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Contiguous gene deletion syndromes Charlotte Alston NCG Mitochondrial Diagnostic Laboratory

Contiguous gene deletion syndromes Charlotte Alston NCG Mitochondrial Diagnostic Laboratory Newcastle upon Tyne. ► What is a contiguous gene deletion syndrome? ► Williams syndrome – a contiguous gene deletion syndrome ► Diagnostic approaches for identifying contiguous gene deletions

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Contiguous gene deletion syndromes Charlotte Alston NCG Mitochondrial Diagnostic Laboratory

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  1. Contiguous gene deletion syndromes Charlotte Alston NCG Mitochondrial Diagnostic Laboratory Newcastle upon Tyne

  2. ► What is a contiguous gene deletion syndrome? ► Williams syndrome – a contiguous gene deletion syndrome ► Diagnostic approaches for identifying contiguous gene deletions Keywords: Williams syndrome; common 1.5Mb deletion; homologous recombination; cardiac defect; elfin facial; elastin gene; FISH; MLPA.

  3. What is a contiguous gene deletion syndrome?

  4. What is a contiguous gene deletion syndrome? “A syndrome caused by a microdeletion that spans two or more genes tandemly positioned along a chromosome”

  5. Examples of contiguous gene deletion syndromes: ► Williams-Beuren syndrome 7q11.23 ► DiGeorge syndrome 22q11.2 ► Wolf-Hirschhorn syndrome 4p16.3 ► Smith-Magenis syndrome 17p11.2

  6. Examples of contiguous gene deletion syndromes: ► Williams-Beuren syndrome 7q11.23 ► DiGeorge syndrome 22q11.2 ► Wolf-Hirschhorn syndrome 4p16.3 ► Smith-Magenis syndrome 17p11.2

  7. ►Williams Beuren syndrome 7q11.23 ► Common 1.5Mb deletion spanning 24-28 genes ► 320-500Kb highly repetitive sequence flanking the Williams critical region ► defective homologous recombination

  8. ►Williams Beuren syndrome 7q11.23 Normal homologous recombination at Williams Beuren syndrome chromosome region (WBSCR)

  9. ►Williams Beuren syndrome 7q11.23 Normal homologous recombination at Williams Beuren syndrome chromosome region (WBSCR)

  10. ►Williams Beuren syndrome 7q11.23 Normal homologous recombination at Williams Beuren syndrome chromosome region (WBSCR)

  11. ►Williams Beuren syndrome 7q11.23 Normal homologous recombination at Williams Beuren syndrome chromosome region (WBSCR)

  12. ►Williams Beuren syndrome 7q11.23

  13. ►Williams Beuren syndrome 7q11.23 Abnormal homologous recombination at Williams Beuren syndrome chromosome region (WBSCR)

  14. ►Williams Beuren syndrome 7q11.23 Abnormal homologous recombination at Williams Beuren syndrome chromosome region (WBSCR)

  15. ►Williams Beuren syndrome 7q11.23

  16. ►Williams Beuren syndrome 7q11.23 Duplication of Williams Beuren syndrome chromosome region (WBSCR)

  17. ►Williams Beuren syndrome 7q11.23

  18. ►Williams Beuren syndrome 7q11.23

  19. ►Williams Beuren syndrome 7q11.23 Deletion of Williams Beuren syndrome chromosome region (WBSCR)

  20. ► Deletion of WBSCR: Williams Beuren syndrome 7q11.23 Prevalence of 1:10,000 births ►dysmorphic facial features ►“Cocktail party” demeanour ►Excessive non-social anxiety ►Preserved vocabulary ►Cardiovascular problems ► supravalvular aortic & renal stenosis ►Transient hypercalcaemia (paediatric)

  21. ►Williams Beuren syndrome 7q11.23 "elfin" facial appearance Flat nose bridge Upturned tip of nose Long philtrum Full lips

  22. ►Williams Beuren syndrome 7q11.23 ► Aortic stenosis

  23. ►Williams Beuren syndrome 7q11.23 ► Renal stenosis Tapering of descending aorta Stenosis at renal arteries

  24. ►Williams Beuren syndrome 7q11.23 Genes involved in common 1.5Mb deletion (95% cases)

  25. ►Williams Beuren syndrome 7q11.23 ► Dissecting the roles of particular genes in WBS pathology is complicated ►Patients with rare deletions are key to dissecting genotype-phenotype relationships ►Hemizygosity of ELN, BAZ1B, CLIP2 and GTF21RD1 linked to classical Williams syndrome. ►Ascertainment bias towards patients with deletions spanning the ELN gene ► defined CV presentation directs WBSCR analysis

  26. ►Williams Beuren syndrome 7q11.23 ELN: Elastin gene. 90-100 % of the WBS patients have hemizygous deletion of ELN => Cause of supravalvular aortic stenosis (SVAS). CLIP2: Encodes cytoplasmic linker protein subunit 2 / 115 (CLIP115); implicated in membranous organelles / microtubules interaction => ? A cause of neurological features of Williams syndrome LIMK1: Encodes LIM Kinase; strongly expressed in brain => some neurological features of WBS FZD9: A homologue of the Drosophila frizzled wnt receptor TBL2: A transducin-beta like gene; putative regulatory role STX1A: Syntaxin 1A; protein plays a central role in neurotransmitter release RFC2: Haploinsufficiency predicted to reduce efficiency of DNA replication => growth deficiency & developmental disturbances FKBP6: Putative function of FKBP6: cellular receptor for immunosuppressants e.g. rapamycin. Deleted in almost all Williams patients.

  27. ►Williams Beuren syndrome 7q11.23 ► Mouse models aimed to elucidate genotype / phenotype correlations; removal of intra- individual variation. ► ~300 species unique genes and ~20% human genes are non-essential in mouse ► homozygous deletion may be tolerated in mouse, yet be lethal in man ► Obvious differences – quadruped mammals may not be best model for musculoskeletal disorder; higher metabolic rate; short lifespan;

  28. ►Williams Beuren syndrome 7q11.23 Mouse model has generated effective models simulating WBS –type defects: Elastin Eln KO creates heart defects in mice -/- lethal smooth muscle cell proliferation +/- viable; hypertensive, elevated serum calcium – facial dysmorphology – aortic & renal stenoses

  29. ►Williams Beuren syndrome 7q11.23 Limkinase1 inactivates cofilin in brain; actin binding putative role in axonal guidance during CNS development Baz1b KO causes heart defects in mice -/- lethal +/- viable; heart defects similar to WS elevated serum calcium

  30. ►Duplication of WBSCR - 7q11.23 ► 50% increase in dosage of affected genes ► No resemblance in clinical presentation vs WBS ►impaired expressive language skills ►absence of WBS facial dysmorphology ► Inversions involving WBSCR ~ phenotypically normal increased risk of duplication/deletion in offspring

  31. Detecting contiguous gene deletions ► FISH ► MLPA

  32. Detecting contiguous gene deletions ► Fluorescent In Situ Hybridisation (FISH) High resolution technique Permits detection of interstitial or subtelomeric microdeletions Metaphase or interphase chromosomes Commercial kits include fluorescently tagged probes specific to a particular locus Probe for Elastin gene in Williams syndrome

  33. Detecting contiguous gene deletions

  34. ► Williams Beuren syndrome 7q11.23 46XXdel7q11.23 Williams syndrome Wildtype sample

  35. ► DiGeorge syndrome 22q11 46XXdup22q11 46XXdel22q11

  36. ► Multiplex ligation-dependent probe amplification (MLPA) Commercially available MLPA kits specific to a given disorder Williams MLPA kit P029-A1 - 12 probes across 7q11.23 include ELN, LIMK1, STX1A - 20 reference probes

  37. ►Multiplex ligation-dependent probe amplification (MLPA) Forward or reverse primer is fluorescently tagged. Each complete ligated probe pair has a unique length. Resulting amplicons can be separated and identified by capillary electrophoresis. Reference probe pairs are included which bind on alternative chromosomes; unaffected by gene or exon deletions at target locus Comparison of a given probe peak area to other normalised probe peak areas (inc. reference probes) allows dosage to be determined.

  38. ► Multiplex ligation-dependent probe amplification (MLPA) Denaturation Probe-DNA Hybridisation Ligation of bound probes PCR of ligated probes Capillary electrophoresis Data analysis; dosage calculated by comparison of peak areas vs other peak areas

  39. References: Osborne LR. Animal models of Williams syndrome. Am J Med Genet C Semin Med Genet. 2010 May 15;154C(2):209-19. Eisenberg DP, Jabbi M, Berman KF. Bridging the gene-behavior divide through neuroimaging deletion syndromes: Velocardiofacial (22q11.2 Deletion) and Williams (7q11.23 Deletion) syndromes. Neuroimage. 2010 Nov 15;53(3):857-69. Pober BR. Williams-Beuren syndrome. N Engl J Med. 2010 Jan 21;362(3):239-52. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002 Jun 15;30(12):e57. MRC Holland MLPA website: http://www.mlpa.com/

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