1. Apparent homozygous deletion identified in Alström syndrome patient Elizabeth Perrott West Midlands Regional Genetics Laboratory

2. 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”

3. 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

4. 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

5. 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

6. 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

7. 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?

8. 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).

9. 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 MIMother of MI Linkage results from Leeds: Clinician has confirmed that this couple are consanguineous

10. 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

11. 8C 10A 10B 10C 8C 10A 10B 10C G C C N A A G 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 MIMother of MI If the parents are homozygous at these SNPs then they are not consanguineous. Patient MI should be a heterozygote at these SNPs.

12. 8C 10A 10B 10C 8C 10A 10B 10C G C C N A A del? G T C G N G G G G G MI Father of MIMother of MI Evidence suggests parents are hemizygous at these SNPs, carrying a deletion on the other allele.

13. 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

14. 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 89 10 11Exon 16 ~65kb ~118kb 8IF-16AR 2kb 10AF-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

15. Dosage analysis assay design Designed four sets of fluorescently-tagged Beckman primers in exon 10 and exon 16 (control exon) FragmentF-primerR-primerSize 10ANew primer-Dye4Existing primer300 10BExisting primerNew primer-Dye4396 10CExisting primerNew primer–Dye3443 16ANew primer–Dye3Existing primer340 Each exon 10 primer was diplexed with exon 16 control primers 25 cycle PCR performed Products analysed by capillary electrophoresis

16. Dosage analysis results MI Mother of MI Father of MI Normal control 10A16A Samples 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

17. Dosage calculations Normal controls Patient MIMother of MIFather of MI 10A/16A1.24:10:10.72:1 (0.58) 0.70:1 (0.56) 10B/16A 0.8:10:10.43:1 (0.54) 0.43:1 (0.53) 10C/16A1.38:10:10.74:1 (0.54) 0.56:1 (0.41) ResultHomozygous deletionHeterozygous 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)

18. 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 910111312161514 9 exonic F16 exonic R Normal: 3454bp Deleted: 745bp

19. RNA studies A ACT G A C T T G T C CAAGAG TC CG A ATG T C AT T C AG AA Exon 9Exon 16 r.7672_10381del; p.Gly2558SerfsX46 FMMI 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

20. 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 2 nd reported case of an ALMS1 deletion Deletions in ALMS1 may be more common than reported Development of ALMS1 MLPA kit?

21. 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

22. 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