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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)
= mental retardation (IQ<70) with causative gene located on X chromosome
Two types:1. Syndromic – MR phenotype, but with
other accompanying features such as dysmorphism and/or other neurological symptoms e.g. ATRX, Rett syndrome
2. Non-syndromic – MR only e.g. Frax E
• 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
Identification of X-linked MR genes
Recent techniques used in 2 papersPaper 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 Genetics 82: 432-443.
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
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
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)
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
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
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
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
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
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