin uptake activity
relative to wild-type VMAT2 (Figure 19). The sensitivity of the assay did not permit
quantification of the uptake kinetics of p.P387L VMAT2, or quantification of the relative
decrease in activity compared with the wild-type protein. In parallel time courses, the
activity of the p.P387L mutant of VMAT2 was indistinguishable from background, as
measured in lysates isolated from vector-transfected Cos7 cultures.
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Figure 19 Vesicular uptake of tritiated serotonin by the wild-type and p.P387L human VMAT2 transiently expressed in a heterologous Cos7 cell system. Inset: Expression of VMAT2 in lysates used for uptake assay. 5-HT, serotonin; wt, wild-type. From New England Journal of Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission.
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3.3 Measurable Residual Transport Activity of VMAT2 p.P387L Protein
Because a complete lack of VMAT2 function would be expected to be lethal on the basis
of mouse models (Wang et al., 1997), the activity of the p.P387L mutant of VMAT2 was
more closely investigated. The specific VMAT inhibitor reserpine was used to better
distinguish VMAT2 transport activity from the nonspecific uptake or retention of
substrate on the filters observed in lysates from vector-transfected cultures.
Microsomal uptake of tritiated serotonin was assayed at the 10-minute timepoint—the
time at which the reaction begins to saturate (Figure 19)—with and without the addition
of 10µM reserpine. The p.P387L mutant VMAT2 exhibited weakly measurable uptake
that was approximately three times the background level measured with the addition of
reserpine (Figure 20). Therefore, the VMAT2 p.P387L mutation reflects a severe, but
not complete, loss of protein function.
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Figure 20 Vesicular uptake of tritiated serotonin by wild-type and p.P387L human VMAT2 transiently expressed in a heterologous Cos7 cell system after 10 minutes with and without the addition of the specific VMAT inhibitor reserpine (10 µM). From New England Journal of Medicine, Rilstone JJ, Alkhater RA, Minassian BA, Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment, 368, 543–50. Copyright © 2013 Massachusetts Medical Society. Reprinted with permission.
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3.4 Subcellular Localization of Protein in Heterologous System
Sucrose gradient centrifugation was performed to compare the subcellular localization of
wild-type VMAT2 protein with that of VMAT2-P387L in the transiently transfected
Cos7 lysates that were used for the serotonin uptake assay. Preliminary data reveals
signal in the majority of gradient fractions for both proteins, with the highest
concentrations of protein in fractions 7–12 (Figure 21). The broad distribution of
VMAT2 protein across gradient fractions is indicative of either broad localization in
multiple microsomal components of the cell, or it is reflective of ER contamination in
multiple fractions. In either case, the lack of confinement of VMAT2 to its expected
compartment—recycling endosomes—is likely a consequence of overexpression in the
transient transfection setting.
Given the expected elution of fractions containing the highest proportion of ER in
fractions 9–13 relative to the earlier elution of recycling endosomes in fractions 4–9
(Figure 16), extensive misfolding of the mutant protein relative to wild-type VMAT2
would be expected to produce a rightward shift in the distribution of VMAT2 across
fractions. No such pattern is observed. This result is therefore inconclusive, but does
suggest no significant difference in localization between the wild-type and mutant
VMAT2 proteins, and likely a lack of specificity of protein localization as a result of
protein overexpression. This preliminary result therefore suggests that the difference in
serotonin uptake between wild-type VMAT2 and VMAT2-p.P387L is not reflective of a
drastic difference in the subcellular distribution of the proteins, but rather a true
difference in transport function. Verification of this observation should be performed
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using an alternative technique such as immunofluorescence. In addition, data regarding
the subcellular localization of VMAT2-P387L in the physiological setting should be
pursued using cell lines with stable expression of the constructs to avoid artifacts of
overexpression.
Figure 21 Sucrose gradient centrifugation of transiently transfected Cos7 lysates
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4 Discussion Direct characterization of the mutant VMAT2 protein in a heterologous cell system in
this study revealed a severe detriment of vesicular transport function. This could be the
result of poor incorporation of the transporter into vesicle membranes or lost transport
activity; data regarding the relative subcellular distribution of wild-type and p.P387L
VMAT2 was inconclusive, but suggestively not the major factor underlying loss of
function.
The p.P387L substitution is immediately adjacent to transmembrane domain 10 (see
Figure 12). Prolines can have significant structural and functional significance in
transmembrane domains because the constraints imposed by their ring structure induce
kinks in α-helices. Prolines are also preferentially located at the central and terminal
regions of transmembrane segments (Cordes et al., 2002). Though prolines constrain the
angle of the peptide backbone, they also induce flexibility in the region through the
disruption of stabilizing hydrogen bonds in upstream amino acids, and have therefore
been implicated as hinge points with possible functional relevance (Cordes et al., 2002).
Prolines are prevalent in the transmembrane domains of ion channels and transport
proteins, but not the transmembrane segments of proteins without transport function,
inspiring a hypothesis that cis-trans isomerization may underlie the conformational
change inherent in the transport process. Additionally, the disruption of hydrogen bonds
in the peptide backbone that is imposed by proline exposes a carbonyl group, providing a
site for cationic ligand binding or proton translocation (Brandl and Deber, 1986, Williams
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and Deber, 1991). Finally, Partridge et al. reported an overrepresentation of proline
substitutions among disease-causing mutations located in transmembrane domains,
indicating a high phenotypic propensity for amino acid substitutions of this nature
(Partridge et al., 2004).
With respect to VMAT2 structure specifically, a hydrogen bond between D400 in
transmembrane domain 10 and Y342 in transmembrane domain 8 was predicted in a
homology model of the rat protein, rVMAT2, and the D400 residue was verified
experimentally to be critical to transport activity (Yaffe et al., 2013). The relative
configuration of transmembrane domain 10 and transmembrane domain 8 is therefore
functionally critical, underscoring the likely importance of proline constraints on the
peptide backbone near the insertion of transmembrane domain 10. This, however, does
not exclude the possibility of a role for the P387 residue in conformational change, ligand
binding of the cationic monoamine substrates, or proton translocation.
Direct biochemical characterization in a heterologous system confirms the functional
significance of the P387L substitution. This functional biochemical evidence—in
combination with genetic linkage data, bioinformatic analysis, and the observed treatment
effect—confirms the causative role of the VMAT2 p.P387L mutation in this autosomal
recessive pediatric neurotransmitter disorder.
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Chapter 5 General Discussion and Future Directions
1 Introduction This thesis presents the identification of a new pediatric neurotransmitter disease and its
causative mutation in the gene encoding the brain vesicular monoamine transporter
(VMAT2; SLC18A2), as well as demonstrating the utility of next-generation sequencing
for the clinical diagnosis of extremely rare diseases.
This discovery extends the spectrum of pediatric neurotransmitter disease
pathophysiology to include defects in the vesicular transport and storage of biogenic
amines. This is the first demonstration of a clinical phenotype directly associated with
VMAT2 deficiency, providing new insight on the physiological consequences of
perturbed monoamine homeostasis in humans.
The research directions and experiments proposed in this chapter are designed to address
open questions stemming from the results presented here pertaining to the role of
VMAT2 in monoamine physiology (Section 2), the pathophysiology of VMAT2
deficiency (Section 3), and the diagnosis and treatment of pediatric neurotransmitter
diseases (Section 4).
In Section 2, a series of experiments are proposed to further probe the dose dependence
of VMAT2 function and its role as a modulator of monoamine homeostasis, leveraging
the P387L variant to demonstrate the effect of reduced VMAT2 function without the
confounding effects of reduced protein levels or off-target effects of drug inhibitors.
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Subcellular localization experiments in relevant cell culture models are proposed to
explore any mechanisms of regulation of VMAT2 in the context of reduced function.
These results will also inform data derived from a proposed mouse model incorporating
the corresponding P390L mutation.
In Section 3, some investigations are proposed to further explore the pathophysiology of
VMAT2 deficiency by obtaining more data in a clinical context. Positron emission
tomography (PET) is proposed to visualize aberrant VMAT2 and dopamine distribution
in vivo. Genetic screening of patient cohorts with neurological disorders is also proposed
to identify additional patients with VMAT2 mutations and better characterize the range of
phenotypes associated with VMAT2 deficiency.
In Section 4, challenges in the diagnosis of VMAT2 deficiency are described and some
methodologies are described that may address the current limitations of clinical
diagnosis. In addition, the promise and challenges of next-generation sequencing for rare
disease diagnosis are outlined.
In sum, this section outlines three different directions for continued research stemming
from the results of this thesis and discusses the relevance of these results to clinical
practice.
2 Identification of the VMAT2 p.P387L Mutation: Impact on Knowledge of Monoamine Physiology
As the first demonstration of a phenotype associated with VMAT2 deficiency in human
patients, the results presented in this thesis confirm that significant loss of VMAT2
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function is associated with a phenotype that is within the spectrum of those caused by
biosynthetic monoamine deficiencies.
The VMAT2 p.P387L variant, which is associated with viability despite corresponding to
a near complete—but not complete—loss of VMAT2 function as measured by functional
assay in a heterologous system, provides an interesting opportunity to further probe the
role of VMAT2 in monoamine physiology and homeostasis. In mice, the complete
knockout of Vmat2 results in absent exocytotic monoamine neurotransmission, and
animals that feed poorly and die within days after birth (Wang et al., 1997, Fon et al.,
1997). By contrast, mice that express just 5% of native Vmat2 levels live to adulthood
and only develop comparatively minor age-related motor deficits over time (Mooslehner
et al., 2001). The phenotypic spectrum of VMAT2 deficiency is therefore broad, and
very large decreases in protein function may be required to result in severe, early onset
motor symptoms. However, there may be differences in the phenotypes caused by lower
protein levels and aberrant protein function, possibly stemming from differences in
VMAT2 trafficking and its relative distribution between readily releasable and recycling
or reserve pools of synaptic vesicles. Future research should clarify the mechanism of
loss of function by confirming whether protein localization to synaptic vesicles is
affected by the mutation in vivo. Appropriate cell culture models can be used to
investigate VMAT2 sorting. These experiments are discussed in Section 2.1.1.
Initial confirmation of reduced uptake activity of a single substrate was considered
sufficient in the present work to confirm the functional effect of the VMAT2 p.P387L
variant. More precise characterization of the p.P387L mutation may be of interest for
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drawing broader insights on VMAT2 structure–function relationships. Proposed
experiments are described in Section 2.1.2.
Because VMAT2 density on synaptic vesicles determines quantal size for monoamine
neurotransmission (Edwards, 2007, Pothos et al., 2000), and its function is therefore
subject to dose dependence, unique insights may be gleaned into monoamine physiology
by investigating the effect of the corresponding Vmat2 p.P390L mutation in a mouse
model that were not derived from studying one of the existing mouse models. The
proposed mouse model is described in Section 2.1.3.
2.1 Future Directions and Experiments
2.1.1 Subcellular Localization of VMAT2 p.P387L
Subcellular localization of VMAT2-P387L can be assessed in relevant cell culture
models using immunofluorescence colocalization and sucrose gradient centrifugation
using relevant cell compartment markers (e.g., synaptophysin for synaptic vesicles). To
avoid the artifacts of overexpression introduced by transient transfection (as in Chapter 4,
Section 3.4), cell lines should be constructed to stably express either wild-type or mutant
VMAT2 protein.
The following section describes the construction of these cell lines. Stable expression of
VMAT2 (wild-type and P387L) was confirmed in both Cos7 cells and the MN9D
mesencephalic cell line. MN9D cells were originally derived as a fusion of embryonic
ventral mesencephalic and neuroblastoma cells, and are extensively used as a model of
dopamine neurons because they express tyrosine hydroxylase and synthesize and release
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dopamine. These cells can be differentiated with exposure to butyric acid, and develop
some morphological characteristics of neurons such as visible processes. Because these
cells possess synaptic vesicles, they have already been used in studies of VMAT2
function, in which it was shown that overexpression of VMAT2 confers additional
protection against MPP+ (Chen et al., 2005). Interestingly, co-expression of
synaptophysin enhanced this effect possibly through increased vesicular capacity. The
co-expression of synaptophysin may therefore also be helpful in these proposed
localization experiments.
2.1.1.1 Generation of Stable Cell Lines Expressing VMAT2 p.P387L
Cell lines were generated to stably express human VMAT2. Independent cell lines were
generated to express wild-type VMAT2 and VMAT2-P387L. Additionally, cell lines
with genomic incorporation of the vector backbone alone were generated for use as
experimental controls.
Stable expression of human VMAT2 was generated in the Cos7 cell line. Cos7 cells were
maintained as described in Chapter 4 (Section 2.2). The pcDNA3.1 vectors containing
wild-type VMAT2 and VMAT2-P387L cDNA sequence, in addition to empty pcDNA3.1
vector, were used in independent transfections. Prior to transfection, the vector was
linearized to optimize genomic incorporation of the construct. Transfection was
performed with Fugene HD transfection reagent (Roche), according to the manufacturer’s
specifications. After 48 hours to allow expression of the neomycin cassette on the vector,
the cells were trypsinized and a series of dilutions from 1:10 to 1:1000 were plated
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independently in DMEM containing 500 µg/mL of G418. The selection medium was
replaced each day with fresh medium until the appearance of visible colonies. Colonies
were transferred to 96-well plates containing trypsin and subsequently diluted into
DMEM + 10% FBS + 500 µg/mL G418. Individual wells were eventually passaged into
larger diameter wells and screened for VMAT2 expression by western blot (Figure 22).
Positive clones were expanded and frozen at –80°C
Figure 22 Expression of wild-type and p.P387L VMAT2 in stable clones of the Cos7 cell line; band at 37 kDa represents GAPDH loading control.
MN9D cells were maintained in custom DMEM media at pH 7.2 containing 10% Fetal
Clone 3 (Hyclone) and 50 U/mL penicillin/streptomycin. They were cultured in flasks at
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37°C and 5% CO2, and fed every other day by aspiration of the media and replacement
with fresh media. Cells were passaged by trypsinization and seeding into new flasks at
plating densities of 200,000 per T75 flask and grown to confluence. The number of
passages was minimized to avoid morphological changes.
Because MN9D cells endogenously express mouse Vmat2, the human VMAT2 protein
was tagged internally with an HA epitope to distinguish the isoforms. Initial attempts to
incorporate N-terminal and C-terminal FLAG and myc tags led to no detectable protein
by western blot. The HA tag was therefore incorporated into the large lumenal loop
immediately after Arg94, as described by Thiriot and Ruoho (Thiriot and Ruoho, 2001).
The tag was incorporated by site-directed mutagenesis using complementary
oligonucleotide sequences that incorporated the HA epitope (Table 18). The transfection
and selection of clones was performed as described for generation of the Cos7 stable cell
lines. Confirmation of human VMAT2 expression was performed by western blot using
an antibody against the HA epitope (Figure 23).
Table 18 Primers for the integration of the HA epitope at Arg94 of VMAT2 using site-direted mutagenesis
Primer Sequence
VMAT2-Arg94-HAepitope-upR AGC GTA ATC TGG AAC ATC GTA TGG GTA TCT GGT AGC ATT CCC GGT G
VMAT2-Arg94-HAepitope-downF TAC CCA TAC GAT GTT CCA GAT TAC GCT GAC CTG ACA CTT CAT CAG
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Figure 23 Expression of wild-type and p.P387L VMAT2 in stable clones of the MN9D cell line
2.1.2 Additional Biochemical Characterization of VMAT2 p.P387L
The precise functional consequences of proline to leucine substitution at residue 387 in
VMAT2 can be further probed by comparing the relative uptake of additional substrates
(i.e., dopamine, norepinephrine, histamine) either directly or through competitive
inhibition of serotonin uptake, and the relative binding of inhibitors (i.e., reserpine,
tetrabenazine). Furthermore, the use of valinomycin (a K+ ionophore) and bafilomycin
(an inhibitor of the vesicular ATPase) can be used to probe the coupling of VMAT2
p.P387L to the electrochemical and proton gradients.
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The recent development of live cell functional assays using fluorescent VMAT2
substrates provide additional options for direct visualization of substrate uptake by live
cell microscopy (Bernstein et al., 2012), or quantitative fluorometric plate assay (Hu et
al., 2013), provide additional options for probing mutant protein function.
2.1.3 Development of a Mouse Model Expression Vmat2 p.P390L
The functional defect of the VMAT2 p.P387L mutation (corresponding to p.P390L in
mouse Vmat2) is expected to dramatically alter dopamine homeostasis in the context of
the brain. Thus, the in vitro characterization presented in Chapter 4 should be
complemented by an in vivo assessment of the physiological consequences of the
mutation. I therefore propose to create a knock-in mouse model to address the role of the
p.P390L mutation in Vmat2 expression and subcellular localization, dopamine
homeostasis, and movement disorder phenotype.
As described in Chapter 1, Sections 2.4 and 2.5, existing mouse models of VMAT2
defect include a Vmat2 knockout mouse and a Vmat2-deficient mouse expressing 5% of
natural protein levels. The knockout mice exhibit normal Mendelian ratios, but neonatal
mice feed poorly, move slowly, and die a few days postnatally (Wang et al., 1997).
Detailed histology of the early postnatal cerebral cortex exhibits an increase in
developmental programmed cell death in these mice, which is partially rescued by MaoA
inhibition (Vmat2-MaoA DKO mice) with increased serotonin levels (Stankovski et al.,
2007). The hypomorpic Vmat2 mouse, by contrast, appears to have sufficient Vmat2
function to overcome this developmental barrier, and these mice exhibit adult onset
nigrostriatal neurodegeneration and deficits in motor function associated with several
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molecular hallmarks of oxidative stress and Parkinson’s Disease (increased alpha-
synuclein, increased levels of cysteinyl adducts) (Caudle et al., 2007, Mooslehner et al.,
2001). Additional phenotypes have been ascribed to heterozygotes of the knockout
allele, including prolonged QT syndrome, an increased rate of sudden death at 2–4
months of age, depressive-like behaviours, and reduced locomotor activity (Fukui et al.,
2007, Itokawa et al., 1999).
Because of the early onset and debilitating motor symptoms of the human disease, the
Vmat2 p.P390L mice would be expected to produce a more severe defect than that
observed with the hypomorphic Vmat2 allele. However, with full survival, normal MRI,
and mostly normal cognitive abilities in human patients harbouring the p.P387L
mutation, the effect of p.P390L is expected to be distinct from the severe developmental
defects exhibited by full Vmat2 knockout in mouse.
Given the potential for dose dependence of Vmat2 function and its multiple substrate
specificities, and that the existing mouse models do not clearly recapitulate the clinical
syndrome of pediatric neurotransmitter disorder, there is value in pursuing additional
studies of Vmat2 function in the mouse. The P390L mutation may represent an allele
with novel properties for vesicular monoamine transport functions, or provide more data
on the dose dependence of Vmat2 function, and would therefore provide a valuable
mouse model in addition to those thus far characterized.
The gene-targeting construct for Vmat2-P390L knock-in mouse model was designed,
constructed, and confirmed. The construction of this gene-targeting vector and the
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planned strategy for generating the mouse model, as well as the experimental plan for the
mouse model, are described below.
2.1.3.1 Design of Gene-Targeting Construct
The gene-targeting strategy for creation of the Vmat2 P390L knock-in mouse is presented
schematically in Figure 24. The c.1169C→T mutation will be introduced into mouse
sequence adjacent to a neomycin selection cassette. The neomycin selection cassette will
be used for the selection of positive recombinants. The cassette will present in the intron
to avoid disruption to Slc18a2 coding sequence. This cassette is also flanked by loxP
recombination sequences. In cells that express the Cre recombinase protein, the loxP
sequences will recombine in vivo and the intervening selection cassette will be excised.
This step will ensure minimal disruption of Slc18a2 expression. The c.1169C→T
mutation would be present at a distance of 1 kb from the neomycin selection cassette. At
this distance, the mutation is expected to be incorporated into the mouse genome in
combination with the selection cassette at a high frequency, generating a high proportion
of neomycin-selected clones that will be positive for the c.1169C→T mutation.
A schematic of the relevant features of the gene-targeting vector is presented in Figure
25. The pGKneolox2DTA vector designed by Soriano was used as the basis for the
construction of this gene-targeting vector (Soriano, 1997), named pGKneolox2DTA-
Vmat2-P390L. The vector was designed to include two long arms of Slc18a2 sequence
to undergo homologous recombination with the mouse genome in mouse embryonic stem
cells. The neomycin selection cassette is present between the flanking arms of Slc18a2
sequence. The c.1169C→T mutation encoding the P390L amino acid substitution was
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introduced into exon 13, present in the downstream arm. The construction of this vector
is discussed in the subsequent section.
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Figure 24 Schematic of incorporation of mouse gene targeting construct into mouse genome. Upper panel, wild type mouse genomic sequence of Slc18a2 exons 11–15 and flanking intronic sequences. Middle panel, mouse genomic sequence after incorporation of the gene-targeting construct by homologous recombination with the long arms of the construct in embryonic stem cells. Slc18a2 c.1169C→T mutation, coding for the P390L amino acid substitution, is present in exon 13.
Lower panel, mouse genomic sequence after expression of Cre recombinase and excision of the selection cassette. PGKneobpA, neomycin selection cassette; loxP, recombination sites targeted by Cre recombinase.
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Figure 25 Gene targeting construct for the introduction of the c.1169C→T mutation into Slc18a2. AmpR, ampicillin
resistance gene; F1_origin, origin of replication; loxP, recombination site for Cre recombinase; NeoR/KanR, neomycin and kanamycin resistance gene (of the PGKneobpA selection cassette); NheI, HindIII, restriction sites for insertion of downstream arm of genomic sequence for recombination; NotI, SacII, restriction sites for insertion of upstream arm of genomic sequence for recombination. Gray boxes, mouse intronic sequence of Slc18a2; black boxes, mouse exonic sequence of Slc18a2.
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2.1.3.2 Construction of Gene-Targeting Construct
Primers were designed to amplify a 1-kb fragment from mouse genomic DNA that encompassed
exon 13 of Slc18a2. This fragment was used to probe the RP23 C57BL/6 BAC library (Table
19). A clone was identified that encompassed the Slc18a2 gene (clone 338J16).
Regions upstream and downstream of the insertion site for the floxed TK-neomycin cassette
were amplified using Roche Long Template (LT) Taq polymerase. The conditions for
amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 57°C for 30s, and
72°C for 2 min; 72°C for 10 min. Primers for amplification incorporated restriction sites for
cloning, and are listed in Table 20. The fragments were initially cloned into pcDNA3.1 for
further subcloning.
After cloning of the downstream fragment into pcDNA3.1, the exon 13 C→T nucleotide
substitution corresponding to P390L was incorporated using the Quikchange Site-Directed
Mutagenesis Kit (Stratagene) according to manufacturer’s specifications. Oligonucleotide
sequences for site-directed mutagenesis are presented in Table 21. The incorporation of the
mutation was confirmed by direct sequencing of the plasmid.
The upstream and downstream fragments were subcloned appropriately into the
pGKneolox2DTA vector to flank the neomycin cassette. The relevant restriction sites used for
subcloning are displayed in the schematic of the gene-targeting vector presented in Figure 25.
The correct orientation and sequence of the fragments were confirmed by direct sequencing
(primer sequences in Table 22).
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Table 19 Primer sequences to amplify a probe for Slc18a2 exon 13 in mouse Primer Sequence Size Slc18a2-MsExon13-Probe-F ATT GAA GCT GAG GGT GGA TG 1018 bp Slc18a2-MsExon13-Probe-R TCC TCT GGA GGG TCA GAA AA
Table 20 Primer sequences to amplify the upstream and downstream arms of the gene-targeting construct Primer Sequence Size Ms-mod-upstream-SacII-F GCC AGG CCG CGG TTC CAG GAT CAG
CCA TTA GG 3677 bp
Ms-mod-upstream-NotI-R TCA GCA GCG GCC GCC AGC ACT TGC AAG AAT GAG G
Ms-mod-downstream-NheI-F
ACG GCT GCT AGC AGG CAT GGC TGA TGA AAC ATT ACA G
5306 bp
Ms-mod-downstream-HindIII-R
TAG CAT AAG CTT AGC AGT TCA CTA CCA GCT GTG ACT C
Table 21 Oligonucleotide sequences for site-directed mutagenesis of gene-targeting construct to introduce the p.P390L variant in the expressed protein Primer Sequence Mouse-Vmat2-P390L Quikchange-F AAA TAT CTA CGG ACT CAT CGC TCT
CAA CTT TGG AGT TGG TTT TGC Mouse-Vmat2-P390L Quikchange-R GCA AAA CCA ACT CCA AAG TTG AGA
GCG ATG AGT CCG TAG ATA TTT
Table 22 Sequencing primer sequences for gene-targeting construct Primer Sequence pGKneolox2DTA-upstream-F GTT TTC CCA GTC ACG ACG TT pGKneolox2DTA-upstream-R GGT TCC GGA TCC ACT AGT TCT pGKneolox2DTA-downstream-F GCT GAT CCG GAA CCC TTA AT pGKneolox2DTA-downstream-R CTA AAG CGC ATG CTC CAG AC Ms-SLC18A2-exon11-F ACC GTG GTG TCC GTG ATT AT Ms-SLC18A2-exon11-R TCA AAG ACC CAG ATC ATT CTC A Ms-SLC18A2-exon12-F TGC ATC TTG GGA CAC ACA GT Ms-SLC18A2-exon12-R GAG CTT CAG TCC CCA CAC TC Ms-SLC18A2-exon13-F GGT CCA CTA GGC CCA GAT AA Ms-SLC18A2-exon13-R CCG TCT GTT CAC TCT TCA CG Ms-SLC18A2-exon14-F CAC AGT CGA TGA CAT CTA GCC Ms-SLC18A2-exon14-F TCT GTA CCA CAT TAA AGT AAA GGT TTT Ms-SLC18A2-exon15-F TGG CTT TCA GGA AAA CCT TTA Ms-SLC18A2-exon15-R GGC CAT GGA GAA ATG ATT G
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2.1.3.3 Gene Targeting
Gene targeting will be accomplished by electroporation of the gene-targeting construct into
embryonic stem (ES) cells of the G4 cell line (129×C57BL/6 F1 hybrid). Clones can be selected
using G418. Primers were designed to amplify probe sequences for screening by Southern blot
(Table 23). Clones will be screened for the presence of both the neomycin cassette and adjacent
sequence to ensure incorporation of the cassette by homologous recombination. The
incorporation of the c.1169C→T mutation will be confirmed by sequencing.
Confirmed clones will be used to inject C57BL/6 blastocysts. Because the presence of the
selection cassette in intron 12 may affect Vmat2 expression, progeny will be bred to C57BL/6
mice expressing Cre recombinase to ensure removal of the cassette.
Experiments in protein expression and subcellular localization, as well as gross histology/MRI
will be performed primarily on F2 progeny. Because of significant strain differences in
monoaminergic phenotypes (Mhyre et al., 2005), for consistency the mice will be backbred for
five generations to C57BL/6 mice before performing behavioral and motor performance tests
(comparable to similar tests in hypomorphic Vmat2 mice), and catecholamine level and MPTP
sensitivity measurements. On the basis of the autosomal recessive human clinical phenotype for
this mutation, the homozygous mutation is not anticipated to be lethal. If this is the case, tests
will be performed on heterozygotes.
Table 23 Primer sequences for generating labelled probes to identify the neomycin selection cassette by Southern blot Primer Sequence Size Neo-Probe-F AGA CAA TCG GCT GCT CTG AT Neo-Probe-R GCG ATG CAA TTT CCT CAT TT Adj-Probe-F AAT GCA GTC CTG CTT GTG TG 880 bp Adj-Probe-R GAG CTT CAG TCC CCA CAC TC
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2.1.3.4 In Vivo and Subcellular Localization
Despite measurement of Vmat2 wild type and P390L levels in heterologous cell culture models,
the regulation of protein expression and localization may depend on factors only present in vivo.
Thus, the quantitation of Vmat2 wild type and P390L levels in mouse brain segments is
necessary to determine the physiologic effect of the mutation.
Relevant areas of the brain should be dissected to measure Vmat2 levels in each (as well as from
whole brain) using western blot of lysates and 3H-TBZ binding assays.
The subcellular localization of VMAT2 in vivo is of paramount importance in determining the
site and regulation of release of neurotransmitter. The cell culture models described in
Section 2.1.1 have significant caveats for the assessment of subcellular localization—Cos7 is not
a neuronal cell model, and in MN9D, the total quantity of available vesicles is diminished. In
dopaminergic axon terminals within the rat striatum, it has been shown that Vmat2
predominantly localizes to small synaptic vesicles (SSVs), but is also present on large dense core
vesicles (LDCVs) (Nirenberg et al., 1997). Using the same methodology, immunogold labeling
and electron microscopy, the subcellular localization of Vmat2 P390L should be studied in
sections from striata of homozygous and wild-type mice. Concurrent labeling of DAT or TH
will distinguish between dopaminergic from serotonergic Vmat2-containing axons.
2.1.3.5 Histology and MRI
MRI represents the best methodology for determining subtle changes in shape and volume of
small brain regions (Chen et al., 2006). MRI will be used on homozygous, heterozygous, and
wild type mice to look for volumetric differences in the cerebral cortex, thalamus, basal ganglia,
striatum, cerebellar cortex, hippocampus, and entorhinal cortex.
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Histology of the substantia nigra and early postnatal cortex will reveal any dopaminergic
neurodegeneration or developmental defects, respectively. Proper assessment of degeneration of
the substantia nigra would involve quantitation of TH-immunopositive fibres. Further, in situ
hybridization should be used to quantify the relative amounts of substance P-positive and
enkephalin-positive terminals in the striatum, to distinguish the effect of the mutation on the
development of the direct and indirect striatal output pathways, respectively.
2.1.3.6 Catecholamine Concentrations
Parkinsonism is associated with a depletion of striatal and whole brain dopamine. Hypomorphic
Vmat2 mice similarly indicate a depletion of monoamines in the adult brain (Mooslehner et al.,
2001). The total content of monoamines (DA, 5-HT, NE) and relevant metabolites (DOPAC,
HVA, 5-HIAA) in whole brains and striata of Vmat2 P390L homozygous, heterozygous, and
wild type mice will be measured using high pressure liquid chromatography with
electrochemical detection (HPLC-EC) of perchloric acid lysates, following established protocols
(Guevara-Guzman et al., 1994). Dopamine turnover can be measured as the ratio of
DOPAC/DA and HVA/DA, and serotonin turnover expressed as 5-HIAA/5-HT. Further, to
measure the amount of available dopamine relevant to neurotransmission, the extracellular
concentration of dopamine in the striatum may be assessed using in vivo microdialysis using
implanted dialysis probes, with fractions analyzed immediately by the same HPLC-EC method.
Each method should be performed on 10–15 mice of each genotype and data analyzed by
Student’s t-test.
2.1.3.7 Motor Performance and Behavioral Testing
The motor performance of Vmat2 P390L mutant mice is the most relevant phenotype. Because
locomotor activity shows vast strain differences (Mhyre et al., 2005), it will be necessary to
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breed these mice to C57BL/6 for 10 generations to ensure homogeneity of the genetic
background. Initially, mice would be examined for evidence of tremor or dystonia (abnormal
postures or twisting movements). Dystonia also manifests as hindlimb retraction or trunk flexion
in response to tail suspension. Abnormal gait should be assessed by pawprint pattern of mice
walking along a narrow runway (measurement of stride length and width). Locomotor hyper- or
hypoactivity can be assessed in open field with measurement of both horizontal locomotion
(crossing) and vertical locomotion (rearing). A beam walking task and accelerating rotarod test
should be performed as sensitive tests of motor coordination and balance. Grip strength tests
should be performed to measure associated muscle weakness. Results can be compared to those
published for the hypomorphic Vmat2 mice, who showed reduced vertical and horizontal
locomotor activity and slower times and more slips in the beam walking task (Mooslehner et al.,
2001). The Vmat2-KO mice do not survive long enough to be tested for motor performance, but
the heterozygotes do show reduced horizontal and vertical locomotor activity in open field
(Fukui et al., 2007).
It is further important to assess behavioral phenotypes of these mice, given the role of VMAT2
in serotonin vesicular storage, and the depression side effects of reserpine treatment in humans.
The mice should be tested in forced swim, learned helplessness, novelty-suppressed feeding, and
open field exploration, as described for the Vmat2-KO heterozygote mice (Fukui et al., 2007).
2.1.3.8 MPTP Resistance
To assess the contribution of the Vmat2 p.P390L mutation to MPTP-induced nigrostriatal
degeneration, homozygous, heterozygous, and wild type mice should be injected
intraperitoneally with the MPTP toxin. After 8 days, animals can be sacrificed and the relative
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sizes of the substantia nigra pars compacta and TH-immunopositive cell counts (vs. control
saline injections) can be assessed in brain sections.
3 VMAT2 Mutation in PND: Impact on Understanding of PND Pathogenesis
The patients treated in this study show a progressive motor phenotype with age, and demonstrate
differential response to treatment on the basis of the age at which treatment was initiated. This
suggests that anatomical differences may underlie the expression of the disease at different ages,
illustrating that monoamine deficiency influences normal neurological development and that the
direct effects of monoamine deficiency are overlaid on abnormal functional architecture in the
basal ganglia. The hypomorphic Vmat2 mouse model exhibited decreased substance P and
increased enkephalin expression, consistent with this hypothesis (Mooslehner et al., 2001). What
is not yet evident, however, is whether there are any downstream neurodegenerative effects of
impaired vesicular monoamine storage over the course of patients’ lifetimes, as were observed in
hypomorphic Vmat2 mice (Caudle et al., 2007). Careful patient follow-up comprising both MRI
and neurological examination should be maintained to monitor this possibility.
The results presented in this thesis have clearly demonstrated a functional effect of the p.P387L
mutation on VMAT2 protein function, but the precise physiological consequences in human
patients remain to be clarified. Studies in a mouse model (as described in Section 2.1.3) may
help clarify the pathophysiological consequences of this mutation relative to complete deletion or
other hypomorphic Vmat2 mice, but there is limited ability to extrapolate these results to human
physiology. Positron emission tomography (PET) is a methodology that would allow direct
visualization of both VMAT2 protein levels and the vesicular retention of monoamines in
patients and control subjects. PET studies are proposed in Section 3.1.1.
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Potential refinement or expansion of the clinical phenotypes associated with VMAT2
dysfunction can be further explored through genetic screening of additional patient cohorts to
identify novel SLC18A2 variants, as proposed in Section 3.1.2.
3.1 Future Directions and Experiments
3.1.1 18F-DOPA and 11C-DTBZ PET scans
Specific VMAT2 ligands have been developed as imaging agents for early diagnosis and
monitoring of neurodegenerative disease and neuropsychiatric conditions with monoaminergic
involvement. Derivatives of tetrabenazine are the primary agents developed for positron
emission tomography (PET). In mice, it was demonstrated that after intravenous injection,
[11C]TBZ is rapidly taken up in the brain where it localizes primarily to the striatum and
hypothalamus, as well as less strongly in the hippocampus, cortex, and cerebellum (DaSilva and
Kilbourn, 1992).
3.1.1.1 [11C]Dihydrotetrabenazine ([11C]DTBZ)
There are several potential limitations to the direct imaging of VMAT2 levels using
tetrabenazine analogs. Because there is evidence of overlap between the binding site of
tetrabenazine and the substrate-binding region of VMAT2, the technique is effectively
visualizing the number of available VMAT2 binding sites in the brain. Competition in binding
between the tetrabenazine analog and natural substrates would therefore make the accuracy of
the technique subject to differences of in vivo monoamine levels. Comparison of [11C]DTBZ
signal between patients with a missense mutation in VMAT2 and control subjects would
therefore be confounded by a relative increase in binding site availability (per VMAT2
molecule) as a result of the drastically decreased availability of natural monoamine substrate. A
quantitative study in rats confirmed a statistically significant increase in [11C]DTBZ binding in
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vivo with significant reduction in brain dopamine levels without a concomitant increase in
protein levels (Kilbourn et al., 2010). Similarly, a human PET study observed a rapid decrease
in [11C]DTBZ binding with the administration of levodopa (exogenous dopamine) (de la Fuente-
Fernandez et al., 2009). Additionally, the natural plasticity of VMAT2 is the subject of some
controversy, and fluctuations in VMAT2 protein levels may exist among healthy individuals and
mask clinically relevant fluctuations in VMAT2 concentrations. Given these confounding
factors, however, a dramatic decrease in VMAT2 signal would clearly indicate mutant protein
instability or aberrant conformation in vivo.
3.1.1.2 6-[18F]fluoro-L-dopa ([18F]-DOPA)
In conjunction with [11C]DTBZ scans, 6-[18F]fluoro-L-dopa ([18F]-DOPA) scans would provide
more information. The retention of [18F]-DOPA for visualization within the synapse is
dependent upon both presynaptic uptake, dopa decarboxylation, and vesicular uptake—namely,
the proper functioning of DAT, AADC, and VMAT2. This imaging substrate would therefore
provide a more holistic visual indication of dopaminergic functioning in these patients, and avoid
the confounding issues discussed above for [13C]DTBZ binding.
The radiation exposure for a PET scan is modest (7–8 mSv). This noninvasive assessment of
both VMAT2 binding and dopaminergic functioning in patients and siblings would provide a
better understanding of the effect of the P387L variant on VMAT2 availability and dopamine
sequestration in vivo.
3.1.2 Screening of Patient Cohorts to Identify Novel SLC18A2 Variants
An additional research direction of interest is genetic screening in patient cohorts to identify
novel variants in SLC18A2 with the aim of refining—and possibly expanding—the phenotypic
spectrum associated with VMAT2 defect. Patients with possible undiagnosed VMAT2
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deficiency may be identified among those previously screened for pediatric neurotransmitter
disease with normal CSF neurotransmitter metabolite profiles. They may also be identified
among patients diagnosed with cerebral palsy (CP) by exclusion. Furthermore, a broader
approach to screening should comprise patients with any phenotype related to biogenic amine
function, including movement disorder, neuropsychiatric phenotypes, addiction phenotypes, and
autonomic dysfunctions. Variants identified through screening should be confirmed through
bioinformatic and functional analyses analogous to those performed in this thesis.
4 VMAT2 Mutation: Impact on Approaches to PND Diagnosis
The clinical investigations presented in Chapter 2 demonstrated the challenges in diagnosis of
VMAT2 deficiency related to limitations in CSF neurotransmitter metabolite analysis as a
conclusive diagnostic for monoamine deficiency. Because the sole conclusive diagnostic for
VMAT2 deficiency at present is genetic analysis, mutation screening of SLC18A2 is
recommended in addition to both CSF and urine neurotransmitter metabolite analysis in
suspected cases of monoamine deficiency. For the comprehensive investigation of suspected
monoamine deficiency, and given the relative ease with which a multigene panel could be
assayed, genetic analysis of all known disorders with primary or secondary neurotransmitter
phenotype should be performed in parallel.
To improve functional confirmation of a genetic diagnosis of VMAT2 deficiency, the
development of methodology to directly measure biogenic amines in CSF is proposed in
Section 4.1.1, and the development of a platelet activity assay for VMAT2 is proposed in
Section 4.1.2.
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Finally, considerations relevant to the clinical utility of next-generation sequencing for the
diagnosis of pediatric neurotransmitter disorders and other rare monogenic diseases is discussed
in Section 4.1.3, and knowledge translation challenges and strategies for the diagnosis of
pediatric neurotransmitter disorders and related diseases are discussed in Section 4.1.4.
4.1 Future Directions and Experiments
4.1.1 Development of Methodology to Measure Biogenic Amines Directly in CSF
The direct measurement of dopamine, serotonin, and norepinephrine in CSF is unfeasible by LC-
MS/MS because it is below the limit of detection of the technique and monoamine
neurotransmitters are less stable than their respective metabolites. The measurement of
metabolites, however, is accompanied by numerous caveats. In cases of defective biosynthesis,
it is reasonable to assume that the metabolites accurately reflect monoamine deficiencies. The
linearity of this response, however, remains to be demonstrated. Transporter defects, by contrast,
effectively influence the turnover of the biogenic amines. The relationship between endogenous
concentrations of amine and metabolite is therefore more complex in these cases.
The biogenic amines can be measured in physiologic samples using commercially available
ultra-sensitive enzyme-linked immunosorbant assay (ELISA) kits according to manufacturer’s
specifications. For example, serotonin can be measured in physiologic samples to a
concentration of 0.05 ng/mL (Labor Diagnostika Nord BA 10-5900).
For this technique, then, the preservation of endogenous amines during CSF collection becomes
paramount. Samples should be collected directly into 0.1% w/v ascorbic acid to prevent
oxidation, and stored immediately at –80°C. Given the risk associated with lumbar puncture, the
collection of control samples will need to occur in the clinic secondary to lumbar puncture
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performed for the diagnosis of unrelated conditions. This will have to be designed prospectively
to ensure proper sample collection in these cases.
Accurate determination of endogenous monoamine concentrations in these patients will provide
better quantitative understanding of the deficiencies in neurotransmission.
4.1.2 Development of VMAT2 Platelet Activity Assay
Because the CSF neurotransmitter metabolite profile appears to be normal in VMAT2
deficiency, alternative strategies are needed to confirm the diagnosis in suspected cases.
Serotonin in the blood is taken up by platelets and released to aid clotting. Both VMAT2 and
SERT are expressed in platelets, and platelets are therefore an accessible peripheral cell type in
which to assay VMAT2 function. As presented in Chapter 2, Section 2.4, AADC enzyme
function is assayed in serum in suspected cases of AADC deficiency.
Some studies have attempted to measure differences in platelet VMAT2 density in various
neuropsychiatric disorders using a tritiated dihydrotetrabenazine ([3H]-TBZOH) binding assay,
with moderate but statistically significant results (Zucker et al., 2002a, Zucker et al., 2002b,
Toren et al., 2005, Schwartz et al., 2005b, Laufer et al., 2005, Schwartz et al., 2005a, Schwartz et
al., 2007, Ben-Dor et al., 2007, Sala et al., 2010, Zalsman et al., 2011a, Zalsman et al., 2011b).
Whereas those studies were intended to probe the regulation of VMAT2 expression as an indirect
indicator of perturbations in monoamine homeostasis, I propose to develop a standardized assay
of tritiated serotonin uptake to assay VMAT2 function. Platelet-rich plasma would be separated
from blood cells by low speed centrifugation, and platelets would be disrupted by sonication in
sucrose buffer. A serotonin uptake assay would be performed on the resulting lysate as
described in Chapter 4, Section 2.3. In this way, a pathogenic loss of VMAT2 function could be
assayed from a blood sample. The caveat of this approach is that previous studies required a
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large blood volume (25 mL) to assay [3H]-TBZOH binding, which is a large blood volume for a
pediatric patient. Guidelines on acceptable blood volumes to be collected from pediatric patients
suggest a maximum of 3 mL/kg within 24 hours (approximately 3.8% of total blood volume)—
prohibiting the use of this test for infants under 8.5 kg (Howie, 2011). Although this test would
provide significantly less risk to the patient than a lumbar puncture, it is specific only to this
particular very rare disease, and would therefore likely be pursued only in the event of a normal
CSF metabolite profile.
4.1.3 Use of Next-Generation Sequencing for Genetic Diagnosis of Rare Monogenic Diseases
Beyond targeted genetic screening of individual candidate genes or multigene panels, the results
presented in this thesis demonstrate that there is now the ability to diagnose a new monogenic
disorder in a single family using genomic methods within a timeframe of relevance to the
treatment of individual patients. At the initiation of this project, the identification of disease-
causing mutations was accomplished by investigating the segregation of particular genomic
regions in association with the inheritance of the disease. Microsatellite markers were still
primarily used, although the availability of single nucleotide polymorphism arrays was
increasing. These techniques were used initially in the present case to identify the disease-
associated locus using a combination of linkage and homozygosity analyses. Subsequently,
mutation screening of a manageable set of candidate genes (selected on the basis of known
function or localization) was performed to identify variants.
Experience and improved resources to pursue these methods allowed the initial identification of
the variant in a matter of months rather than years, as was the timeframe for gene hunting during
the previous decade; however, the clinical utility of this information was limited by its
identification in only a single family. Without functional confirmation from the identification of
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an unrelated family exhibiting a similar phenotype and harboring a variant in the same gene,
there was an increased burden of proof for confirming the functional relevance of the variant
before translating these insights to the clinic. Namely, we pursued the confirmation of a
functional effect of the variant, the absence of the variant in a significant number of unaffected
family members, and the absence of further variants in the region. The success of these
approaches in finding the causative mutation in a single family was dependent upon the size of
the pedigree, the mode of inheritance, and the number of affected individuals—a significant
limitation for genomic diagnosis by these methods in isolated cases that present to a clinic. In
addition, de novo mutations cannot be identified by positional cloning approaches.
The more recent availability of next-generation sequencing methodology, including whole-
exome sequencing, addresses many limitations of gene hunting using linkage and association
with genomic markers with respect to clinical diagnosis. First, sequence results for the whole
genome can be obtained very quickly and cost is no longer strictly prohibitive. A list of genetic
variants can be generated for a single patient and compared with databases of presumably benign
variants present in an expanding number of healthy individuals. In a small family with more
than one affected individual, shared variants can be identified and subsequently screened in
unaffected family members. The list of variants can then be filtered on the basis of the proposed
mode of inheritance (e.g., the presence of two variants in a single gene for an autosomal
recessive disorder). One recent review estimated that 234 novel rare disease genes have been
discovered by either whole-exome or whole genome sequencing (Boycott et al., 2014).
Whole-exome sequencing of the proband in the present case allowed the exclusion of exonic
mutations in other genes within the shared homozygous region, as well as the exclusion of
candidate genes throughout the genome. The ability to exclude the involvement of other
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candidate genes increased our confidence in the involvement of the SLC18A2 variant in the
disease.
Awareness of the pediatric neurotransmitter disorders has increased in the clinical community,
but the majority of neurologists who may encounter such a patient will not have prior experience
with the phenotypic breadth of this group of rare disorders. The identification of putative
causative variants by whole genome or whole-exome sequencing—if not conclusive—can focus
the search for related cases in the literature, and thereby aid differential diagnosis. For example,
one treatable pediatric neurotransmitter disorder, sepiapterin reductase deficiency (see Chapter 1,
Section 1.4.2)—which is very severe when untreated—was recently diagnosed by whole genome
sequencing in a pair of siblings who had long remained undiagnosed and therefore untreated
because of the common difficulties in precise diagnosis of rare diseases. Diagnosis allowed
treatment and recovery of these children (Bainbridge et al., 2011).
Whereas the clinical utility of genomic diagnosis has been illustrated here, many limitations
remain. Sequence data is high resolution, and therefore it is necessary to filter and interpret a
large dataset to derive a clinically meaningful subset. This would be beyond the expertise of
many clinicians. There will therefore need to be an infrastructure in place before whole-exome
sequencing and data interpretation can become routine, including clinical geneticists who receive
specific training and specialization in the interpretation of next-generation sequencing data, and
continuous improvement to bioinformatic methods for filtering variants. Notably, a Canadian
research consortium (Finding of Rare Disease Genes [FORGE] Canada) providing such
centralized infrastructure recently reported the identification of disease-causing variants in 146
of 264 disorders under study (Beaulieu et al., 2014).
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Additionally, the success of the technique would vary with the inferred mode of inheritance. In
the present case, an autosomal recessive mode of inheritance in a consanguineous family
narrowed the search to homozygous variants. Without clear consanguinity, compound
heterozygous mutations in a single gene would be expected. A dominant mode of inheritance, in
contrast, would require examining an extensive list of heterozygous variants, which would be
facilitated by parallel sequencing of both parents and available siblings. De novo dominant
mutations, however, may be more straightforward to identify with the provision of parental
genome information. In both cases, genetic diagnosis would necessitate the availability of
family members, their consent to provide their genomes, and would incur additional costs. This
also produces a unique challenge in seeking reimbursement, with respect to defining the minimal
number of patient and family genomes required for a particular diagnostic application.
Whereas whole-exome sequencing is suitable for the discovery of protein-coding variants, other
genetic mechanisms are increasingly recognized to have phenotypic consequences, such as
splicing variants, promoter mutations, and epigenetic modifications. Mutations outside the
exome may be better addressed as the cost of whole-genome sequencing decreases and becomes
competitive with the cost of whole-exome sequencing. However, the availability of predictive
bioinformatics tools with sufficient confidence to identify disease-causing variants with
regulatory significance will need to be developed before this becomes feasible in a clinical,
rather than research, setting.
An additional challenge inherent in using whole-exome or whole-genome methods for clinical
diagnosis derives from the vast quantity of data generated by each individual test. Multiple
testing of this magnitude increases the number of incidental findings, making it difficult to
identify true causative mutations. Attributing confidence to identified variants will involve the
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incorporation of functional data, through understanding of the underlying pathophysiology, that
of related diseases, or metabolic data derived through other clinical investigations or
metabolomic screening performed in conjunction with genomics. Additional ethical challenges
may result from the identification of incidental findings with clinical significance that are
unrelated to the reason for testing. For these reasons, initial analyses based on disease-targeted
subsets of data may have a role in clinical utilization of next-generation sequencing.
In summary, although cost and time are no longer prohibitive factors to the clinical use of
genomics for the diagnosis of rare diseases in individual patients and small kindreds, significant
challenges remain in the implementation. However, the use of next-generation sequencing
technologies allow a large volume of information to be brought to bear on diagnostic challenges
that were previously not amenable to investigation by a uniform methodology.
4.1.4 Knowledge Translation Challenges and Strategies for PND Diagnosis
There are nine pediatric neurotransmitter disorders among more than 80 identified inborn errors
of metabolism with neurological correlates (van Karnebeek and Stockler, 2012). As with
VMAT2 deficiency, many of these diseases are readily treatable with already available
medications, diet, or vitamin or co-factor supplementation. The challenge lies in accurate and
timely diagnosis. Individual disorders are exceedingly rare, and the rate of discovery of these
disorders has increased significantly with the advance of genomic technologies to identify
disease-causing mutations in single families. The volume of data, combined with the rate of
increase of the dataset and the potential relevance of extremely recent data, places a significant
information burden on individual clinicians who may encounter such patients infrequently in
their career. Given the rarity of individual disorders, this situation is no less challenging for
specialists. The huge potential impact of a correct diagnosis on patient outcome emphasizes the
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importance of improving the ability of clinicians to access the appropriate information as a
standard part of clinical practice.
The publication of research findings in peer-reviewed literature is an inefficient and ineffective
strategy for the communication of relevant information to the primary users of the information.
Although clinicians are familiar with the language and presentation of research findings in the
literature and many have relevant journal subscriptions, the information is not organized in a
consistent and meaningful way. Database searches, such as PubMed, are often incomplete
because they rely on keyword searches, and consistency in the presentation of concepts is not
ensured. Linkages between related studies on the basis of citations are useful, but biased by the
perspectives of individual authors. As a result of these factors, information presented in the
scientific literature is not properly synthesized within a broader body of knowledge. The mere
availability of data to potential knowledge users is not sufficient to ensure its application in
practice. For information with such critical value to patients’ lives, the traditional avenues for
dissemination of research findings are no longer acceptable.
The Canadian Institutes of Health Research (CIHR) define knowledge translation as “a dynamic
and iterative process that includes synthesis, dissemination, exchange, and ethically-sound
application of knowledge to improve the health of Canadians, provide more effective health
services and products, and strengthen the health care system (CIHR, 2013).” Lavis provides a
framework for knowledge translation in the form of five questions: “What should be transferred
to decision makers?”, “To whom should research knowledge be transferred?”, “By whom should
research knowledge be transferred?”, “How should research knowledge be transferred?”, and
“With what effect should research knowledge be transferred?” (Lavis et al., 2003). This section
aims to discuss these elements in the context of improving the timely diagnosis of rare diseases.
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The challenge for PNDs and inborn errors of metabolism—and more broadly, all rare diseases—
is the synthesis of disparate and rapidly expanding information in a manner that can be directly
used by clinicians. Van Karnebeek and colleagues produced a synthesis of information for
inborn errors of metabolism in the form of a systematic literature review of inborn errors of
metabolism that present with intellectual disability as a major feature (van Karnebeek and
Stockler, 2012). In this manner, they arranged a vast collection of information (features and
treatment of 81 separate disorders) around a single unifying principle—the clinical presentation
of intellectual disability. In doing so, they presented the information in a context relevant to its
use.
Beyond synthesis, the manner of delivery of information is key. Van Karnebeek et al. further
derived a two-step diagnostic protocol to identify underlying inborn errors of metabolism
presenting with intellectual disability. The protocol was implemented through a web and mobile
app, Treatable-ID (http://www.treatable-id.org/) (van Karnebeek et al., 2012). Information on
the 81 diseases is presented in multiple ways: biochemical categories, neurologic and non-
neurologic signs and symptoms, diagnostic investigations, therapies, effects on outcomes, and
available evidence. A disease page for each condition connects clinicians to online resources and
primary literature. In this way, the information synthesized by the systematic review is made
intuitively navigable in a real-life clinical context. The use of web and mobile technology makes
the information broadly accessible and allows for updates to be made at the pace of discovery.
This is a model of the approach to be applied more broadly for clusters of rare diseases. Tools
should be developed that arrange information about rare conditions around their presentation,
providing consolidated diagnostic strategies and treatment information. In the case of VMAT2
deficiency and other PNDs, the primary presentation is movement disorder rather than
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intellectual disability (although there is overlap), and a focus may be placed on motor features,
with respect to the phenotypic spectrum of PNDs and age-specific presentation. Because
movement disorder is a highly specialized field, physician education materials (diagrams and
video) may be integrated into the app to enable them to better classify the relevant disease
features. Finally, it is key that any diagnostic app provide links to primary information sources
and contact information for affiliated clinicians and investigators to promote collaboration. A
long-term vision of an integrated diagnostic tool, rather than a collection of independent apps,
should be pursued for use in regular clinical practice. Furthermore, the adoption of this
technology should be aggressively advertised through medical associations, international
meetings, and physician education programs.
Beyond improving the resources available to physicians, consideration should be given to
developing a framework for presenting analogous information in a publicly accessible manner.
Information accessibility in both format and language fosters patient and caregiver agency in the
diagnostic process; providing a framework for information curation and presentation may better
focus the existing epidemic of internet self-diagnosis. In recognizing the multiple demands on a
clinician’s time, the availability of well-organized rare disease information to patients (and their
families and caregivers) can leverage the motivation of these stakeholders to explore potential
diagnoses. In conjunction with the clinical judgment of the physician through targeted
discussions, this would reflect an evolution toward a collaborative model of rare disease
diagnosis in which information may be more efficiently utilized in the clinical setting through the
involvement of multiple stakeholders in the diagnostic process, made possible through effective
knowledge translation.
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5 Concluding Statement This thesis described a new autosomal recessive pediatric neurotransmitter disorder discovered
in eight cousins of a consanguineous Bedouin family in Saudi Arabia, and identified its causative
mutation in the vesicular monoamine transporter 2 (VMAT2; SLC18A2). Biochemical
confirmation of loss of function underscored the importance of vesicular transport in monoamine
homeostasis, extending the spectrum of known pediatric neurotransmitter diseases and providing
the first demonstration of mutation in VMAT2 causing a clinical phenotype. Unremarkable CSF
monoamine metabolite findings demonstrate the inability of this single test to diagnose all
monoamine deficiencies, and prompt reconsideration of the diagnostic strategy.
For pediatric neurotransmitter disorders and other inborn errors of metabolism, precise
and timely diagnosis leads to significantly better patient outcomes with the availability of simple
and effective treatments for many. The rapid identification of causative mutations in single
families with previously undiagnosed rare genetic disease is now possible with the advancement
of next-generation sequencing technologies for whole-genome and whole-exome sequencing.
Diagnostic challenges in genomic data interpretation and dissemination of research findings
remain to be addressed.
150
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