Uncovering obsessive-compulsive disorder risk genes in a pediatric cohort by high-resolution analysis of copy number variation

Background

Obsessive-compulsive disorder (OCD) is a heterogeneous neuropsychiatric condition, thought to have a significant genetic component. When onset occurs in childhood, affected individuals generally exhibit different characteristics from adult-onset OCD, including higher prevalence in males and increased heritability. Since neuropsychiatric conditions are associated with copy number variations (CNVs), we considered their potential role in the etiology of OCD.

Methods

We genotyped 307 unrelated pediatric probands with idiopathic OCD (including 174 that were part of complete parent-child trios) and compared their genotypes with those of 3861 population controls, to identify rare CNVs (<0.5 % frequency) of at least 15 kb in size that might contribute to OCD.

Results

We uncovered de novo CNVs in 4/174 probands (2.3 %). Our case cohort was enriched for CNVs in genes that encode targets of the fragile X mental retardation protein (nominal p?=?1.85?×?10^?03; FDR=0.09), similar to previous findings in autism and schizophrenia. These results also identified deletions or duplications of exons in genes involved in neuronal migration (ASTN2), synapse formation (NLGN1 and PTPRD), and postsynaptic scaffolding (DLGAP1 and DLGAP2), which may be relevant to the pathogenesis of OCD. Four cases had CNVs involving known genomic disorder loci (1q21.1-21.2, 15q11.2-q13.1, 16p13.11, and 17p12). Further, we identified BTBD9 as a candidate gene for OCD. We also sequenced exomes of ten “CNV positive” trios and identified in one an additional plausibly relevant mutation: a 13 bp exonic deletion in DRD4.

Conclusions

Our findings suggest that rare CNVs may contribute to the etiology of OCD.

     

Expression of <Emphasis Type="Italic">CHRFAM7A</Emphasis> and <Emphasis Type="Italic">CHRNA7</Emphasis> in neuronal cells and postmortem brain of HIV-infected patients: considerations for HIV-associated neurocognitive disorder

Despite the recent advances in antiretroviral therapy, human immunodeficiency virus type 1 (HIV-1) remains a global health threat. HIV-1 affects the central nervous system by releasing viral proteins that trigger neuronal death and neuroinflammation, and promotes alterations known as HIV-associated neurocognitive disorders (HAND). This disorder is not fully understood, and no specific treatments are available. Recently, we demonstrated that the HIV-1 envelope protein gp120_IIIB induces a functional upregulation of the α7-nicotinic acetylcholine receptor (α7) in neuronal cells. Furthermore, this upregulation promotes cell death that can be abrogated with receptor antagonists, suggesting that α7 may play an important role in the development of HAND. The partial duplication of the gene coding for the α7, known as CHRFAM7A, negatively regulates α7 expression but its role in HIV infection has not been studied. Hence, we studied both CHRNA7 and CHRFAM7A regulation patterns in various gp120_IIIB in vitro conditions. In addition, we measured CHRNA7 and CHRFAM7A expression levels in postmortem brain samples from patients suffering from different stages of HAND. Our results demonstrate the induction of CHRNA7 expression accompanied by a significant downregulation of CHRFAM7A in neuronal cells when exposed to pathophysiological concentrations of gp120_IIIB. Our results suggest a dysregulation of CHRFAM7A and CHRNA7 expressions in the basal ganglia from postmortem brain samples of HIV+ subjects and expand the current knowledge about the consequences of HIV infection in the brain.     

The contribution of 7q33 copy number variations for intellectual disability

Copy number variations (CNVs) at the 7q33 cytoband are very rarely described in the literature, and almost all of the cases comprise large deletions affecting more than just the q33 segment. We report seven patients (two families with two siblings and their affected mother and one unrelated patient) with neurodevelopmental delay associated with CNVs in 7q33 alone. All the patients presented mild to moderate intellectual disability (ID), dysmorphic features, and a behavioral phenotype characterized by aggressiveness and disinhibition. One family presents a small duplication in cis affecting CALD1 and AGBL3 genes, while the other four patients carry two larger deletions encompassing EXOC4, CALD1, AGBL3, and CNOT4. This work helps to refine the phenotype and narrow the minimal critical region involved in 7q33 CNVs. Comparison with similar cases and functional studies should help us clarify the relevance of the deleted genes for ID and behavioral alterations.     

The complete chloroplast genomes of two <Emphasis Type="Italic">Sinalliaria</Emphasis> species and species delimitation (Brassicaceae)

Sinalliaria is a new small genus of Brassicaceae with only two species (S. grandifolia and S. limprichtiana) endemic in eastern China. Because of their limited distribution, identifying the species and sources of S. grandifolia and S. limprichtiana are important for biodiversity conservation and ecological preservation. The chloroplast genome sizes of Sinalliaria grandifolia and S. limprichtiana were 154,113 and 154,060 bp in length. Both genomes contain a large single copy region of 83 k bp and a small single copy region of 17 k bp, which were separated by a pair of 26,253 bp (S. grandifolia) and 26,275 bp (S. limprichtiana) inverted repeat regions. They both contained 129 genes, including 85 protein-coding genes (78 PCG species), seven ribosomal RNA genes (four rRNA species) and 37 tRNA genes (30 tRNA species). Phylogenetic analyses of five chloroplast genome sequences clustered two Sinalliaria grandifolia individuals and three S. limprichtiana individuals into two highly supported monophyletic groups. These complete chloroplast genomes laid a good foundation for biodiversity conservation, population genomics, molecular and ecological studies of Sinalliaria.     

Systematic Prioritization and Integrative Analysis of Copy Number Variations in Schizophrenia Reveal Key Schizophrenia Susceptibility Genes

Schizophrenia is a common mental disorder with high heritability and strong genetic heterogeneity. Common disease-common variants hypothesis predicts that schizophrenia is attributable in part to common genetic variants. However, recent studies have clearly demonstrated that copy number variations (CNVs) also play pivotal roles in schizophrenia susceptibility and explain a proportion of missing heritability. Though numerous CNVs have been identified, many of the regions affected by CNVs show poor overlapping among different studies, and it is not known whether the genes disrupted by CNVs contribute to the risk of schizophrenia. By using cumulative scoring, we systematically prioritized the genes affected by CNVs in schizophrenia. We identified 8 top genes that are frequently disrupted by CNVs, including NRXN1, CHRNA7, BCL9, CYFIP1, GJA8, NDE1, SNAP29, and GJA5. Integration of genes affected by CNVs with known schizophrenia susceptibility genes (from previous genetic linkage and association studies) reveals that many genes disrupted by CNVs are also associated with schizophrenia. Further protein-protein interaction (PPI) analysis indicates that protein products of genes affected by CNVs frequently interact with known schizophrenia-associated proteins. Finally, systematic integration of CNVs prioritization data with genetic association and PPI data identifies key schizophrenia candidate genes. Our results provide a global overview of genes impacted by CNVs in schizophrenia and reveal a densely interconnected molecular network of de novo CNVs in schizophrenia. Though the prioritized top genes represent promising schizophrenia risk genes, further work with different prioritization methods and independent samples is needed to confirm these findings. Nevertheless, the identified key candidate genes may have important roles in the pathogenesis of schizophrenia, and further functional characterization of these genes may provide pivotal targets for future therapeutics and diagnostics.Key words: schizophrenia, copy number variation, prioritization, integrative analysis, NRXN1, CHRNA7     

<Emphasis Type="Italic">DAT1</Emphasis> and <Emphasis Type="Italic">DRD4</Emphasis> genes involved in key dimensions of adult ADHD

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder often persisting in adulthood. Genetic studies of ADHD mainly focused on the Dopamine Transporter (DAT1) and the Dopamine Receptor 4 (DRD4) genes. Nevertheless, polymorphisms of these genes explain only a small fraction of the assigned risk, suggesting that intermediate dimensions and environmental factors should also be considered. We investigated in 77 adult ADHD subjects compared to 474 controls, how polymorphisms within the genes coding for DAT1 (40-bp VNTR in 3′UTR), the Dopamine Receptor 2 (DRD2) (rs1799732) and DRD4 (48-bp VNTR in exon 3), may modulate the expression of the disorder. By genotyping DAT1, we detected a new 9.5R allele showing a deletion of 40 bp and also an insertion of 19 bp compared to the 10R allele. This novel allele was found to be significantly protective for ADHD (p < 0.0001). Another significant difference was found in the distribution of DRD4 48-bp VNTR 6R allele when comparing patients and controls (p = 0.0007). In addition significant results were also found for DAT1 9.5R allele, which was associated with impulsiveness (p = 1.98 × 10^?4) and trait anger scores (p = 7.66 × 10^?4). Moreover, impulsiveness scores were partly modulated by an interaction between the DRD4 48-bp VNTR 6R allele and childhood maltreatment (p = 0.01), however, this result did not resist correction for multiple comparisons. Altogether, our results show the putative involvement of DAT1 and DRD4 genes in the aetiology of ADHD with a main role in modulation of key dimensions of the disorder.     

<Emphasis Type="Italic">ADCY5</Emphasis>-related dyskinesia presenting as familial myoclonus-dystonia

We describe a family with an autosomal dominant familial dyskinesia resembling myoclonus-dystonia associated with a novel missense mutation in ADCY5, found through whole-exome sequencing. A tiered analytical approach was used to analyse whole-exome sequencing data from an affected grandmother-granddaughter pair. Whole-exome sequencing identified 18,000 shared variants, of which 46 were non-synonymous changes not present in a local cohort of control exomes (n = 422). Further filtering based on predicted splicing effect, minor allele frequency in the 1000 Genomes Project and on phylogenetic conservation yielded 13 candidate variants, of which the heterozygous missense mutation c.3086T>G, p. M1029R in ADCY5 most closely matched the observed phenotype. This report illustrates the utility of whole-exome sequencing in cases of undiagnosed movement disorders with clear autosomal dominant inheritance. Moreover, ADCY5 mutations should be considered in cases with apparent myoclonus-dystonia, particularly where SCGE mutations have been excluded. ADCY5-related dyskinesia may manifest variable expressivity within a single family, and affected individuals may be initially diagnosed with differing neurological phenotypes.     

Expression Analysis of <Emphasis Type="Italic">CYFIP1</Emphasis> and <Emphasis Type="Italic">CAMKK2</Emphasis> Genes in the Blood of Epileptic and Schizophrenic Patients

Schizophrenia and epilepsy are two prevalent neurological disorders with high global burden to the society. Genome-wide studies have identified potential underlying causes for these neurological diseases. In the present case-control study, we have assessed expression of CYFIP1 and CAMKK2 genes in the blood samples of epileptic and schizophrenic patients compared with healthy subjects. A total of 180 subjects including 40 epileptic patients, 50 schizophrenic patients, and 90 healthy individuals participated in the study. Expression of the mentioned genes was measured using TaqMan real-time PCR. The results demonstrated a significant upregulation of CYFIP1 gene expression in epileptic patients (P?=?0.029). CAMKK2 was downregulated in female schizophrenic patients compared with female healthy individuals (P?=?0.048). These results may provide new insight into the pathogenesis of epilepsy and schizophrenia and suggest these genes as potential therapeutic targets for these neurological disorders. Future studies should evaluate these results in larger cohorts of patients.     

The complete chloroplast genome sequence of <Emphasis Type="Italic">Sophora japonica</Emphasis> var. <Emphasis Type="Italic">violacea</Emphasis>: gene organization and genomic resources

Sophora japonica var. violacea is an important ornamental plant, but its wild populations are now in danger, and need urgent conservation. In the present study, we sequenced the complete chloroplast genome of S. japonica var. violacea and used these data to assess genomic resources. The S. japonica var. violacea chloroplast genome is 158,837 bp in length, and includes a pair of inverted repeats of 25,398 bp that are separated by small and large single copy regions of 19,058 and 88,983 bp, respectively. The chloroplast genome of S. japonica var. violacea encodes 112 different genes, including 78 protein-coding genes, 30 transfer RNAs (tRNA) and 4 ribosomal RNAs (rRNA). We identified a total of 171 simple sequence repeat markers in the chloroplast genome. A phylogenetic tree constructed based on 78 protein-coding genes of 42 angiosperms showed that the position of S. japonica var. violacea is on the base of Papilionoideae. Overall, the results of this study will contribute to better support of the evolution, molecular biology and genetic improvement of S. japonica var. violacea.     

Characterization of the complete chloroplast genome of <Emphasis Type="Italic">Pinus uliginosa</Emphasis> (Neumann) from the <Emphasis Type="Italic">Pinus mugo</Emphasis> complex

Taxonomic status of endangered peat-bog pine, Pinus uliginosa (Neumann) classified within the Pinus mugo complex, still remains to be elucidated. Here we present a complete chloroplast genome of P. uliginosa, to aid resolve its complex systematical position. The total genome size was 119,877 bp in length and contained a total of 112 genes, including 73 protein-coding genes, 35 tRNAs, and four rRNAs. The most of genes occur as a single copy. Five tRNA genes were duplicated from two to four times. Eighteen genes contain one intron, with a single gene containing two introns. No large inverted repeats were identified. The overall G?+?C content of P. uliginosa chloroplast genome is 38.5%.     

Characterization of the complete chloroplast genomes of two sister species of <Emphasis Type="Italic">Paeonia</Emphasis>: genome structure and evolution

The two tree peony species, namely, Paeonia ludlowii (Stern & G. Taylor) D. Y. Hong and P. delavayi Franch, belongs to the section Moutan Paeonia (Paeoniaceae). They are the only sources of yellow pigment in tree peony cultivar breeding. P. ludlowii has been listed as “critically endangered”, whereas P. delavayi has been listed as “near threatened” species according to the China Species Red List. The complete chloroplast genome sizes of P. ludlowii and P. delavayi are 152,687 and 154,405 bp respectively. Both contain a 17,056 bp long small single copy region (SSC). The large single copy region (LSC) in P. ludlowii is 84,613 bp, whereas the inverted repeat regions (IRs) are 25,644 bp. In addition, LSC in P. delavayi is 86,142 bp, whereas the IRs is 25,650 bp. The genomes of the two species encode the same set of 134 genes, including 86 protein-coding genes, 8 ribosomal RNA genes and 40 transfer RNA genes. Phylogenetic analysis revealed that all five Paeonia species clustered together, and P. ludlowii and P. delavayi are most closely related to each other. These newly characterized chloroplast genomes will provide essential data for the further conservation of P. ludlowii and P. delavayi.     

The complete chloroplast genome of <Emphasis Type="Italic">Lilium taliense</Emphasis>, an endangered species endemic to China

The complete chloroplast genome of Lilium taliense which is a rare and endangered species native to China was determined in this study. The chloroplast genome is 153,055 bp long and consists of a pair of inverted repeat regions (26,543 bp), one large single copy region (82,458 bp), and one small single copy region (17,511 bp). The genome contains 132 genes, including 86 protein-coding genes, 38 transfer RNA gens and eight ribosomal RNA genes. In addition, 188 simple sequence repeats (SSRs) were identified in the L. taliense chloroplast genome. Maximum likelihood phylogenetic analysis with the reported chloroplast genomes revealed that L. taliense was closely related to L. fargesii and L. superbum.     

The phenotypic spectrum of <Emphasis Type="Italic">ARHGEF9</Emphasis> includes intellectual disability,focal epilepsy and febrile seizures

Mutations or structural genomic alterations of the X-chromosomal gene ARHGEF9 have been described in male and female patients with intellectual disability. Hyperekplexia and epilepsy were observed to a variable degree, but incompletely described. Here, we expand the phenotypic spectrum of ARHGEF9 by describing a large Ethiopian-Jewish family with epilepsy and intellectual disability. The four affected male siblings, their unaffected parents and two unaffected female siblings were recruited and phenotyped. Parametric linkage analysis was performed using SNP microarrays. Variants from exome sequencing in two affected individuals were confirmed by Sanger sequencing. All affected male siblings had febrile seizures from age 2–3 years and intellectual disability. Three developed afebrile seizures between age 7–17 years. Three showed focal seizure semiology. None had hyperekplexia. A novel ARHGEF9 variant (c.967G>A, p.G323R, NM_015185.2) was hemizygous in all affected male siblings and heterozygous in the mother. This family reveals that the phenotypic spectrum of ARHGEF9 is broader than commonly assumed and includes febrile seizures and focal epilepsy with intellectual disability in the absence of hyperekplexia or other clinically distinguishing features. Our findings suggest that pathogenic variants in ARHGEF9 may be more common than previously assumed in patients with intellectual disability and mild epilepsy.     

Polymorphisms in <Emphasis Type="Italic">CAMKK2</Emphasis> may predict sensory neuropathy in African HIV patients

HIV-associated sensory neuropathy (HIV-SN) is the most common neurological condition associated with HIV. HIV-SN has characteristics of an inflammatory pathology caused by the virus itself and/or by antiretroviral treatment (ART). Here, we assess the impact of single-nucleotide polymorphisms (SNPs) in a cluster of three genes that affect inflammation and neuronal repair: P2X7R, P2X4R and CAMKK2. HIV-SN status was assessed using the Brief Peripheral Neuropathy Screening tool, with SN defined by bilateral symptoms and signs. Forty-five SNPs in P2X7R, P2X4R and CAMKK2 were genotyped using TaqMan fluorescent probes, in DNA samples from 153 HIV⁺ black Southern African patients exposed to stavudine. Haplotypes were derived using the fastPHASE algorithm, and SNP genotypes and haplotypes associated with HIV-SN were identified. Optimal logistic regression models included demographics (age and height), with SNPs (model p?<?0.0001; R ²?=?0.19) or haplotypes (model p?<?0.0001; R ²?=?0.18, n?=?137 excluding patients carrying CAMKK2 haplotypes perfectly associated with SN). Overall, CAMKK2 exhibited the strongest associations with HIV-SN, with two SNPs and six haplotypes predicting SN status in black Southern Africans. This gene warrants further study.     

Characterization of the complete chloroplast genome sequence of <Emphasis Type="Italic">Cardamine macrophylla</Emphasis> (Brassicaceae)

Cardamine macrophylla is a perennial herb with short-lived seeds. Here, the complete chloroplast genome (plastome) of C.macrophylla was obtained by Illumina sequencing technology. The plastome is 155,393 bp in length, including a large single copy (LSC) region of 84,492 bp, a small single copy (SSC) region of 17,957 bp and a pair of inverted repeats (IRs) of 26,472 bp. The genome contained 131 genes, including 85 protein-coding genes (78 PCG species), 37 tRNA genes (30 tRNA species), eight rRNA genes (four rRNA species) and one pseudogene. Among these genes, 15 genes harbored a single intron and three genes harbored two introns. Seven tRNA, five protein-coding and all four rRNA genes were completely duplicated in the IR regions. The GC content of C. macrophylla plastome is 36.4%. A total of 26,470 codons were detected in 85 protein-coding genes of C. macrophylla plastome. Phylogenetic analysis demonstrated that the all Cardamine species formed a monophyletic clade, and C. macrophylla was closely related to C. impatiens.     

Characterization of the complete plastid genome of <Emphasis Type="Italic">Quercus tarokoensis</Emphasis>

Quercus tarokoensis is an endemic species to Taiwan, China. The complete plastid genome of Q. tarokoensis was assembled from Illumina pair-end sequence reads. The whole plastome was 161,355 bp in length and presented a quadripartite structure consisting of two copies of inverted repeat (IR) regions (25,860) separated by a large single copy region (90,602 bp) and a small single copy region (19,033 bp). The plastome of Q. tarokoensis encoded a total of 134 genes, including 86 protein-coding genes (79 PCG species), 40 tRNA genes (33 tRNA species), and 8 rRNA genes (4 rRNA species). The overall GC content of Q. tarokoensis plastome is 36.8%. A maximum likelihood phylogenetic analysis based on 33 complete plastomes revealed that Q. tarokoensis was collectively sister to a clade of (Quercus variabilis, (Quercus dolicholepis, Quercus baronii)) with high support.     

The complete chloroplast genome of the dove tree <Emphasis Type="Italic">Davidia involucrata</Emphasis> (Nyssaceae), a relict species endemic to China

The dove tree, Davidia involucrate, with its distinguishing pair of white bracts, is endemic to China. Here, we reported the complete chloroplast (cp) genome sequence and the cp genomic features of D. involucrata. The D. involucrata cp genome was 169,196 bp long, with 129 genes comprising 83 protein-coding genes (PCG), 40 tRNA genes, and six rRNA genes. The majority of the gene species occurred as a single-copy, while 18 gene species occurred in double copies, including six PCG species (ndhB, rpl2, rpl23, rps7, rps15, and ycf2), eight tRNA species (trnH-GTG, trnL-CAA, trnI-CAT, trnV-GAC, trnL-GAV, trnA-UGC, trnN-GTT, and trnR-ACG) and all four rRNA species (rrn4.5, rrn5, rrn16, and rrn23). A neighbor-joining (NJ) phylogenetic tree was reconstructed with sequences of complete cp, revealing that D. involucrata belongs to the asterids. The complete cp genome of D. involucrata will be useful for further investigations and the conservation of this endemic relict woody plant.     

The complete chloroplast genome of <Emphasis Type="Italic">Lilium cernuum</Emphasis>: genome structure and evolution

Lilium cernuum is endangered and was listed in the category of key protected wild plants in China. In this study, we report the complete chloroplast genome of L. cernuum using Illumina paired-end sequencing. The entire chloroplast genome maps as a circular molecule of 152,604 bp built with a quadripartite organization: two inverted repeats (IRs) of 26,481 bp separated by a large single copy (LSC) sequence of 82,058 bp and a small single copy (SSC) sequence of 17,584 bp. The chloroplast genome contains the conservative structure present in most Liliaceae chloroplasts composing of 79 protein-coding genes, 30 tRNAs and 4 rRNAs. Among these genes, 15 harbor a single intron, and 2 contain a couple of introns. A maximum likelihood phylogenomic analysis showed that L. cernuum was closely related to L. tsingtauense and L. hansonii that belonged to the family Liliaceae.     

A novel gain-of-function mutation in the <Emphasis Type="Italic">ITPR1</Emphasis> suppressor domain causes spinocerebellar ataxia with altered Ca<Superscript>2+</Superscript> signal patterns

We report three affected members, a mother and her two children, of a non-consanguineous Irish family who presented with a suspected autosomal dominant spinocerebellar ataxia characterized by early motor delay, poor coordination, gait ataxia, and dysarthria. Whole exome sequencing identified a novel missense variant (c.106C>T; p.[Arg36Cys]) in the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor gene (ITPR1) as the cause of the disorder, resulting in a molecular diagnosis of spinocerebellar ataxia type 29. In the absence of grandparental DNA, microsatellite genotyping of healthy family members was used to confirm the de novo status of the ITPR1 variant in the affected mother, which supported pathogenicity. The Arg36Cys variant exhibited a significantly higher IP₃-binding affinity than wild-type (WT) ITPR1 and drastically changed the property of the intracellular Ca²⁺ signal from a transient to a sigmoidal pattern, supporting a gain-of-function disease mechanism. To date, ITPR1 mutation has been associated with a loss-of-function effect, likely due to reduced Ca²⁺ release. This is the first gain-of-function mechanism to be associated with ITPR1-related SCA29, providing novel insights into how enhanced Ca²⁺ release can also contribute to the pathogenesis of this neurological disorder.