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