CNTNAP2 and NRXN1 Are Mutated in Autosomal-Recessive Pitt-Hopkins-like Mental Retardation and Determine the Level of a Common Synaptic Protein in Drosophila 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 André Reis,1 Annette Schenck,2 and Anita Rauch1,7 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 The American 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 1Institute of Human Genetics, Friedrich Alexander University Erlangen-Nuremberg, 91054 Erlangen, Germany; 2Department 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; 3Unita Operativa Medicina Molecolare, Azienda Ospedaliera Universitaria Senese, Policlinico S. Maria alle Scotte, 53100 Siena, Italy; 4Department of Clinical Genetics, Odense University Hospital, 5000 Odense C, Denmark; 5Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton, SO16 5YA, UK; 6Department of Clinical Genetics, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands; 7Institute 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. Journal of Human Genetics 85, 655–666, November 13, 2009 655 mailto:czweier@humgenet.uni-erlangen.de molecular pathogenesis. We therefore utilized the fruit fly Drosophila melanogaster as a model and collected data that point to a common synaptic link between these two genes. Subjects and Methods Patients Our study group consisted of 179 patients, including two sibling pairs, who were referred for TCF4 testing because of severe MR and variable additional features reminiscent of the PTHS spectrum, such as microcephaly, dysmorphic facial gestalt, or breathing anomalies. TCF4 mutational testing revealed normal results in all of these patients. Ethics approval for this study was obtained from the ethics committee of the Medical Faculty, University of Erlangen-Nuremberg, and informed consent was obtained from parents or guardians of the patients. Molecular Karyotyping Molecular karyotyping was performed in 48 patients with the Affymetrix 500 K SNP Array and in 12 patients with the Affymetrix 6.0 SNP Array, in accordance with the supplier’s instructions. In the index patient of family 1, hybridization was performed with an Affymetrix GeneChip Mapping 500K SNP array, and the second affected patient and both parents were analyzed with the Affyme- trix GeneChip Mapping 250K Nsp SNP array. Copy-number data were analyzed with the Nexus software (Biodiscovery) and the Affymetrix Genotyping Console 3.0.2 software. Molecular karyo- typing in patients 2 and 3 was performed with the Affymetrix GeneChip Mapping 6.0 array platform, and copy-number data were analyzed with the Affymetrix software Genotyping Console 3.0.2. The identified copy-number variants (CNVs) were sub- mitted to the Decipher database (patient 1a, 250902; patient 2, 250903; patient 3, 250904). Mutational Screening DNA samples from 177 patients, derived from peripheral-blood or lymphoblastoid cell lines, were screened for CNTNAP2 (NM_014141) and NRXN1 (NM004801) mutations by unidirec- tional direct sequencing of the coding exons 1–24 of CNTNAP2 and the coding exons 2–22 of NRXN1, including intronic flanking regions (ABI BigDye Terminator Sequencing Kit v.3; Applied Biosystems), with the use of an automated capillary sequencer (ABI 3730; Applied Biosystems). Mutations were confirmed with an independent PCR and bidirectional sequencing. Primer pairs can be found in Table S1, available online. For splice-site predic- tion, the online tools NNSPLICE 0.9 and HSF V2.3 were used. FISH and MLPA Fluorescence in situ hybridization (FISH) analysis was performed in family 1 with the directly Cy3-labeled bacterial artificial chro- mosome (BAC) clone RP4-558L10 on metaphase spreads, in accor- dance with standard protocols. Probes for all coding exons of CNTNAP2 were designed and MLPA reaction was performed in accordance with the guidelines of MRC-Holland. The deletion in patient 3 was confirmed with MLPA with the use of a probe within exon 2 and a control probe within exon 12 of NRXN1. Probe sequences are listed in Table S2. Analysis of Relationship The relationship of individuals within family 1 was analyzed, with a four-generation family with known relationships used as back- 656 The American Journal of Human Genetics 85, 655–666, Novem ground, with the Graphical Representation of Relationships (GRR) software.13 For GRR, we selected, from Affymetrix 250K arrays, 10,000 randomly distributed autosomal SNPs with a minimal minor allele frequency of 0.2 in Europeans. For each pair of indi- viduals, GRR calculates over the 10,000 markers the identical- by-state (IBS) mean and standard deviation. The graphical plot of IBS mean versus IBS standard deviation facilitates distinguishing between relationships such as parents and offspring, siblings, half siblings, and cousins, as well as identical or unrelated individuals. Additionally, SNP genotypes around CNTNAP2 were analyzed in the family members. Drosophila Genes and Lines Drosophila orthologs of TCF4, NRXN1, and CASPR2 (daughterless [CG5102], Nrx-1 [CG7050], and Nrx-IV [CG6827]) were identified by the ENSEMBLE genome browser or by the reciprocal BLAST best-hit approach. Two RNAi lines, to Nrx-IV and daughterless, respectively, were obtained from the Vienna Drosophila Research Center (VDRC) and gave consistent phenotypes. VDRC lines no. 9039 (Nrx-IV) and no. 51297 (daughterless) were utilized for further analysis. RNA interference was induced with the UAS- Gal4 system. The w1118 line (VDRC no. 60000) was used as a control, representing the same genetic background as the RNAi lines. Flies were raised at 28�C for maximum efficiency of knock- down. The Nrx-I overexpression line pUAST-Nrx-I was obtained from Wei Xie from Nanjing, China. Gal4 driver lines and the inducible Nrx-IV overexpression line P(EP)Nrx-IVEP604 were obtained from the Bloomington stock center. Quantitative Real-Time PCR RNA extraction from 333 L3 larvae of each genotype was per- formed with the RNeasy Lipid Tissue Kit (QIAGEN) in accordance with the supplier’s protocols. cDNA synthesis was performed with iScript (Biorad). Quantitative real-time PCR was performed with the Power SYBR Green PCR Master Mix on a 7500 Fast Real-Time PCR System (Applied Biosystems), and results were normalized to the endogenous control actin. Primer sequences can be found in Table S3. Immunostaining and Data Acquisition We harvested 10–18 hr embryos and fixed them with 3.7% PFA for 20–25 min. All primary antibodies—anti-elav (labels nuclei of all neurons), antibody 22c10 (sensory nervous system), antibody BP102 (axon tracts, central neuropile region), anti-fas II (motor- and central pioneer axons), and antibody nc82 (anti-bruchpilot, synaptic active zones) (all from the Developmental Studies Hybridoma Bank [DHSB])—were used in a 1:100 dilution. Late stage (16/17), nc-82-labeled embryos were assigned to one of three phenotypic categories: strong peripheral staining, moderate stain- ing, and weak or residual staining, respectively. We performed statistical analysis of 69 wild-type (WT) embryos (w1118) and 73 Nrx-IV knockdown embryos from three independent experiments with a chi-square test and a Fisher’s-exact test to obtain p values. The images for peripheral synaptic staining were obtained with a Zeiss Apotome. Brains were dissected from L3 larvae and fixed for 30 min in 3.7% PFA. Pictures of WT and mutant brains were acquired with the use of the same microscope settings. Intensities of nc82 immu- nostainings were measured with Image J within two fields at two standardized positions in each CNS, one in the upper third and one in the lower third of the ventral nerve cord. The average of ber 13, 2009 these two values was normalized to the average of controls for comparison of results from independent experiments. A total of 45 w1118 brains from six independent experiments, 11 elav-Gal4:: Nrx-I brains from two independent experiments, 19 double- elav-Gal4::Nrx-I brains from three independent experiments, 12 elav-Gal4::Nrx-IV brains from two independent experiments, and 21 double-elav-Gal4::Nrx-IV brains from four independent experiments were measured. p values were obtained with a Wil- coxon test for two samples for comparison to the WT. Type 1b neuromuscular junctions (NMJs) of muscle 4 were analyzed after dissection of L3 larvae and fixation in 3.7% PFA for 30 min. Costaining was performed with nc82 and DLG (both from DHSB) or HRP (Jackson Immuno Research) antibodies in a dilution of 1:500. NMJ pictures were stacked in ImageJ and pro- cessed in Adobe Photoshop. Numbers of active zones and branches were manually counted in an animated stack, and total synaptic area was determined by ImageJ. A total of 21 WT NMJs, 19 overexpression Nrx-I NMJs, and 20 overexpression Nrx-IV NMJs from at least two independent experiments were counted. For the evaluation of branches, 27 WT NMJs, 25 overexpression Nrx-I NMJs, and 20 overexpression Nrx-IV NMJs from three exper- iments were counted. We performed statistical evaluation with the Wilcoxon test for two samples, comparing each of the genotypes to the WT. The antibody against NrxIV was obtained from Chris- tian Klämbt, Münster, Germany.14 Secondary antibodies for all stainings were either Alexa 568- or Alexa 488-labeled antibodies against mouse or rabbit (Molecular Probes). All data were acquired blind to the evaluated phenotype. Results Identification of Recessive Deletions and Mutations in CNTNAP2 and NRXN1 Molecular karyotyping led to the identification of a homo- zygous deletion of exons 2–9 within the CNTNAP2 gene on chromosome 7q35-q36.1 in a sibling pair of European origin (P1a and P1b), formerly published as possible clinical cases of Pitt-Hopkins syndrome.15 This deletion was confirmed by FISH analysis (Figure S2) and MLPA (data not shown) and is predicted to be in frame but result in the loss of several functional domains (Figure 1A, Figure 2A, and Figure S1). Consanguinity of the parents had been denied,15 and no indication for consanguinity was found by analysis of relationship with the use of the information of 10.000 SNPs. However, when the SNPs within and around CNTNAP2 were analyzed, they showed homozygosity in both children, indicating an allele of common ancestry. By subsequent mutational screening of CNTNAP2 in a larger cohort of 177 additional TCF4- mutation-negative patients, we identified a third patient of European origin (P2) with compound heterozygosity for the splice mutation IVS10-1G>T and a partial in-frame deletion of exons 5–8, identified by molecular karyotyping (Figure 1B and Figure 2A) and confirmed with MLPA. The splice-site mutation resulted in lack of recognition of the splice acceptor site by two splice-site-prediction programs and is therefore predicted to result in loss of exon 10, leading to a frame shift, and the deletion is predicted to result in the loss of two laminin G domains. The splice- The America site mutation was not found in 384 control chromosomes, and no CNTNAP2 deletion was found in 667 molecularly karyotyped control individuals. In both families, the parents were heterozygous carriers of one of the respective defects. In another European patient of our cohort who had a very similar phenotype (P3), we identified a heterozygous 180 kb deletion within the NRXN1 gene on chromosome 2p16.3, spanning exons 1–4, including the start codon. This deletion was inherited from the healthy mother, but no deletions affecting the coding region of NRXN1 were found in 667 molecularly karyotyped healthy controls. Subsequent sequencing of NRXN1 in this patient revealed a stop mutation in exon 15 on the second allele, which was inherited from the healthy father (Figure 1C and Figure 2B). Both mutations are predicted to result in loss of the so-called alpha-isoform of NRXN1, one of two NRXN1 isoforms that are transcribed from alternative promoters. The presumably remaining shorter beta- isoform (Figures 2B and 2C) appears not to be sufficient to ensure normal function, which is in accordance with findings in alpha-neurexin knockout mice.16 Mutational screening of NRXN1 in our study cohort did not reveal any additional defects. Clinical Characterization As far as data are available, birth measurements of all patients (P1a, P1b, P2, and P3) were normal. Further growth development was also normal, apart from short stature in the siblings from family 1 and additional microcephaly in one of them. All four patients with recessive defects in CNTNAP2 or NRXN1 showed severe MR with lack of speech or with speech limited to single words (P1b), whereas motor milestones were normal or only mildly delayed, with a walking age of 2 years in P3. Episodes of hyperbreathing occurred in all patients, and seizures with an age at onset between 4 months and 30 months were observed in P1a, P1b, and P2. Additional variable anomalies were cerebellar hypoplasia, autistic behavior, and stereotypic movements. Apart from a wide mouth with thick lips in P1a and P1b and a wide mouth in P3, no specific facial dysmorphisms were noted (Figure 1D). Parents of all patients were healthy, and the deceased sister of the father of P3 was said to have had epilepsy and mild MR. P1a and P1b have been described in detail by Orrico et al.,15 and an overview of clinical details of all patients is shown in Table 1. Lack of NRXN1 and CNTNAP2 expression in blood or fibroblasts (Bakkaloglu et al.17 and data not shown) precluded functional studies on human material. Analysis of CNTNAP2 and NRXN1 Orthologs Nrx-IV and Nrx-I in Drosophila Although a synaptic role for NRXN1 is known, this has not yet been established for CNTNAP2. However, the high simi- larity of clinical phenotypes caused by defects in the two genes suggested a potential common molecular contribu- tion. To address this, as well as a further possible connection n Journal of Human Genetics 85, 655–666, November 13, 2009 657 Figure 1. Pedigrees and Results of Molecular Karyotyping (A) Pedigree of family 1, with two affected children and homozygous deletion of CNTNAP2 affecting exons 2–9. Both parents are hetero- zygous carriers of the deletion. Results are from molecular karyotyping with Affymetrix 250K SNP arrays and analysis with the Genotyp- ing Console 3.0.2 software (Affymetrix). The deletion-flanking SNPs in the 500K array of P1a are SNP_A-1991616 (145,562,641 Mb; UCSC Human Genome Browser version 18 [hg18]) and SNP_A-1991672 (146,730,410 Mb; hg18), with a maximal deletion size of 1,167,269 bp and a minimal size of 1,146,016 bp (Nexus software). (B) Pedigree of P2, with one affected child. The patient harbors an in-frame 180 kb deletion affecting CNTNAP2 exons 5–8 and a splice- site mutation in the splice donor site of exon 11. Results of molecular karyotyping data from the Affymetrix 6.0 SNP array were analyzed with the Genotyping Console 3.0.2 software, showing a deletion from CN_1217185 (146,387,354 Mb; hg18) to SNP_A-4269862 (146,566,863 Mb; hg18). See Figure S1 for SNP copy-number profiles. 658 The American Journal of Human Genetics 85, 655–666, November 13, 2009 with TCF4, we utilized Drosophila as a model organism. All three genes, TCF4, NRXN1, and CNTNAP2, are highly conserved in evolution and have orthologs in Drosophila. We initially hypothesized that the TCF4 ortholog daughterless might regulate Nrx-I and Nrx-IV as transcrip- tional targets. Knockdown of daughterless to 60% of WT levels by the use of two different ubiquitous driver lines (promoter-Gal4 lines that regulate inducible RNAi alleles; see Subjects and Methods) resulted in pupal lethality, con- firming the importance of daughterless for fly development (C) Pedigree of P3, with a compound-heterozygous deletion of NRXN1 exons 1–4 and a stop mutation in exon 15. Results of molecular karyotyping data from the Affymetrix 6.0 SNP array were analyzed with the Genotyping Console 3.0.2 (Affymetrix), showing a 113 kb deletion between CN_864223 (51,001,003 Mb; hg18) and CN_864269 (51,113,677 Mb; hg18). (D) Clinical pictures of P2, with a compound-heterozygous deletion and mutation in CNTNAP2 and of patient 3, with a compound- heterozygous deletion and mutation in NRXN1. Apart from a wide mouth in patient 3, no specific dysmorphisms are noted. Figure 2. Structure of CNTNAP2 and NRXN1 (A) Schematic drawing of the genomic structure of CNTNAP2 with color coding for domain-coding exons and localization of mutations and deletions. Black bars represent deletions. Abbreviations are as follows: SP, signal peptide; DISC, discoidin-like domain; LamG, lam- inin-G domain; EGF, epidermal growth factor-like domain; FIB, fibrinogen-like domain; TM, transmembran region; PDZPB, PDZ- domain-binding site ; F1, family 1; P2, patient 2; Amish, homozygous mutation in the Amish population, published by Strauss et al.36 (B) Schematic drawing of the genomic structure of a-NRXN1 and b-NRXN1 with color coding for domain-coding exons and localization of the mutation and deletion in patient 3, the deletion being represented by a black bar. Abbreviations are as follows: SP, signal peptide; LamG, laminin-G domain; EGF, epidermal growth factor-like domain; TM, transmembrane region; PDZPB, PDZ-domain-binding site. (C) Schematic drawing of the domain structure of neurexins, CASPR2, and CASPR in humans and Drosophila. In contrast to CASPR, CASPR2 contains a PDZ-domain-binding site but lacks a PGY repeat region, rich in proline, glycine, and tyrosine residues. Both neurexin I and CASPR2/Nrx-IV contain PDZ-domain-binding sites at their intracellular C terminus but differ in the presence of discoidin-like and fibrinogen-like domains and in the order of laminin-G domains. The American Journal of Human Genetics 85, 655–666, November 13, 2009 659 Table 1. Phenotype in Patients with CNTNAP2 and NRXN1 Mutations Siblings Patients P1a P1b P2 Amish36 (N ¼ 9) P3 Mutations CNTNAP2 deletion of exons 2–9, homozygous CNTNAP2 deletion of exons 2–9, homozygous CNTNAP2 deletion of exons 5–8 þ IVS10-1G>T CNTNAP2 c.3709delG, homozygous NRXN1 deletion of exons 1–4 þ p.S979X Age 20 yrs 15 yrs 11 yrs 1–10 yrs 18 yrs Sex f m f not reported f Parents healthy healthy healthy not reported healthy Birth weight Length OFC 3700 g 51 cm 34.5 cm not known 3700 g at term not reported 3450 g normal Height