Jinong Feng

High frequency of neurexin 1β signal peptide structural variants in patients with autism

Neuroligins are postsynaptic membrane cell-adhesion molecules which bind to beta-neurexins, a family of proteins that act as neuronal cell surface receptors. To explore the possibility that structural variants in the beta-neurexin genes predispose to autism, the coding regions and associated splice junctions of three beta-neurexin genes were scanned with detection of virtually all mutations-SSCP (DOVAM-S) in 72 Caucasian patients with autism. In addition, segments of the neurexin 1beta gene were sequenced in 131 additional Caucasian and 61 Afro-American patients with autism from South Carolina and the Midwest. Two putative missense structural variants were identified in the neurexin 1beta gene in four Caucasian patients with autism and not in 535 healthy Caucasian controls (4/203 vs. 0/535, P=0.0056). Initial family data suggest that incomplete penetrance may occur. In addition, no structural variant was found in the neurexin 2beta gene and the neurexin 3beta gene. In the context of all available data, we conclude that mutations of the neurexin 1beta gene may contribute to autism susceptibility.

Analysis of the neuroligin 3 and 4 genes in autism and other neuropsychiatric patients

SIR—Jamain et al reported a frameshift and a mis sense mutation in the X-linked neuroligin 4 (NLGN4, MIM# 300427) and neuroligin 3 (NLGN3, MIM# 300336) genes, respectively, in Swedish families with autism. A frameshift mutation in NLGN4 appeared de novo in the mother, cosegregated with an affected brother with Asperger syndrome and was absent in a normal brother. This frameshift mutation was not present in 600 unrelated control X-chromosomes. A missense mutation in NLGN3, R451C, was found in the mother and two sibs, one with autism and another with Asperger syndrome, but no other relatives were studied. It was not found in 300 unrelated control X-chromosomes. Laumonnier et al reported a large French family in which 10 males had nonspecific X-linked mental retardation, two had autism and one had pervasive developmental disorder. All affected patients were found to have the same frameshift mutation (1253delAG) in the NLGN4 gene. One obligate female carrier had mild mental retardation. The NLGN3 and NLGN4 genes map to Xq13 and Xp22.3, respectively. The NLGN3 gene spans 32 kb, and the NLGN4 gene spans 338 kb. NLGN3 has eight exons, encoding two alternatively spliced isoforms of 828 and 848 amino acids. The NLGN4 gene contains six exons and codes for a protein of 816 amino acids. All neuroligins contain an N-terminal hydrophobic sequence with the characteristics of a cleaved signal peptide followed by a large esterase homology domain, a highly conserved single transmembrane region, and a short cytoplasmic domain. To better understand the relationship between the NLGN4 gene and autism, the coding regions and associated splice junctions of the NLGN4 gene were scanned for mutations with DOVAM-S (Detection of Virtually All Mutations-SSCP) and direct sequencing in the following subjects: 148 unrelated patients with autism (76 Midwest US Caucasians and 72 Portuguese Caucasians; 122 males and 26 females), 48 patients without autism, including 24 Midwest US Caucasian patients with attention deficit hyperactivity disorder (ADHD) and 24 UK Caucasian patients with DSM-IV Bipolar I Disorder (BPD), as well as 48 Portuguese healthy control subjects. The Portuguese autistic patients were diagnosed using DSM-IV criteria, the Autism Diagnostic Interview-Revised (ADI-R) and the Childhood Autism Rating Scale (CARS). Idiopathic subjects were included in the study after clinical assessment and screening for known medical and genetic conditions associated with autism (fragile X, chromosomal disorders, neurocutaneous syndromes, metabolic disorders, infectious diseases). Neuropsychological evaluation was performed using the Ruth Griffiths Mental Developmental Scales or the Wechsler Intelligence Scale for Children (WISC), depending on the patient’s age. The Midwest autistic patients were diagnosed as described previously. Putative missense mutations were identified once each in the NLGN4 gene in four separate autistic patients (Table 1). G99S and K378R were found in unrelated Portuguese patients. V403M and R704C were found in unrelated Midwest patients. G99, K378 and V403 are located in the esterase domain and R704 is located in the cytoplasmic domain. Three of the structural changes, K378R, V403M and R704C, occur in asymptomatic mothers, while G99S occurs in a mother with learning disability. Comprehensive mutation scanning of 48 Portuguese healthy controls and sequencing of the appropriate exons in 288 healthy controls including 96 Portuguese and 192 Midwest US Caucasians (144 males and 192 females; 528 X-chromosomes total) did not reveal these four missense variants or any other structural changes (4/148 vs 0/336, P1⁄4 0.009 or 4/174 vs 0/528, P1⁄4 0.004, when the Fisher exact test is performed with patients or X-chromosome alleles, respectively). In addition, no structural variants were found in a pilot experiment performed on patients with ADHD and BPD (24 of each). Patient #1 has a younger brother with a diagnosed language disability and a global developmental quotient below the mean (Ruth Griffiths Mental Developmental Scales score of 89), who also carries the G99S variation. Their mother, who is heterozygous for the variation, had a documented learning disability. As also found by Laumonnier et al, sequence variation in NLGN4 may be segregating with autism and cognitive disability in this family. Patient #3 had an affected brother with V403M. He had three other unaffected sibs, including a sister without the variant and two brothers with V403M who had normal social function (making friends easily), normal school performance and no attentional problems, consistent with an absence of cosegregation between this variant and any phenotype. The three unaffected children had Social Communication Questionnaire (SCQ) (Lifetime) scores of 0. Proband #3 was specifically of Irish descent. In all, 50 normal female controls of Irish descent were sequenced, none of them had V403M. Molecular Psychiatry (2005) 10, 329–335 & 2005 Nature Publishing Group All rights reserved 1359-4184/05

SNPs in human miRNA genes affect biogenesis and function

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Neurexin 1α structural variants associated with autism

MicroRNAs (miRNAs) are 21-25-nucleotide-long, noncoding RNAs that are involved in translational regulation. Most miRNAs derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm. Sequence variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have profound affects on miRNA biogenesis and function. In the context of analyzing the roles of miRNAs in Schizophrenia and Autism, we defined at least 24 human X-linked miRNA variants. Functional assays were developed and performed on these variants. In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of processing. Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature miRNA sequence may drive the evolution of new microRNAs by altering their biological function. Finally, the miRNAs examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases.

MECP2 structural and 3′-UTR variants in schizophrenia, autism and other psychiatric diseases: A possible association with autism

Neurexins are presynaptic membrane cell-adhesion molecules which bind to neuroligins, a family of proteins that are associated with autism. To explore the possibility that structural variants in the neurexin alpha genes predispose to autism, the coding regions and associated splice junctions of the neurexin 1alpha gene were sequenced in 116 Caucasian patients with autism and 192 Caucasian controls. Five ultra-rare structural variants including a predicted splicing mutation were found in patients with autism and absent in 10,000 control alleles. Only one ultra-rare structural variant was found in controls (5/116 vs. 1/192; P=0.03, Fishers exact test, one-sided). In the context of all available data, the ultra-rare structural variants of the neurexin 1alpha gene are consistent with mutations predisposing to autism.

Evidence for X-Chromosomal Schizophrenia Associated with microRNA Alterations

Mutations in the gene coding for methyl‐CpG‐binding protein 2 (MECP2) cause Rett syndrome (RTT) and have also been reported in a number of X‐linked mental retardation syndromes. Furthermore, putative mutations recently have been described in a few autistic patients and a boy with language disorder and schizophrenia. In this study, DNA samples from individuals with schizophrenia and other psychiatric diseases were scanned in order to explore whether the phenotypic spectrum of mutations in the MECP2 gene can extend beyond the traditional diagnoses of RTT in females and severe neonatal encephalopathy in males. The coding regions, adjacent splicing junctions, and highly conserved segments of the 3′‐untranslated region (3′‐UTR) were examined in 214 patients, including 106 with schizophrenia, 24 with autism, and 84 patients with other psychiatric diseases by detection of virtually all mutations‐single strand conformation polymorphism (SSCP) (DOVAM‐S). To our knowledge, this is the first analysis of variants in conserved regions of the 3′‐UTR of this gene. A total of 5.2 kb per haploid gene was analyzed (1.5 Mb for 214 patients). A higher frequency of missense and 3′‐UTR variants was found in autism. One missense and two 3′‐UTR variants were found in 24 patients with autism versus one patient with a missense change in 144 ethnically similar individuals without autism (P = 0.009). These mutations suggest that a possible association between MECP2 mutations and autism may warrant further study.

Identification of high risk DISC1 structural variants with a 2% attributable risk for schizophrenia

Background Schizophrenia is a severe disabling brain disease affecting about 1% of the population. Individual microRNAs (miRNAs) affect moderate downregulation of gene expression. In addition, components required for miRNA processing and/or function have also been implicated in X-linked mental retardation, neurological and neoplastic diseases, pointing to the wide ranging involvement of miRNAs in disease. Methods and Findings To explore the role of miRNAs in schizophrenia, 59 microRNA genes on the X-chromosome were amplified and sequenced in males with (193) and without (191) schizophrenia spectrum disorders to test the hypothesis that ultra-rare mutations in microRNA collectively contribute to the risk of schizophrenia. Here we provide the first association of microRNA gene dysfunction with schizophrenia. Eight ultra-rare variants in the precursor or mature miRNA were identified in eight distinct miRNA genes in 4% of analyzed males with schizophrenia. One ultra-rare variant was identified in a control sample (with a history of depression) (8/193 versus 1/191, p = 0.02 by one-sided Fishers exact test, odds ratio = 8.2). These variants were not found in an additional 7,197 control X-chromosomes. Conclusions Functional analyses of ectopically expressed copies of the variant miRNA precursors demonstrate loss of function, gain of function or altered expression levels. While confirmation is required, this study suggests that microRNA mutations can contribute to schizophrenia.

Mutations in the dystrophin gene are associated with sporadic dilated cardiomyopathy.

The causes of schizophrenia remain elusive. In a large Scottish pedigree, a balanced translocation t(1;11) (q42.1;q14.3) disrupting the DISC1 and DISC2 genes segregates with major mental illness, including schizophrenia and unipolar depression. A frame-shift carboxyl-terminal deletion was reported in DISC1 in an American family, but subsequently found in two controls. A few common structural variants have been associated with less than a 2-fold increased risk for schizophrenia, but replication has not been uniform. No large scale case-control mutation study has been performed. We have analyzed the regions of likely functional significance in the DISC1 gene in 288 patients with schizophrenia and 288 controls (5 megabases of genomic sequence analyzed). Six patients with schizophrenia were heterozygous for ultra-rare missense variants not found in the 288 controls (p=0.015) and shown to be ultra-rare by their absence in a pool of 10,000 control alleles. We conclude that ultra-rare structural variants in DISC1 are associated with an attributable risk of about 2% for schizophrenia. In addition, we confirm that two common structural variants (Q264R and S704C) elevate the risk for schizophrenia slightly (odds ratio 1.3, 95% CI: 1.0-1.7). DISC1 illustrates how common/moderate risk alleles suggested by the HapMap project might be followed up by resequencing to identify genes with high risk, low frequency alleles of clinical relevance.

Analysis of the neuroligin 4Y gene in patients with autism.

Dilated cardiomyopathy (DCM) is the major indication for heart transplantation. Approximately 30% of all DCM is thought to be inherited, while 70% is sporadic. Mutations in the dystrophin gene have been associated with the uncommon X-linked form of DCM. We hypothesized that missense mutations and other less severe mutations of the dystrophin gene might predispose to the common form of sporadic DCM. To test this hypothesis, 22kb of genomic dystrophin DNA was scanned with DOVAM-S in each of the 22 patients with sporadic DCM, including all 79 coding sequences and splice junctions, as well as six alternative exon 1 dystrophin isoforms (484kb, total). Three putative new mutations (IVS5+1 G>T, K18N, and F3228L) and seven polymorphisms were identified. The splice site mutation IVS5+1 is predicted to cause skipping of exon 5, which is within a region containing an actin binding site. The missense mutations occur at amino acids that display substantial evolutionary conservation. Screening of 236 control individuals failed to identify these three mutations. The three patients with putative mutations had CK-MM (creatine kinase, skeletal muscle) levels greater than 250 units while the 14 patients without mutations for which CK-MM were available had values ranging from 20 to 200. The first comprehensive mutation scanning of the exons and splice junctions of the dystrophin gene in patients with sporadic DCM presents the evidence that point mutations are associated with sporadic DCM without clinical evidence of skeletal myopathy. It may be prudent to measure CK-MM in all patients with dilated cardiomyopathy to identify candidates at high risk for dystrophin mutations.

ATM missense mutations are frequent inpatients with breast cancer

Frameshift and missense mutations in the X-linked neuroligin 4 (NLGN4, MIM♯ 300427) and neuroligin 3 (NLGN3, MIM♯ 300336) genes have been identified in patients with autism, Asperger syndrome and mental retardation. We hypothesize that sequence variants in NLGN4Y are associated with autism or mental retardation. The coding sequences and splice junctions of the NLGN4Y gene were analyzed in 335 male samples (290 with autism and 45 with mental retardation). A total of 1.1 Mb of genomic DNA was sequenced. One missense variant, p.I679V, was identified in a patient with autism, as well as his father with learning disabilities. The I679 residue is highly conserved in three members of the neuroligin family. The absence of p.I679V in 2986 control Y chromosomes and the high similarity of NLGN4 and NLGN4Y are consistent with the hypothesis that p.I679V contributes to the etiology of autism. The presence of only one structural variant in our population of 335 males with autism/mental retardation, the unavailability of significant family cosegregation and an absence of functional assays are, however, important limitations of this study.