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Identification of X-linked mental retardation genes

Identification of X-linked mental retardation genes. Cat Yearwood St. George’s, London. Keywords. Mental retardation Syndromic Non-syndromic Sequencing Array-CGH Protein-truncating mutations Candidate gene Segregation studies Expression in brain. X-linked mental retardation (XLMR).

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Identification of X-linked mental retardation genes

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  1. Identification of X-linked mental retardation genes Cat Yearwood St. George’s, London

  2. Keywords • Mental retardation • Syndromic • Non-syndromic • Sequencing • Array-CGH • Protein-truncating mutations • Candidate gene • Segregation studies • Expression in brain

  3. X-linked mental retardation (XLMR) = mental retardation (IQ<70) with causative gene located on X chromosome Two types: • Syndromic – MR phenotype, but with other accompanying features such as dysmorphism and/or other neurological symptoms e.g. ATRX, Rett syndrome • Non-syndromic – MR only e.g. Frax E

  4. Identification of X-linked MR genes • Excess of males in the population who are affected with mental retardation (male:female ratio of 1.3:1) • Likely that genes on X chromosome have a role • Many XLMR genes already identified using traditional techniques such as positional cloning, translocation breakpoint mapping, candidate gene analysis and cytogenetic studies • But, likely to be more as many MR families with inheritance suggestive of an X-linked disorder with no mutations in known XLMR genes

  5. Recent techniques used in 2 papers Paper 1 – uses systematic sequencing approach Tarpey et al., 2007. Mutations in UPF3B, a member of the nonsense mediated mRNA decay complex, cause syndromic and non-symdromic mental retardation. Nature Genetics 39 (9): 1127-1133. Paper 2 – uses X chromosome array-CGH Froyen et al., 2008. Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation. The American Journal of Human Genetics82: 432-443.

  6. Paper 1 – X-chromosome sequencing • Part of larger study using high-throughput sanger sequencing to sequence coding regions of majority of X chromosome genes (>700 in total) • Carried out sequencing in probands of >200 MR families compatible with X linkage and who did not have mutations in known XLMR genes or any cytogenetic abnormalities • When protein truncating mutations identified, futher work was done to determine pathogenicity i.e. segregation studies and sequencing of normal controls • Interestingly found many genes in which truncating mutations did not segregate with disease in family and/ or were also found in normal controls, suggesting that a proportion of genes on the X-chromosome can be lost with no ill-effect • Identified 9 XLMR genes in total, this particular paper is about one of them UPF3B

  7. UPF3B mutations • 3 PTC mutations identified in 3 different families with syndromic MR • Sequencing of UPF3B gene in 118 probands from a new cohort of XLMR families identified a missense mutation in 1 family with non-syndromic MR (100% conserved residue therefore likely to be important for function of protein) • UPF3B = UPF3 regulator of nonsense transcripts homolog B (yeast) • Protein involved in nonsense-mediated mRNA decay

  8. RNA and Protein studies • Nonsense-mediated decay of significant proportion of mRNA transcripts occurred (RT-PCR used to measure expression levels) • Looked at 3 genes that are known targets of NMD – compared patients and controls – 1 of 3 genes showed significant increase in expression suggesting impairment of NMD • Western blotting using lymphoblastoid cell lines showed absence of UPF3B protein in 2 individuals from 2 families (other 2 families not tested)

  9. Phenotype • 2 PTC families had XLMR with marfanoid habitus (LFS phenotype) • 3rd PTC family had FG phenotype (MR, macrocephaly, hypotonia, imperforate anus, facial dysmorphism) • Missense family had non-syndromic MR • LFS and FG phenotypes thought to be allelic as previous studies found mutations in MED12 gene in both phenotypes. • Evidence suggests that mutations of UPF3B alter NMD of some mRNAs leading to phenotypes varying from non-syndromic MR to LFS and FG phenotypes

  10. Paper 2 – X chromosome array • X-chromosome specific array (nearly 2000 genomic clone probes, 80kb resolution) • Tested 300 probands picked from same large cohort used by paper 1 and another XLMR cohort • One of findings was that 5 families had overlapping microduplications of Xp11.22 that segregated with disease • Duplications of genes have previously been shown to be pathogenic e.g. MECP2 duplication in males with severe MR, another XLMR gene • This paper investigated Xp11.22 microduplications further

  11. Xp11.22 microduplications • Characterised breakpoints to determine region of overlap using 20 sets of primers for region and real-time PCT – determine which products were duplicated and which were not • Using real-time PCR to screen for duplication in another XLMR cohort found 1 additional duplication (B), none found in 350 normal controls • Region of overlap contained 4 genes: • SMC1A, RIBC1, HSD17B10 and HUWE1 and microRNAs mir-98 and let-7f-2 within the HUWE1 gene • FISH deduced that duplication was tandem

  12. Determining candidate gene(s) • RIBC1 not expressed in brain (in silico analysis) • SMC1A only partially duplicated in one family and when mRNA expression in the proband was quantified, using RT-PCR to obtain cDNA followed by real-time PCR, it was found that amount of transcript was not increased • HSD17B10 and HUWE1 both ubiquitously expressed with high expression in brain and significant increase in mRNA expression detected for both, therefore candidate genes • HSD17B10 has 1 previously described splicing mutation in XLMR case in literature • Sequencing study in paper 1 found 3 families with HUWE1 missense mutations that changed highly conserved residues and were not found in 750 normal controls

  13. Conclusions for Paper 2 • Evidence suggests that duplications which include HSD17B10 and HUWE1 are associated with non-syndromic XLMR • HUWE1 point mutations also associated with XLMR • Results from global expression studies using an exon expression array suggest that HUWE1 might be the major contributor to phenotype • Would need to find duplication of 1 gene without the other to confirm this

  14. Further Reading • Raymond and Tarpey, 2006. The genetics of mental retardation. Human Molecular Genetics 15 (2): R110-R116 • Froyen et al., 2007. Detection of genomic copy number changes in patients with idiopathic mental retardation by high resolution X-array-CGH: important role for increased gene dosage of XLMR genes. Human Mutation 28 (10): 1034-1042 • Tarpey et al., 2009. A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. Nature Genetics 41(5): 535-543 • Whibley et al., 2010. Fine-scale survey of X chromosome copy number variants and indels underlying intellectual disability. American Journal of Human Genetics 87: 173-188

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