Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing

Abstract

Background: Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation.

Methods: We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases.

Results: We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients' clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders.

Conclusions: With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism.

Keywords: autism; causative; high-throughput sequencing; intellectual disability; mutation.

Figure 1

Inherited disrupting splice variant in DEAF1: what contribution to the phenotype? (A) Pedigree showing the maternally inherited splice variant in DEAF1 (c.290-3C>G); (B) prediction scores for the effect of the herein described variant on splicing (prediction scores for acceptor splice sites (ASS) as computed by SpliceSite Finder, MaxEntScan, NNsplice, GeneSplicer and Human Splicing Finder for the consensus ASS with either the wild-type or the mutated allele); (C) localisation of the variant, and its resulting effect on splicing in vitro (minigene construct): 90% of abnormal transcripts: 80% with entire exon #2 skipped, and 10% using the alternative ASS c.290-16 both leading to a frameshift and a premature stop codon.

Figure 2

Truncating variants not or ambiguously co-segregating with ID. (A) Pedigree of patient APN-13 carrying a frameshift variant in SRPX2 (c.602del, p.Ala201Valfs*10) demonstrating the likely inheritance from the asymptomatic (deceased) maternal grandfather, yet a putative germinal mosaicism of a de novo mutation cannot be excluded.; (B) predicted functional domains of SRXP2 from Pfam indicating locations of the herein identified mutation (in red) and those previously described. DUF4174: domain of unknown function; (C) pedigree showing the non-segregating nonsense variant in SHROOM4 (c.3772C>T; p.Gln1258*) in the family of patient APN-86; (D) location of the premature stop codon, which would disrupt the ASD2 domain of the protein.

Figure 3

Patient carrying two probably damaging missense variants in HCFC1 and ATRX: one causative mutation and one modifier variant? (A) Family tree of patient APN-113: proband carries a missense mutation in HCFC1 (c.218C>T; p.Ala73Val) already reported in two patients with cblX as well as another missense variant in ATRX (c.1013C>G, p.Ser338Cys). Both variants are maternally inherited, absent from the unaffected brother, but carried by the younger brother who died of sudden death at 2 months old; (B) associated predictions for the recurrent pathogenic missense mutation in HCFC1 and the possible modifier in ATRX, showing putative pathogenicity and moderate nucleotide conservation; (C) representation of HCFC1 and its domains: kelch domains (K1–K5), Fn3 (fibronectin type 3), basic domain, HCF-proteolysis repeats (HCF-pro), acidic domain and nuclear localisation signal (NLS) domain. The initial mutations involved in milder non-syndromic ID are indicated above: a regulatory variant in the 5′UTR of HCFC1 was identified by targeted massive parallel resequencing in a family with probable X-linked ID (XLID) (MRX3), which had for long remained unsolved. This variant was disrupting the functional binding site of the transcription factor YY1 within the HCFC1 promoter region, leading to an upregulation of its expression in lymphoblastoid cells. Subsequent screening of additional unsolved families identified one single co-segregating missense variant (c.674G>A; p.Ser225Asn) in HCFC1. The phenotype of both patients was rather mild: non-syndromic mild to moderate ID. In the bottom are indicated the mutations recently described in cblX patients; (D) the ATRX missense variant is located close but outside of the hotspot for disease-causing missense mutations (in the zinc-finger binding domain, in green) reported in patients with ATRX mutations, in red: mutations reported independently in at least two patients. Mutations are indicated when affecting residues 1–700 (reported in OMIM, ClinVar or in75), the rest of the protein is not represented; (E) Clinical information regarding APN-113 and previous genetic explorations, including X-inactivation status in the mother. CblX patients present with the same very severe phenotype: severe ID, early infantile epilepsy, choreoathetosis, microcephaly and more variable muscular hypotonia. Three of them are reported with early death in infancy; (F) biochemical abnormalities observed in the two affected brothers are similar to those previously observed.