ARTICLE CNTNAP2 and NRXN1 Are Mutated in Autosomal-Recessive Pitt-Hopkins-like Mental Retardation and Determine the Level of a Common Synaptic Protein in Drosophila Christiane Zweier, 1,2, * Eiko K. de Jong, 2 Markus Zweier, 1 Alfredo Orrico, 3 Lilian B. Ousager, 4 Amanda L. Collins, 5 Emilia K. Bijlsma, 6 Merel A.W. Oortveld, 2 Arif B. Ekici, 1 Andre ´ Reis, 1 Annette Schenck, 2 and Anita Rauch 1,7 Heterozygous copy-number variants and SNPs of CNTNAP2 and NRXN1, two distantly related members of the neurexin superfamily, have been repeatedly associated with a wide spectrum of neuropsychiatric disorders, such as developmental language disorders, autism spectrum disorders, epilepsy, and schizophrenia. We now identified homozygous and compound-heterozygous deletions and mutations via molecular karyotyping and mutational screening in CNTNAP2 and NRXN1 in four patients with severe mental retardation (MR) and variable features, such as autistic behavior, epilepsy, and breathing anomalies, phenotypically overlapping with Pitt-Hopkins syndrome. With a frequency of at least 1% in our cohort of 179 patients, recessive defects in CNTNAP2 appear to significantly contribute to severe MR. Whereas the established synaptic role of NRXN1 suggests that synaptic defects contribute to the associated neuropsychiatric disor- ders and to severe MR as reported here, evidence for a synaptic role of the CNTNAP2-encoded protein CASPR2 has so far been lacking. Using Drosophila as a model, we now show that, as known for fly Nrx-I, the CASPR2 ortholog Nrx-IV might also localize to synapses. Overexpression of either protein can reorganize synaptic morphology and induce increased density of active zones, the synaptic domains of neurotransmitter release. Moreover, both Nrx-I and Nrx-IV determine the level of the presynaptic active-zone protein bruchpilot, indicating a possible common molecular mechanism in Nrx-I and Nrx-IV mutant conditions. We therefore propose that an analogous shared synaptic mechanism contributes to the similar clinical phenotypes resulting from defects in human NRXN1 and CNTNAP2. Introduction The etiology of severe mental retardation (MR) is heteroge- neous, and, despite a significant number of identified disease genes, 1 the majority of cases, especially nonsyn- dromic cases, remain unsolved. 2 Many of the currently known MR-related genes are involved in neurogenesis and neuronal migration, and awareness of the implication of synaptic organization and plasticity in MR has only recently begun to rise. 3,4 In 2007, haploinsufficiency of the basic helix-loop-helix (bHLH) transcription factor 4 (TCF4) was identified as causative for Pitt-Hopkins syn- drome (PTHS [MIM 610954]), a severe MR disorder with variable additional anomalies, such as breathing anomalies, epilepsy, and facial dysmorphism including a beaked nose and a wide mouth with a cupid’s-bow- shaped upper lip. 5,6 TCF4 belongs to the E-protein family of bHLH transcription factors, which bind as homo- and heterodimers to E-box consensus sequences in promoters of target genes. 7 Like other E-proteins, TCF4 shows a broad expression pattern and a high expression in the CNS. 8,9 After the identification of the underlying gene in 2007, approximately 50 patients have been reported, 5,6,8–11 demonstrating the importance of a diagnostic test for the increased recognition and appreciation of a previously clinically underdiagnosed condition. Because of a similar severe degree of MR, commonly associated seizures, and microcephaly, PTHS has evolved as an important differen- tial diagnosis to the two most common syndromic disor- ders in severe MR, Rett (MIM 312750) and Angelman (MIM 105830) syndromes. 11 Because only 12% of patients referred to us with suspected PTHS showed mutations in TCF4 (Zweier et al. 11 and unpublished data), the clinically relatively homogenous group of 179 TCF4-mutation-nega- tive patients, including two sibling pairs, represented a suit- able study cohort for searching for additional candidate genes for overlapping disorders. Through molecular karyotyping and mutational anal- ysis, we indeed identified recessive defects in two genes, CNTNAP2 and Neurexin I (NRXN1), in patients with a very similar severe MR disorder and variable additional symptoms, such as seizures and breathing anomalies, resembling Pitt-Hopkins syndrome. In light of the shared phenotype that characterizes our patients with recessive CNTNAP2 and NRXN1 defects, and on the basis of the theme of overlapping phenotypes being caused by genes that are linked with each other in molecular networks, 12 we further aimed to address the hypothesis of a common 1 Institute of Human Genetics, Friedrich Alexander University Erlangen-Nuremberg, 91054 Erlangen, Germany; 2 Department of Human Genetics, Nijme- gen Centre for Molecular Life Sciences, Donders Institute for Brain, Cognition and Behaviour & Radboud University Nijmegen Medical Centre, 6525 GA Nijmegen, The Netherlands; 3 Unita Operativa Medicina Molecolare, Azienda Ospedaliera Universitaria Senese, Policlinico S. Maria alle Scotte, 53100 Siena, Italy; 4 Department of Clinical Genetics, Odense University Hospital, 5000 Odense C, Denmark; 5 Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton, SO16 5YA, UK; 6 Department of Clinical Genetics, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands; 7 Institute of Medical Genetics, University of Zurich, 8603 Zurich-Schwerzenbach, Switzerland *Correspondence: czweier@humgenet.uni-erlangen.de DOI 10.1016/j.ajhg.2009.10.004. ª2009 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 85, 655–666, November 13, 2009 655