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Apparent homozygous deletion identified in Alström syndrome patient

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

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Apparent homozygous deletion identified in Alström syndrome patient

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  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. 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? Discussion of results

  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 235 135 138 206 ALMS gene 241 131 142 214 ALMS gene 235 135 148 214 ALMS gene 241 131 142 214 ALMS gene 235 131 142 214 ALMS gene 235 Linkage results from Leeds: Father of MI Mother of MI Patient MI 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 C G G C G G G A G A A G C C G G C G T T T G G G A A G A N N N N N N G G Father of MI Mother of MI 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 C G G C G del? del? del? G A del? del? del? del? G del? C del? T del? del? G G del? del? del? del? A del? N del? del? N del? G del? Father of MI Mother of MI 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. ~118kb 8IF-16AR 2kb No amplification 10AF-10CR Exon 8 9 10 11 Exon 16 ~65kb 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 • 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) • Each exon 10 primer was diplexed with exon 16 control primers • 25 cycle PCR performed • Products analysed by capillary electrophoresis

  16. 10A 16A Normal control MI Mother of MI Father of MI Dosage analysis results Samples tested twice with 25 normal controls in total No exon 10 peaks present for MI All exon 10 peaks for parents showed reduced peak height compared to normals

  17. Dosage calculations (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

  18. Normal: 3454bp Deleted: 745bp 9 exonic F 16 exonic R 9 10 11 12 13 14 15 16 RNA studies • 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

  19. F M MI 1kb 500bp 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 RNA studies • Product visible on agarose gel (~750bp) • No normal product visible Normal RNA = 3,454bp • Sequencing revealed exons 10-15 missing • Deletion results in creation of protein termination codon r.7672_10381del; p.Gly2558SerfsX46

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

  21. ? ? 10 11 15 Exon 16 9 13.1kb 34.3kb 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 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

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