Transcript Elizabeth Perrott

Apparent homozygous deletion
identified in Alström syndrome patient
Elizabeth Perrott
West Midlands Regional
Genetics Laboratory
Alström syndrome
“An autosomal recessive disease
characterised by cone-rod retinal dystrophy,
cardiomyopathy and type 2 diabetes mellitus”
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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
Genetics
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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
–
Full
Detects mutations in 25-40% of individuals
gene screen of coding regions of ALMS1 in 35
fragments available from Jan 2008
Case study
Patient MI: Asian female, born in 1999
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–
–
–
–
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
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Clinician requested DNA be sent to Professor Barrett’s research lab
no pathogenic mutations identified – failed to amplify exon 10
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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
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Inhibition of exon 10 amplification
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8C variant pathogenic
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An unidentified mutation causing phenotype
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Homozygous deletion
1) How frequent are ALMS1 deletions?
2) Are the couple consanguineous?
1) How frequent are ALMS1 deletions?
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Literature:
One case of homozygous exon 9 deletion in ALMS1
in a patient presenting with dilated cardiomyopathy
– consanguineous family (1/79 mutations reported)
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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?
Linkage results
from Leeds:
Father of MI
Mother of MI
131
135
131
135
142
138
142
148
214
206
214
214
ALMS gene
ALMS gene
ALMS gene
ALMS gene
235
241
235
241
Patient MI
131
131
142
142
214
214
ALMS gene
ALMS gene
235
235
Clinician has
confirmed that this
couple are
consanguineous
Analysis of parental samples
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Both parents showed normal alleles for fragments
10A, 10B and 10C
– Parents carry at least one copy of exon 10
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All SNPs in exon 10 are homozygous in parents
– Parents may be hemizygous for these SNPs and
carry a heterozygous deletion of exon 10
Father of MI
Mother of MI
8C
C
G
10A
G
G
G
G
T
G
T
G
10C N
N
8C
C
G
10A
A
A
C
C
G
A
G
A
10B
10C N
N
10B
MI
G
G
A
G
C
G
G
A
T
G
N
N
If the parents are
homozygous at these
SNPs then they are not
consanguineous.
Patient MI should be a
heterozygote at these
SNPs.
Father of MI
Mother of MI
8C
C
G
10A
G
del?
G
del?
T
G
del?
del?
10C N
del?
8C
C
G
10A
A
del?
C
del?
G
A
del?
del?
10B
10C N
del?
10B
MI
G
G
del?
del?
del?
del?
del?
del?
del?
del?
del?
del?
Evidence suggests parents are
hemizygous at these SNPs,
carrying a deletion on the
other allele.
Possible methods to confirm deletion
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PCR + seq using newly-designed primers
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Dosage analysis in parents using QF-PCR
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MLPA
– No Alström MLPA kit avaliable
Microarrays
– Not sufficient coverage of the ALMS1 gene
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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
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8IF-16AR
10AF-10CR
Exon 8
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9
~118kb
2kb
10
~65kb
No amplification
11
RNA studies using exonic primers
– Will identify exons deleted from mRNA transcript
– Will reduce size of region to cover
– Need fresh blood sample
Exon 16
Dosage analysis assay design
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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
a
a
l
l
Dosage analysis results
D
y
e
S
n
10A
0
50
100
150
200
357.17
16A
250
1 0 04 5 0
1 5 05 0 0
200 550
Normal control
a
45000
70000
6 5 0 3 5 0 7 0 04 0 0
S iz e ( n t)
450
500
450
500
550
i g
i g
n
a
80000
n
50000
250 600 300
MI
l
l
No exon 10 peaks
present for MI
17000
16000
15000
14000
1318.77
3000
12000
11000
1 0 0 0 0 357.16
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
300
03 5 0
5 04 0 0
S iz e ( n t)
i g
i g
Samples tested twice with
25 normal controls in total
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
S
0
0
0
0
0
0
0
0
0
0
0
0
0
0
e
0
0
0
0
0
0
0
0
0
0
0
0
0
0
y
0
0
0
0
0
0
0
0
0
0
0
0
0
0
D
4
3
2
1
0
9
8
7
6
5
4
3
2
1
n
1
1
1
1
1
40000
All exon 10 peaks for
parents showed reduced
peak height compared to
normals
60000
357.27
357.23
S
S
35000
50000
318.94
e
y
20000
30000
D
D
e
318.96
25000
40000
y
30000
15000
20000
10000
10000
5000
0
0
0
50
100
150
200
250
300 0
Mother of MI
350 50
S iz e ( n t)
400100
450150
500200
550250
600300
650350 700400
S iz e ( n t)
Father of MI
550
6
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)
Homozygous deletion
Heterozygous deletion
Heterozygous deletion
Result
(sample ratio/average ratio of normal controls)
Results consistent with the presence of a heterozygous deletion
in both parents
Fresh sample requested for RNA studies
RNA studies
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RNA + DNA extracted from fresh blood sample
cDNA prepared
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Deletion expected to encompass exons 10-15
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Amplification performed using exonic primers in
exons 9 and 16
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Normal: 3454bp
9 exonic F
16 exonic R
Deleted: 745bp
9
10
11
12 13 14 15
16
RNA studies
F
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M
MI
Normal RNA =
3,454bp
Product visible on agarose
gel (~750bp)
No normal product visible
1kb
500bp
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Sequencing revealed exons
10-15 missing
Exon 9
Exon 16
A ACT G A C T T G T C CAAG AG TC CG A ATG T C AT T C AG AA
Deletion results in creation of
protein termination codon
r.7672_10381del; p.Gly2558SerfsX46
Conclusions
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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
Characterisation of breakpoints
?
?
10
9
34.3kb
11
15
Exon 16
13.1kb
Minimum deletion size: 69kb
Maximum deletion size: 117kb
How did the deletion arise?
 Unequal homologous recombination of repetitive elements?
– reports of Alu elements causing homozygous deletions in
consanguineous families in other diseases
Acknowledgements
West Midlands Regional Genetics Laboratory
Pauline Rehal
Richard Barber
Jennie Bell
Fiona Macdonald
Sequencing team
Department of Medical and Molecular Genetics,
University of Birmingham
Tim Barrett
Chris Ricketts