The development of microarray-based technologies for comparative genomic hybridization (array-CGH) analysis has enabled the detection of submicroscopic microdeletions or microduplications, also referred to as copy number variations (CNVs). Recently, this approach has been widely used in neurologic and psychiatric disorders, including mental retardation (MR),^1-3 autism spectrum disorders (ASDs),^4-7 and schizophrenia.^8-11 Findings from these studies suggested that several genes involved in similar neurodevelopmental pathways may be associated with these conditions. However, so far only rare structural variants, sometimes present in a single case, have been identified. Therefore, it is difficult to decipher which of these variations are causative, which are risk factors, and which are only rare polymorphisms unrelated to any pathologic phenotype. De novo rearrangements are usually considered pathogenic, but this argument (which is acceptable for rare large rearrangements detectable by conventional cytogenetics) should be considered with caution for smaller CNVs, for which a high mutation rate is expected. Indeed, it has been estimated that a de novo segmental deletion occurs in 1 per 8 newborns and a de novo segmental duplication in 1 per 50 newborns, with most of these rearrangements being benign polymorphic variants.¹² Therefore, the disease association of CNVs has to be tested systematically by comparing the frequency of each candidate CNV in cases and in healthy control subjects. Given the low frequency of each CNV, this would require the study of huge series, achievable only in the context of forthcoming meta-analyses. Other problems arise because ascertainment of most of the published samples, initially recruited for linkage studies, is biased toward multiplex cases and because control samples, when present, are generally composed of subjects not screened for the studied pathologic conditions. The objectives of the present study were (1) to provide an estimate of the collective frequency of a set of recurrent or overlapping CNVs in 3 different groups of cases compared with controls and (2) to assess whether each CNV is present in more than 1 clinical category.

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.

Truncating mutations were found in the PHF8 gene (encoding the PHD finger protein 8) in two unrelated families with X linked mental retardation (XLMR) associated with cleft lip/palate (MIM 300263). Expression studies showed that this gene is ubiquitously transcribed, with strong expression of the mouse orthologue Phf8 in embryonic and adult brain structures. The coded PHF8 protein harbours two functional domains, a PHD finger and a JmjC (Jumonji-like C terminus) domain, implicating it in transcriptional regulation and chromatin remodelling. The association of XLMR and cleft lip/palate in these patients with mutations in PHF8 suggests an important function of PHF8 in midline formation and in the development of cognitive abilities, and links this gene to XLMR associated with cleft lip/palate. Further studies will explore the specific mechanisms whereby PHF8 alterations lead to mental retardation and midline defects.