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.