apparent homozygous deletion identified in alström syndrome patient elizabeth perrott west midlands...
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Apparent homozygous deletion identified in Alström syndrome patient
Elizabeth Perrott
West Midlands Regional Genetics Laboratory
Alström syndrome
First described by C.H. Alström in Sweden in 1959 Prevalence is less than 1/100,000 ~450 cases have been identified 30 known Alström families in the UK
“An autosomal recessive disease characterised by cone-rod retinal dystrophy, cardiomyopathy and type 2 diabetes mellitus”
Genetics
ALMS1 gene identified in 2002 23 exons, exon 8, 10 and 16 very large ALMS1 protein is of unknown function Frameshift, nonsense and missense
mutations Mutation hotspots are in exons 8, 10 and 16 No genotype-phenotype correlations
Testing at WMRGL
Local expertise in Alstrom syndrome– NSCAG clinics– research laboratory screening ALMS1
Confirmation of mutations identified by research laboratory
Partial gene screen of exons 10, 16 and part of exon 8 available
– Detects mutations in 25-40% of individuals
Full gene screen of coding regions of ALMS1 in 35 fragments available from Jan 2008
Case study
Patient MI: Asian female, born in 1999– Cone dystrophy – registered blind– Cardiomyopathy (diagnosed 3 months)– Developmental delay– Mild truncal obesity – difficult to take blood– Weight gain and insatiable appetite
DNA forwarded to Leeds for linkage analysis on this family
Clinician requested DNA be sent to Professor Barrett’s research lab no pathogenic mutations identified – failed to amplify exon 10
Request to WMRGL to perform partial gene screen on patient MI Exons 10, 16 and part of 8 were sequenced
Results of partial screen
No known pathogenic mutations identified
Homozygous missense variant (c.3386C>G; p.Ala1129Gly) identified in exon 8C -Not reported on databases or in literature-Variant is of unknown significance-parents both heterozygous for variant
Repeat analysis failed to amplify any product for any of the 3 fragments of exon 10
Discussion of results
Inhibition of exon 10 amplification
8C variant pathogenic
An unidentified mutation causing phenotype
Homozygous deletion
1) How frequent are ALMS1 deletions?
2) Are the couple consanguineous?
1) How frequent are ALMS1 deletions?
Literature:One case of homozygous exon 9 deletion in ALMS1 in a patient presenting with dilated cardiomyopathy – consanguineous family (1/79 mutations reported)
Other labs:“only seen patients with SNPs or small deletions, but none in which we have suspected that one or more exons have been deleted” (Douglas Friday, Senior Application Scientist, Centogene GmbH).
2) Are parents consanguineous?
131
142
214
ALMS gene
235
131
142
214
ALMS gene
235
131
142
214
ALMS gene
235
135
138
206
ALMS gene
241
131
142
214
ALMS gene
235
135
148
214
ALMS gene
241
Patient MI
Father of MI Mother of MI
Linkage results from Leeds:
Clinician has confirmed that this couple are consanguineous
Analysis of parental samples
Both parents showed normal alleles for fragments 10A, 10B and 10C– Parents carry at least one copy of exon 10
All SNPs in exon 10 are homozygous in parents– Parents may be hemizygous for these SNPs and
carry a heterozygous deletion of exon 10
8C
10A
10B
10C
8C
10A
10B
10C
G
C
C
N
A
AG
G
C
N
A
A
T
C
G
N
G
G
T
G
G
N
G
G
G
G
C
N
A
A
T
G
G
N
G
G
MI
Father of MI Mother of MI
If the parents are homozygous at these SNPs then they are not consanguineous.
Patient MI should be a heterozygote at these SNPs.
8C
10A
10B
10C
8C
10A
10B
10C
G
C
C
N
A
Adel?
G
del?
del?
del?
del?
T
C
G
N
G
G
del?
G
del?
del?
del?
del?
del?
G
del?
del?
del?
del?
del?
G
del?
del?
del?
del?
MI
Father of MI Mother of MI
Evidence suggests parents are hemizygous at these SNPs, carrying a deletion on the other allele.
Possible methods to confirm deletion
PCR + seq using newly-designed primers
Dosage analysis in parents using QF-PCR
MLPA– No Alström MLPA kit avaliable
Microarrays– Not sufficient coverage of the ALMS1 gene
Possible methods to confirm deletion
PCR + sequencing using newly-designed primers– How big could deletion be?– No primers for 9,11,12,13,14,15
Exon 8 9 10 11 Exon 16
~65kb
~118kb8IF-16AR
2kb10AF-10CR No amplification
RNA studies using exonic primers– Will identify exons deleted from mRNA transcript – Will reduce size of region to cover– Need fresh blood sample
Dosage analysis assay design
Designed four sets of fluorescently-tagged Beckman primers in exon 10 and exon 16 (control exon)
Fragment F-primer R-primer Size
10A New primer-Dye4 Existing primer 300
10B Existing primer New primer-Dye4 396
10C Existing primer New primer–Dye3 443
16A New primer–Dye3 Existing primer 340
Each exon 10 primer was diplexed with exon 16 control primers
25 cycle PCR performed Products analysed by capillary electrophoresis
Dosage analysis results
0100020003000400050006000700080009000
1000011000120001300014000150001600017000
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700
Dy
e
Si
gn
al
Size (nt)
357.17
MI
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700
Dy
e
Si
gn
al
Size (nt)
318.96
357.23
Mother of MI
0
10000
20000
30000
40000
50000
60000
70000
80000
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700
Dy
e
Si
gn
al
Size (nt)
318.94
357.27
Father of MI
0100002000030000400005000060000700008000090000
100000110000120000130000140000
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700
Dy
e
Si
gn
al
Size (nt)
318.77
357.16
Normal control
10A 16ASamples tested twice with 25 normal controls in total
All exon 10 peaks for parents showed reduced peak height compared to normals
No exon 10 peaks present for MI
Dosage calculations
Normal controls
Patient MI Mother of MI Father of MI
10A/16A 1.24:1 0:1 0.72:1(0.58)
0.70:1(0.56)
10B/16A 0.8:1 0:1 0.43:1(0.54)
0.43:1(0.53)
10C/16A 1.38:1 0:1 0.74:1(0.54)
0.56:1(0.41)
Result Homozygous deletion Heterozygous deletion Heterozygous deletion
Results consistent with the presence of a heterozygous deletion in both parents Fresh sample requested for RNA studies
(sample ratio/average ratio of normal controls)
RNA + DNA extracted from fresh blood sample cDNA prepared
Deletion expected to encompass exons 10-15
Amplification performed using exonic primers in exons 9 and 16
RNA studies
9 10 11 1312 161514
9 exonic F 16 exonic RNormal: 3454bp
Deleted: 745bp
RNA studies
A ACT G A C T T G T C CAAG AG TC CG A ATG T C AT T C AG AA
Exon 9 Exon 16
r.7672_10381del; p.Gly2558SerfsX46
F M MI
1kb
500bp
Normal RNA = 3,454bp
Product visible on agarose gel (~750bp)
No normal product visible
Sequencing revealed exons 10-15 missing
Deletion results in creation of protein termination codon
Conclusions
MI has a homozygous deletion including exons 10-15 predicted to result in truncated protein
Confirms clinical diagnosis of Alström syndrome Both parents carry the deletion
– 25% risk to future pregnancies Testing can now be offered to family members
2nd reported case of an ALMS1 deletion Deletions in ALMS1 may be more common than
reported Development of ALMS1 MLPA kit?
Further work
How did the deletion arise? Unequal homologous recombination of repetitive elements?
– reports of Alu elements causing homozygous deletions in consanguineous families in other diseases
Characterisation of breakpoints
9 10 11 15 Exon 16
? ?
34.3kb 13.1kb
Minimum deletion size: 69kb
Maximum deletion size: 117kb
Acknowledgements
West Midlands Regional Genetics LaboratoryPauline RehalRichard BarberJennie BellFiona MacdonaldSequencing team
Department of Medical and Molecular Genetics,University of BirminghamTim BarrettChris Ricketts