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.
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We thank all the families who participated in this research. We also thank Susanne Freier, Ines Müller, Rien Blok, Carolien Oosterhoud, Roel Brandts, Demis Tserpelis, Evelyn Douglas, Lynne Hobson, Pia Winter and Sara Ekvall for excellent technical assistance; Dagmar Wieczorek, Vincent Desportes, Jean-Paul Bonnefont, Valerie Biancalana, Daniel Amram, Stanislas Lyonnet, Jacqueline Vigneron, Veronica Cusin, Philippe Jonveaux, Laurence-Olivier Faivre, Lydie Burglen, Yves Alembik, Sophie Scheidecker, Aurelia Jacquette, Delphine Heron, Cyril Goizet, Marie Ange Delrue, Didier Lacombe, Odile Boute, Andre Megarbane, Anne Moncla, Brigitte Gilbert Tiong Tan, Sharron Townshend, Michael Gabbett, Zornitza Stark, Mac Gardner, Joanne Dixon, Ian Glass, Martin Delatycki, Salim Aftimos, Felicity Collins, Paul James, George McGillivray, Kate Pope, Judith Allanson, Claude Moraine, Christine Francannet, Annick Toutain, James Lespinasse, Brigitte Gilbert, Isabelle Mortemousque, Anne Moncla, Etienne Mornet, Albert David, Connie Schrander-Stumpel, Christine de Die-Smulders, Yvonne Arens, Eric Smeets, Dominique Marcus-Soekarman, Ute Moog, Bert Smeets, Marzena Wiśniewska, Renata Glazar, Maciej Robert Krawczyński, Barbara Leube, Magdalena Nawara, Agnieszka Charzewska, Irma Järvelä, Barbara Oehl-Jaschkowitz, Marco Henneke, Peter Propping, Gholamali Tariverdian, Almuth Caliebe, Dorothea Wand, Jürgen Mücke, Eva Seemanova, Birgit Zirn, Jörg Müsebeck, Rudolf Mallmann, Christiane Zweier, Rami Abou Jamra and Anita Rauch for contributing families with XLID. We would like to thank the Exome Aggregation Consortium and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at http://exac.broadinstitute.org/about. This work was financed by a grant of the German Ministry of Education and Research through the MRNET (to HHR); the EU FP7 project GENCODYS, grant number 241995; the Max Planck Innovation Funds (to HHR), NWO VENI grant: 91666017 to SGMF, grant 2009(2)-81 from the Dutch Brain Foundation to AdB, WCH Foundation and Australian NHMRC to JG, grant from Fondation pour la Recherche Médicale (FRM, J Chelly-Equipe FRM 2013: DEQ2000326477). Web Resources 1000 Genomes Project Consortium, http://www.1000genomes.org/data. SplazerS, http://www.seqan.de/projects/splazers.html. SnpStore, http://www.seqan.de/projects/snpstore.html.
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