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Although the etiology of obsessive-compulsive disorder (OCD) is largely unknown, it is accepted that OCD is a complex disorder. There is a known bi-directional interaction between the gut microbiome and brain activity. Several authors have reported associations between changes in gut microbiota and neuropsychiatric disorders, including depression or autism. Furthermore, a pediatric-onset neuropsychiatric OCD-related syndrome occurs after streptococcal infection, which might indicate that exposure to certain microbes could be involved in OCD susceptibility. However, only one study has investigated the microbiome of OCD patients to date. We performed 16S ribosomal RNA gene-based metagenomic sequencing to analyze the stool and oropharyngeal microbiome composition of 32 OCD cases and 32 age and gender matched controls. We estimated different α- and β-diversity measures and performed LEfSe and Wilcoxon tests to assess differences in bacterial distribution. OCD stool samples showed a trend towards lower bacterial α-diversity, as well as an increase of the relative abundance of Rikenellaceae, particularly of the genus Alistipes, and lower relative abundance of Prevotellaceae, and two genera within the Lachnospiraceae: Agathobacer and Coprococcus. However, we did not observe a different Bacteroidetes to Firmicutes ratio between OCD cases and controls. Analysis of the oropharyngeal microbiome composition showed a lower Fusobacteria to Actinobacteria ratio in OCD cases. In conclusion, we observed an imbalance in the gut and oropharyngeal microbiomes of OCD cases, including, in stool, an increase of bacteria from the Rikenellaceae family, associated with gut inflammation, and a decrease of bacteria from the Coprococcus genus, associated with DOPAC synthesis. This project was supported by grants from the Spanish ministry of science and innovation (MICINN; SAF2013-49108-R); the Carlos III Health Institute (PI16/00950, PI18/00856, PI19/01184); FEDER funds (‘A way to build Europe’) and by the Agency of University and Research Funding Management of the Catalan Government (2014 SGR 1672 and 2017 SGR 738). We thank CERCA Programme / Generalitat de Catalunya for institutional support. LD was supported by a Severo Ochoa grant (SVP-2013-068066), MA was supported by the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia Grant co-funded by the European Social Fund (ESF) “ESF, Investing in your future” (2017 FI_B 00327), DSC was supported by a grant from the MICINN (BES-2014-069814) and RR was supported by a fellowship from the Health Department of the Generalitat de Catalunya through the PERIS 2016-2020 program (SLT002/16/00310).

Common single-nucleotide polymorphisms (SNPs) account for a large proportion of the heritability of obsessive-compulsive disorder (OCD). Co-ocurrence of OCD and schizophrenia is commoner than expected based on their respective prevalences, complicating the clinical management of patients. This study addresses two main objectives: to identify particular genes associated with OCD by SNP-based and gene-based tests; and to test the existence of a polygenic risk shared with schizophrenia. The primary analysis was an exon-focused genome-wide association study of 370 OCD cases and 443 controls from Spain. A polygenic risk model based on the Psychiatric Genetics Consortium schizophrenia data set (PGC-SCZ2) was tested in our OCD data. A polygenic risk model based on our OCD data was tested on previous data of schizophrenia from our group. The most significant association at the gene-based test was found at DNM3 (P=7.9 × 10(-5)), a gene involved in synaptic vesicle endocytosis. The polygenic risk model from PGC-SCZ2 data was strongly associated with disease status in our OCD sample, reaching its most significant value after removal of the major histocompatibility complex region (lowest P=2.3 × 10(-6), explaining 3.7% of the variance). The shared polygenic risk was confirmed in our schizophrenia data. In conclusion, DNM3 may be involved in risk to OCD. The shared polygenic risk between schizophrenia and OCD may be partially responsible for the frequent comorbidity of both disorders, explaining epidemiological data on cross-disorder risk. This common etiology may have clinical implications. The study was supported by Fundación María José Jove, Fondo Europeo de Desarrollo Regional (FEDER), Xunta de Galicia; and by grants from the Instituto de Salud Carlos III FIS PI13/01136 to AC, CP11/00163 to JC, PI13/00918 to JMM, PI14/00413 to PA; the Generalitat de Catalunya AGAUR 2014 SGR-1138; the Spanish MINIECO SAF2013-49108- R Plan Estatal; and the European Commission 7th Framework Program, Project N. 262055 (ESGI) to XE. LD is supported by a Severo Ochoa fellowship of the Spanish MINIECO.

Background The human chromosome 8p23.1 region contains a 3.8–4.5 Mb segment which can be found in different orientations (defined as genomic inversion) among individuals. The identification of single nucleotide polymorphisms (SNPs) tightly linked to the genomic orientation of a given region should be useful to indirectly evaluate the genotypes of large genomic orientations in the individuals. Results We have identified 16 SNPs, which are in linkage disequilibrium (LD) with the 8p23.1 inversion as detected by fluorescent in situ hybridization (FISH). The variability of the 8p23.1 orientation in 150 HapMap samples was predicted using this set of SNPs and was verified by FISH in a subset of samples. Four genes (NEIL2, MSRA, CTSB and BLK) were found differentially expressed (p

Attention-deficit/hyperactivity disorder (ADHD) is the most common psychiatric condition with onset in childhood, and in more than 50% of cases it persists into adulthood as a chronic disorder. Over five million methylphenidate (MPH) prescriptions were issued in the USA in 2003, mostly for children. A previous report [R.A. El-Zein, S.Z. Abdel-Rahman, M.J. Hay, M.S. Lopez, M.L. Bondy, D.L. Morris and M.S. Legator Cytogenetic effects in children treated with methylphenidate, Cancer Lett. 230 (2005) 284-291.] described the induction of chromosome abnormalities by MPH in children treated for three months, contrary to most of the in vitro and in vivo studies reported since then. We present new relevant information concerning the cytogenetic effects of MPH in children and adults. We include a prospective sample of 12 children and 7 adults with a new diagnosis of ADHD and naive to MPH. We analyzed the cytogenetic effects on peripheral lymphocytes before and three months after starting MPH therapy. The cytogenetic analyses included a cytokinesis-block micronucleus (CBMN) assay, a sister chromatid exchange (SCE) analysis and the determination of chromosome aberrations (CA). Following the same strategy and analyzing the same cytogenetic endpoints that were investigated in the original report [R.A. El-Zein, S.Z. Abdel-Rahman, M.J. Hay, M.S. Lopez, M.L. Bondy, D.L. Morris and M.S. Legator Cytogenetic effects in children treated with methylphenidate, Cancer Lett. 230 (2005) 284-291.], we found no evidence of increased frequency of micronuclei, sister chromatid exchanges or chromosome aberrations induced by MPH in children and adult populations. MPH treatment of children and adults with ADHD resulted in no significant genomic damage (as suggested by the three endpoints studied), results that do not support a potential increased risk of cancer after exposure to MPH.

Neurofibromatosis type 1 (NF1) is one of the commonest autosomal dominant disorders in man, affecting 1 in 3500 people. Consensus clinical criteria were defined in 19871 and revised and updated in 1997.2 Cafe au lait spots, axillary freckling, dermal neurofibromas, and Lisch nodules of the iris are the most common manifestations of this disorder. Most of the clinical symptoms of the disease are age dependent and considerable phenotypic variability has been described both between and within families.3,4 This genetic disorder is caused by mutations in the NF1 gene, one of the largest human genes, composed of 60 exons and spanning more than 300 kb of genomic DNA.5 The determination of the NF1 mutational spectrum has been complex owing to the large number of coding exons and the considerable mutational heterogeneity. Until recently, most diagnostic laboratories just offered linkage analysis for NF1 patients, which excluded diagnosis of the 50% of de novo cases. The use of techniques based on the analysis of NF1 mRNA greatly facilitated the number of mutations identified and NF1 screening efficiency, depicting a mutational NF1 spectrum.6–8 These studies highlighted the importance of splicing defects in molecular NF1 pathology and, despite most patients bearing unique mutations, they suggested the recurrence of several mutations. Here we present our experience with the direct analysis of the whole NF1 coding region in 474 unrelated subjects suspected of having NF1. Mutations have been identified in 189 patients, 85 of them bearing recurrent mutations. ### Patients and families Four hundred and seventy-four unrelated subjects suspected of having NF1 were analysed for mutations in the NF1 gene. Included in these 474 cases are 80 NF1 patients studied previously.6 Clinical data confirming NF1 diagnostic criteria were available in 201 (42%) of the subjects studied and in the remaining cases either no …

A novel polymorphism (6376 G/T) in intron 7 (17) of the human PROC gene has been identified by direct DNA sequencing. Restriction analysis with the use of mutagenic primers indicate that the allele frequencies are 0.17 (allele T) and 0.83 (allele G), with a calculated heterozygosity of 28%.

Anticoagulant protein S (PS) deficiency is a known risk factor for thrombophilia. The structure and high allelic heterogeneity of the PS gene (PROS1), together with the presence of a 97% homologous pseudogene, complicates PROS1 analysis. We have optimized a simple, fast, and non-isotopic Single-Strand Conformation Analysis (SSCA or SSCP) method for PROS1 mutation detection. This is accomplished through the analysis of the single-stranded and heteroduplex DNA fragments corresponding to 15 PCR segments that include part of the 5'-upstream region and the 15 PROS1 exons with their intron boundaries. To standardize the method, 13 known PROS1 mutations or allele variants in 10 different fragments were analyzed under different electrophoretic conditions. The results indicated that, using a combination of two different electrophoretic settings, all the allele variants could be detected as a single-strand band shift and/or by the presence of a heteroduplex. This method was used to analyze the PROS1 gene in 31 propositi with different types of PS deficiency and thrombosis. Ten different cosegregating mutations, seven of which are novel (143C-G, L-27H, G96X, M599T, P626L, 1418delA, and 1877delT), were identified in the five families suffering from type I or quantitative PS deficiency and in four of the nine families with coexistence of type I and type III phenotypes. No clearly co-segregating PROS1 mutations were identified in any of the 17 type III propositi analyzed, although eight of them were heterozygotes for the uncommon P460 allele of the S/P460 variant. Furthermore, five apparently neutral allelic variants, three of which are novel (-296C-T, 182G-C and T57S), were identified in a normal control, two type I/III and two type III PS-deficient pedigrees.

DNA sequence analysis of the protein S gene (PROS1) in 22 Spanish probands with type I or III PS deficiency, has allowed the identification of 10 different mutations and 2 new sequence variants in 15 probands. Nine of the mutations, 8 of which are novel, cosegregate with type I or quantitative PS deficiency in 12 of the 13 pedigrees analyzed. One of these mutations (Q238X) also cosegregates with both type I and III PS-deficient phenotypes coexisting in a type I/III pedigree. Another mutation identified in a pedigree with these two PS phenotypes is the missense mutation R520G, present in the homozygous form in the type I propositus and in the heterozygous form in his type III relatives. By contrast, no cosegregating PROS1 mutation has been found in any of the six families with only type III phenotypes. Three of these families, as well as the two families with type I and I/III phenotypes where no other PROS1 mutation has been identified, segregate the P allele of the S460P variant, although this allele does not always cosegregate with the deficient phenotype. From these results we conclude that while mutations in PROS1 are the main cause of type I PS deficiency, the molecular basis of the type III phenotype is probably more complex, with many cases not being explained by a PROS1 mutation.

RNA-based studies are an important tool for the identification and functional characterization of mutations underlying inherited disease. These studies could in principle be compromised by 'aberrant splicing' (the generation of alternatively spliced transcripts lacking any obvious function) during normal expression of the genes under investigation. Using a highly sensitive RT-PCR assay, we show here that aberrant splicing is a frequent occurrence during expression of the protein C (PROC) and protein S (PROS) genes. Aberrantly spliced transcripts were present in different cell types including liver, the main expressing tissue for both protein C and protein S. In an attempt to compare individual mRNA splicing patterns, PROC and PROS RNA from easily accessible cells of different healthy control individuals was studied. However, variation between different RT-PCR assays from the same individual precluded both the relative quantitation of the aberrant transcripts and the analysis of interindividual differences. Our findings are consistent with the notion that a low level of aberrantly spliced transcripts are routinely generated during PROC and PROS gene expression. The possibility that these transcripts may complicate the RT-PCR analysis of pathological transcripts must be taken into account when RNA-based strategies of disease analysis are considered.

SummaryThe multifactorial character of thrombotic disease is shown in a Spanish pedigree in which the propositus, with recurrent deep vein thrombosis, inherited the factor V R/Q506 mutation, the prothrombin 20210G/A variant and type III Protein S deficiency. Among 14 relatives carrying one or two of these three risk factors, thrombosis is present in a heterozygote for R/Q506 and in another for 20210G/A, who also had slightly positive antiphospholipid antibodies. Type I PS deficiency was also found in a young asymptomatic woman. PROS1 analysis showed coexistence of type III and type I PS deficiency to be associated with heterozygosity and homozygosity, respectively, for the P460 or PS Heerlen allele of the S/P460 variant. Analysis of PS values in this and other pedigrees segregating this variant revealed that not only free but also mean total PS levels are slightly but significantly lower in the SP460 heterozygotes than in the SS460 homozygotes. These findings strongly suggest a role of the P460 variant in the expression of the PS deficient phenotype.

We report the results of protein C gene (PROC) analysis in a Spanish family with hereditary PC deficiency characterized by the presence of three siblings with PC anticoagulant activity levels clearly below 50% of normal and PC antigen and amidolytic activities between 50 and 75% of normal. Their parents are first cousins and have PC levels between 50 and 80% of normal. Sequence analysis of the whole coding sequence of the PROC gene revealed that the three siblings are double homozygotes for a G to A transition at nucleotide 3203 that replaces arginine 87 by histidine (R87H) and for another G to A transition at nucleotide 7054, in intron 7 (7054G --> A). Both parents and one sister were found to be double heterozygotes for these two mutations. Screening for the intronic mutation in a control group and RT-PCR cDNA studies from ectopically transcribed mRNA indicated that 7054G --> A is most likely a rare but neutral DNA variant. These results and the fact that heterozygosity for the missense R87H mutation has also been found associated with a slightly decreased PC anticoagulant activity in another Spanish family, lead us to conclude that homozygosity for R87H is responsible for the PC deficient phenotype in these three siblings.