X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4−/− mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

We ascertained a multi-generation Malaysian family with Joubert syndrome (JS). The presence of asymptomatic obligate carrier females suggested an X-linked recessive inheritance pattern. Affected males presented with mental retardation accompanied by postaxial polydactyly and retinitis pigmentosa. Brain MRIs showed the presence of a "molar tooth sign," which classifies this syndrome as classic JS with retinal involvement. Linkage analysis showed linkage to Xpter-Xp22.2 and a maximum LOD score of 2.06 for marker DXS8022. Mutation analysis revealed a frameshift mutation, p.K948NfsX8, in exon 21 of OFD1. In an isolated male with JS, a second frameshift mutation, p.E923KfsX3, in the same exon was identified. OFD1 has previously been associated with oral-facial-digital type 1 (OFD1) syndrome, a male-lethal X-linked dominant condition, and with X-linked recessive Simpson-Golabi-Behmel syndrome type 2 (SGBS2). In a yeast two-hybrid screen of a retinal cDNA library, we identified OFD1 as an interacting partner of the LCA5-encoded ciliary protein lebercilin. We show that X-linked recessive mutations in OFD1 reduce, but do not eliminate, the interaction with lebercilin, whereas X-linked dominant OFD1 mutations completely abolish binding to lebercilin. In addition, recessive mutations in OFD1 did not affect the pericentriolar localization of the recombinant protein in hTERT-RPE1 cells, whereas this localization was lost for dominant mutations. These findings offer a molecular explanation for the phenotypic spectrum observed for OFD1 mutations; this spectrum now includes OFD1 syndrome, SGBS2, and JS.

We report on a large family in which a novel X-linked recessive mental retardation (XLMR) syndrome comprising macrocephaly and ciliary dysfunction co-segregates with a frameshift mutation in the OFD1 gene. Mutations of OFD1 have been associated with oral-facial-digital type 1 syndrome (OFD1S) that is characterized by X-chromosomal dominant inheritance and lethality in males. In contrast, the carrier females of our family were clinically inconspicuous, and the affected males suffered from severe mental retardation, recurrent respiratory tract infections and macrocephaly. All but one of the affected males died from respiratory problems in infancy; and impaired ciliary motility was confirmed in the index patient by high-speed video microscopy examination of nasal epithelium. This family broadens the phenotypic spectrum of OFD1 mutations in an unexpected way and sheds light on the complexity of the underlying disease mechanisms.

Recently, a truncating mutation of the UBE2A gene has been observed in a family with X-linked mental retardation (XLMR) (1). The three affected males had similar phenotypes, including seizures, obesity, marked hirsutism and a characteristic facial appearance. Here, we report on two families with a total of seven patients and a clinically very similar syndromic form of XLMR. Linkage analysis was performed in the larger of these families, and screening several positional candidate genes revealed a G23R missense mutation in the UBE2A gene. Subsequent UBE2A screening of a phenotypically similar second family revealed another missense mutation, R11Q, again affecting an evolutionarily conserved amino acid close to the N-terminus of the protein. SIFT and PolyPhen analyses suggest that both mutations are pathogenic, which is supported by their absence in 168 healthy controls. Thus, both missense and truncating mutations can give rise to a specific, syndromic form of XLMR which is identifiable in a clinical setting.