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