neurogenetics made easy

Neurogenetics made easyNeurogenetics made easyEnza Maria Valente, MD, PhD

Dept of Molecular Medicine, University of PaviaNeurogenetics, IRCCS Mondino Foundation

Enza Maria Valente, MD, PhDDept of Molecular Medicine, University of Pavia

Neurogenetics, IRCCS Mondino Foundation

Talk outlineTalk outline

• How geneticists can help neuroradiologists• Genetic testing: when, which and why

• Genetic testing today• genome-wide strategies for mutation detection

• How neuroradiologists can help geneticists• practical examples• the importance of collaboration

How geneticists can help neuroradiologists

Genetic testing: when, which and why

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Why genetics is important?Why genetics is important?

for families

assessment of recurrence risksearly prenatal diagnosis

testing relatives (e.g. carrier testing)new therapies, clinical trials etc…

for patients

improved diagnosisprognostic indications

indications for management and treatment

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A quick refresh about mendelian inheritanceA quick refresh about mendelian inheritance

autosomal recessive two mutated copies of the gene are required to cause the diseasehealthy heterozygous parents transmit both mutated copies of the gene to 25% offspringhorizontal transmission (multiple affected individuals in one generation)can also present in sporadic cases

everything that is not acquired (such as infective, toxic, vascular, traumatic etc…) MUST recognize a genetic basis

autosomal dominantone mutated copy of a gene is sufficient to cause the diseasefor diseases reducing fitness, the mutation usually occurs de novo during parental meiosis sporadic casesfor diseases non reducing fitness, the mutation is transmitted vertically from one affected parent to 50% offspring

X-linkedrecessive: hemizygous male patients inherit one mutated copy of a gene on the X chrom. from the heterozygous mother (healthy or minimally affected) – also de novodominant: heterozygous females are affected while in hemizygous males the mutation is lethal or gives rise to much more severe phenotypes – also de novo

Decision-making flowchart for genetic testingDecision-making flowchart for genetic testing

Indication to genetic testing

pre-test genetic counselling and informed consent

test selection

laboratory selection

GENETIC TESTING

negative or ambiguous results

implications for management, prenatal diagnosis,

counselling for relatives

diagnostic reassessment?further genetic testing?

research testing?

post-test genetic counselling

positive results

validation of diagnostic suspicion (clinical / imaging) prognostic indications (genotype-phenotype correlates)

Indications to diagnostic genetic testingIndications to diagnostic genetic testing

Leigh syndrome (mitochondrial)onset < 1 yearsevere neurological picturetypical basal ganglia lesions

Neurofibromatosis 1 (NF1 gene)café-au-lait spots, axillary frecklescutaneous neurofibromasoptic gliomas, cranial tumours, other MRI lesions

Joubert syndromecongenital ataxiavariable organ involvementtypical “molar tooth sign”

Indications to diagnostic genetic testingIndications to diagnostic genetic testing

reproductive risks, carrier testing and prenatal diagnosisspecific treatments, therapeutic trials

++ severe disorders with full penetranceNBIANeuronal Ceroid LipofuscinosesLysosomal storage disorders

diagnosis of conditions without clear clinical or imaging cluesdifferential diagnosis among distinct conditions with

overlapping phenotypes

Early onset pure cerebellar atrophy• AT (ATM)• AOA1 (APTX)• SCA29 (ITPR1) …

Poretti et al, Neuropediatrics 2015; Vedolin et al, AJNR 2013

PLAN

ITPR1AOA1AT

Infantile NCLInfantile NCL

rationale and procedure of the testinheritance, natural history, and management of the diseaseimplications for the health and reproductive choices of the relativespotential benefits, disadvantages, and consequences for the future health, employment, and insurance prospects of the individualreferral for psychosocial support when indicated

an appropriately trained person (clinical or medical geneticist, genetic counsellor, or genetic nurse) offers the patient and his family accurate and comprehensive information on:

genetic testing must be considered an integrated service, preceded and followed by specific genetic counseling

Genetic counsellingGenetic counselling

results, especially when positive, should also be rendered in the context of genetic counselling

mutation(s) already identified in affected family membersgenetic testing specifically targeted to confirm the familial mutation(s)

Selection of testSelection of test

genetic and molecular homogeneitya specific mutation causes most cases of a disease (e.g.

Friedreich’s Ataxia GAA expansion in the FXN gene)standardized genetic testing, high sensitivity

genetic and/or molecular heterogeneity (commonest!)The same disease can be caused by different mutations and different mutational mechanisms involving one or even many genes

ataxia-telangiectasia (ATM, very large gene, many mutations)Joubert syndrome (>40 genes known to date)

Genetic testing today

genome-wide strategies for mutation detection

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Genetic strategies: high resolution arrayGenetic strategies: high resolution array

array studies (CNVs)standard cytogenetics

Ordered pool of genomic sequences (oligos – CGHarray or SNPs –SNParray), which physical position on the genome is known

The whole genome is analyzed in a single experimentResolution depends on the length of DNA probes and the average distance

between two adjacent probes on the genome

on the same chip you can simultaneously test for:- copy number variations on the whole genome (dels-dups)

- regions of homozygosity by descent

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Dandy Walker syndrome – chrom. 3q25 deletionDandy Walker syndrome – chrom. 3q25 deletion

Grinberg et al, Nat Genet 2004; Ferraris et al, OJRD 2013

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Dandy Walker syndrome – chrom. 6p25 deletionDandy Walker syndrome – chrom. 6p25 deletion

- large deletions: nearly all haveDWS (but 1 normal!!!)

- small deletions or duplications:CVH – mega cisterna magna

- heterozygous point mutations: Axenfeld-Rieder syndrome + mild CVH

Aldinger et al, Nature Genet 2009

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Genetic strategies: next generation sequencing (NGS)Genetic strategies: next generation sequencing (NGS)

linkage studies bioinformatics

high-throughput, cost- and time-effective simultaneous resequencing of large portions of the genome

Current diagnostic applications:• target resequencing of panels of genes or clinical exome hundreds of genes of

our choice or about 4000-7000 genes known to cause inherited disorders• mutation screening in clinically and genetically heterogeneous disorders

• whole exome sequencing the coding regions of the >20.000 genes in our genome• novel gene identification (even in small families/sporadic)• «fishing» for diagnosis in patients with limited clinical-imaging clues

Caution in interpretation of NGS resultsCaution in interpretation of NGS results

mutation permanent change in the nucleotide sequence

polymorphism variant with frequency >1%

assumed as pathogenic assumed as benign

confusionboth replaced by

variant

pathogenic likely pathogenic

of uncertain significance (VUS)

likely benign

benign

with respect to a condition and inheritance pattern (e.g., c.1521_1523delCTT (p.Phe508del), pathogenic, cystic fibrosis, autosomal recessive).

Comprehensive pre- and post-test counselling is essential

Incidental findings in NGS testingIncidental findings in NGS testing

findings that have potential health or reproductive

importance discovered in the course of conducting research

but beyond the aims of the study

sequencing laboratories should actively seek and report pathogenic variants in 56 genes associated with 24 conditions, all with evidence that early intervention can prevent or ameliorate severe adverse medical outcomes

examples:- BRCA1-2: breast / ovarian cancer- LDLR: hypercholesterolemia- PKP2: arithmogenic cardiomiopathy

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Joubert syndrome (>40 genes)Joubert syndrome (>40 genes)

NGS-based screening in 400 JS probands

Gene-phenotype correlatesGene-phenotype correlates

negative43%

C5orf429%

CEP2908%

AHI16%

CC2D2A6%

TMEM676%

KIAA05863%

RPGRIP1L3%

OFD12%

INPP5E2%

MKS12%

CSPP12%

TMEM2161%

B9D11%

TMEM2371%

other genes6%

Cerebello-oculo-renal

Pure JS (++ retinopathy)

Variablephenotypes

JS + liver

JS + kidney

- Pure JS- JS + JATD + OFD

- Pure JS- OFDVI

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PCH GENE LOCUS CLINICAL FEATURES

PCH1 EXOSC3, VRK1TSEN54, RARS2

9p1314q32

Onset at birthaxonal motor neuropathy

PCH2TSEN54TSEN2TSEN34

17q253p2519q13

Onset at birth, dyskinesias / choreavermis less hypoplastic than hemispheres(dragonfly appearance)

PCH3 Not known 7q11-q21 Onset at birth, optic atrophyhearing impairement

PCH4/PCH5 TSEN54 17q25 Fetal onset, severe progression, myoclonus

PCH6 RARS2 6q15 Onset at birth, mitochondrial defects

PCH7 TOE1 ambiguous genitalia

PCH8 CHMP1A 16q24 onset at birth

CASK CASK Xp11 progressive microcephaly, ID, deafnessDragonfly/butterfly, normal CC

PCH9 AMPD2 1p13 unspecific clinical features, death by age 10CC hypoplasia, «8» shape of vermis (axial cut)

PCH10 CPL1 11q12 Slow onset, progressive spasticity/seizuresAbsent or delay speech

Ponto-Cerebellar Hypoplasias: clinical and genetic classificationPonto-Cerebellar Hypoplasias: clinical and genetic classification

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PCH screening – Italian cohortPCH screening – Italian cohort

• unique inclusion criterion: PCH confirmed on neuroimaging

• 61 probands analyzed• 44 genes

CASK mutations

Males:missense or splice

Males:mosaic loss of function

severe missense

Females:het loss of function

Males:hemiz. loss of function

progressive microcephalyvariable PCHmoderate to severe DD/IDvery limited languageseizures, short stature, scoliosissensory-neural hearing lossbutterfly (also dragonfly)normal corpus callosum

DD/IDnystagmusseizures (not severe)(short stature)(tremor, hypotonia)(dysmorphism)

most severe clinical presentationsevere to profound DDrapidly progressive microcephalycortical atrophy / hypomielinationsevere intractable seizures(congenital heart defects, contractures)often lethal

neg39%

CASK34%

TSEN5416%

EXOSC35%

RARS22%

VLDLR2%

PPM2/ATP2B32%

Burglen 2012, Saitsu 2012, Moog 2015

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«Shrunken cerebellum»: non-progressive cerebellar atrophy«Shrunken cerebellum»: non-progressive cerebellar atrophy

Mild developmental delayCerebellar AtaxiaNormal cognitive development or

mild intellectual deficitClinical improvement in timeNo progression of atrophy at

neuroimaging

18 months

6 years

several genes involved

PMM2Congenital Defectof Glycosilation IA:both atrophy and hypoplasia

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The same genes cause late onset SCA and early onset SCARThe same genes cause late onset SCA and early onset SCAR

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… and things can get even more complicated!… and things can get even more complicated!

SCA5: dominant, late onset, non LOF

SCAR14: autosomal recessive, congenital onset, intellectual impairment, LOF

R480W: dominant (de novo), congenital onset, intellectualimpairment

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two brothers, 12 and 7 years oldMild psychomotor delay, IDAtaxiaNystagmusNot progressive cerebellar atrophy

Mother Father Probandaffected

sib CTRL

Rigidity and multifocal seizure syndrome / lethalMicrocephaly, RigidityMultifocal seizuresApnea and bradycardiaDeath in infancy (3-21 m)MRI: normal / Frontal hypoplasia / cerebro-cerebellar atrophymilder, non lethal form reported in few cases

Same gene, multiple phenotypes = BRAT1Same gene, multiple phenotypes = BRAT1

p.V214Gfs*189; WT p.T465T; WT

p.V214Gfs*189; p.T465T p.V214Gfs*189; p.T465T How pediatric neuroradiologists can help geneticists

a few practical examplesthe importance of collaboration

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Why neuroimaging is important for genetics?Why neuroimaging is important for genetics?

a good neuroimaging classification is essential to:- address certain patients to specific genetic testing

- group homogeneous patients for identification of novel genes

• clinical features can be unspecific (especially at onset) and indistinguishable among distinct disorders• the same disorder may present wide clinical heterogeneity• definite clear-cut correlations are emerging between certain gene mutations and specific neuroimaging patterns

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- early onset, rapid progression- extrapyramidal signs (dystonia, parkinsonism), pyramidal signs- bulbar involvement- mental deterioration, dementia- epilepsy, retinopathy, optic atrophy, acanthocythosis…

eye of the tiger sign

Imaging- atrophy of basal ganglia and substantia nigra- iron deposition in the basal ganglia

100% correlation with PANK2 mutations

Peculiar imaging patterns aiding genetic diagnosisPeculiar imaging patterns aiding genetic diagnosis

Pantothenate Kinase Associated Neurodegeneration (PKAN)

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Tubulinopathies

• variable neurological presentation•malformations involving:• basal ganglia• commissural tracts• cerebellum (+/- cysts), brainstem• cerebral cortex (lys, pachigyria, PMG)

Tubulin genes (dominant)

Poretti-Boltshauser syndrome

• ataxia, ID, language impairment•myopia or retinal dystrophy• diffuse cerebellar dysplasia• cerebellar cysts• square-like IV ventricle• splayed SCP LAMA1 (recessive)

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cerebellar agenesis + pancreatic agenesis

• severe IUGR, microcephaly, death in infancy• dysmorphisms• neonatal diabetes, near absence of subcutaneous fat•marked cerebellar hypoplasia / agenesis

PTF1A (recessive)


Chudley-McCullough Syndrome

• profound sensorineural hearing loss• (mild motor/cognitive delay)• cerebellar dysplasia• hydrocephalus, partial CC agenesis• frontal PMG, gray matter heterotopias

GPSM2 (recessive)

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GPR56-related PMG

- hypotonia, cerebellar and pyramidal signs- severe ID- frontoparietal or diffuse polymicrogyria- cerebellar dysplasia with cysts- patchy white matter anomalies- thin corpus callosum- mild pontine hypoplasiaGPR56 (recessive)

horizontal gaze palsy + progressive scoliosis

• congenital absence of horizontal gaze• severe progressive scoliosis• absence of decussation of corticospinal and somatosensory axonal tracts in the medulla

ROBO3 (recessive)


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the importance of collaborations

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MRI pattern recognition for gene discoveryMRI pattern recognition for gene discovery

WES of the two probands pathogenic recessive mutations in GSX2

Toresson et al, Development 2000

2 unrelated sporadic patients with unspecific clinical features (DD, tetraparesis, hyperkinetic movements) but identical and highly peculiar MRI pattern:

control patient 1

hypothalamic-midbrain fusion

absence of putamina

absence of olfactory tracts

KO mouse model fully recapitulatesCNS malformationsseen in patients

control Gsh2 ko mouse (E16.5)

De Mori et al, Brain 2019

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MRI pattern recognition for gene discoveryMRI pattern recognition for gene discovery

2 sisters with unspecific clinical features (DD, tetraparesis, intellectual defect) and MRI pattern resembling a tubulinopathy

tubulin genes all NEGATIVE WES homozygous missense mutation in TTL gene –Tubulin Tyrosin Ligase

manuscript in preparation

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Still hunting the causative genes…Still hunting the causative genes…

romboencephalosynapsis pontine tegmental cap dysplasia

• very rare conditions, only few patients described • extremely peculiar, well recognizable malformations• diagnosis made on a neuroradiological basis• no candidate genes, no shared chromosomal rearrangements• it is possible to speculate that sporadic cases are due to de novo mutations in a single gene or in genes involved in the same pathway

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What can we do with other “mysterious” defects?What can we do with other “mysterious” defects?

• defects that are clinically and neuroradiologically heterogeneousdiagnosis is descriptive, nosology is scarce• e.g. some primary brainstem anomalies, cerebellar cortical dysplasias, “macrocerebellum”• it is mandatory to homogeneously CLASSIFY these cases according to strict criteria similar defects may recognize a similar genetic basis(related to specific developmental pathways)• collaborations are important!

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Networking is crucial!Networking is crucial!

IRCCS Medea

IRCCS Besta

Univ. Brescia

IRCCS Gaslini

IRCCS Stella Maris

IRCCS Santa Lucia, Univ.

Sapienza, Univ. Cattolica, OPBG

Univ. Federico II

Univ. Messina

IRCCS Mondino

Univ. Verona

Univ. Padova

neurogenetics made easy

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