Sequencing all X-chromosomal exons ('X-exome') in 405 families with X-linked intellectual disability (XLID) identified seven novel XLID genes but left a substantial residue — including about 75 families with four or more affected males in separate sibships — without a molecular diagnosis. The objective is to determine the remaining molecular causes of strongly X-linked families that remain undiagnosed after exome-level testing, in particular pathogenic non-coding and regulatory variation, and to establish the relative contributions of the candidate explanations.

Background

Intellectual disability (ID) is defined by significantly impaired cognitive function (intelligence quotient below about 70) and adaptive deficits with onset in childhood, and affects 1–2% of the population. Because males carry only one copy of the X chromosome, a single damaging variant there cannot be compensated by a healthy second copy, so X-linked intellectual disability (XLID) was historically the first genetic form of ID to be dissected: by 2014, linkage mapping, copy-number analysis (detection of deletions and duplications, for example by array comparative genomic hybridization, a microarray technique), and candidate-gene Sanger sequencing had implicated more than 100 X-chromosomal genes. Fragile X syndrome, caused by a CGG repeat expansion in the FMR1 gene, accounts for roughly 15% of XLID families. Yet even after two decades of gene discovery, more than half of all families showing the classical signature of X-linkage — multiple affected males in different sibships (sibling groups) connected through unaffected female carrier mothers — had no molecular diagnosis.

Hu and colleagues (Molecular Psychiatry, 2016) attacked this gap with 'X-exome' sequencing: massively parallel sequencing of essentially all protein-coding exons of the X chromosome (protein-coding exons make up roughly 1–2% of the genome) in the index males of 405 such unresolved families. They found pathogenic variants in established XLID genes in about 20% of the families, and likely causative variants in seven novel validated genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM, USP27X — most of them involved in ubiquitin-dependent protein turnover, transcriptional regulation, or synaptic signaling) and two candidate genes (CDK16, a cyclin-dependent kinase, and TAF1, a core component of the transcription machinery). Even after this screen a large residue remained undiagnosed, including 75 families with four or more affected males in separate sibships — the families whose statistical evidence for X-linkage is strongest. The authors enumerated candidate explanations: (1) technical failure of exon enrichment or coverage; (2) pathogenic variants in non-coding or not-yet-annotated regions of the X chromosome; (3) families that are in fact autosomal rather than X-linked; (4) rare missense variants of uncertain significance (VUS — sequence changes whose protein-level impact cannot be predicted) that are actually pathogenic; (5) multigenic causation; and (6) variants in undiscovered regulatory elements (DNA sequences that control when and where genes are active but do not encode protein). They argued that whole-genome sequencing (WGS), which also reads intronic and intergenic DNA, combined with resequencing of linkage intervals (the genome regions co-inherited by affected relatives) and better computational annotation of non-coding functional elements, was needed to close the gap; one precedent existed, a regulatory non-coding variant causing overexpression of the transcriptional regulator HCFC1 in a non-syndromic XLID family.

Subsequent studies have characterized the functional roles of the seven novel genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and of the two candidate genes (CDK16 and TAF1). The genetic basis of the unresolved families, however, still has no systematic explanation: whole-genome sequencing and systematic reanalysis resolve a minority of such families and uncover individual pathogenic non-coding variants, repeat expansions, and misclassified autosomal inheritance, and roughly a quarter to a third of XLID families — including the majority of intellectual-disability probands even under trio whole-genome sequencing (sequencing the affected individual and both parents) — still lack a diagnosis.

Problem Statement

Determine the molecular causes of the families with strong evidence for X-linked intellectual disability that remain without a genetic diagnosis after comprehensive X-exome sequencing — the roughly 75 families with four or more affected males in separate sibships described by Hu et al. (2016) being the best-documented subset of this general class — and establish the relative contributions of the candidate explanations: residual technical and coverage limitations; pathogenic non-coding or regulatory variants, including repeat expansions and variants in yet-to-be-annotated regions of the X chromosome; misclassified autosomal or multigenic inheritance; and genuinely pathogenic rare missense variants of uncertain significance. The remaining causes are sought by identifying the disease-causing variant(s) in as many such families as possible and by determining the functional consequences of the variants found.

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