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 papers Paper 1 – uses systematic sequencing approach Tarpey et al., Mutations in UPF3B, a member of the nonsense mediated mRNA decay complex, cause syndromic and non-symdromic mental retardation. Nature Genetics 39 (9): Paper 2 – uses X chromosome array-CGH Froyen et al., 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:

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) 3 rd 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, The genetics of mental retardation. Human Molecular Genetics 15 (2): R110- R116 Froyen et al., 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): Tarpey et al., A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. Nature Genetics 41(5): Whibley et al., Fine-scale survey of X chromosome copy number variants and indels underlying intellectual disability. American Journal of Human Genetics 87: