Chiara Magri

Role of allelic variants of FK506-binding protein 51 (FKBP5) gene in the development of anxiety disorders.

Anxiety disorders exhibit remarkably high rates of comorbidity with major depressive disorder (MDD). Mood and anxiety disorders are considered stress‐related diseases. Genetic variations in the co‐chaperone FK506‐binding protein 51, FKBP5, which modulates the function of glucocorticoid receptors, have been associated with an increased risk for the development of posttraumatic stress disorder, but data regarding its role in MDD are controversial. The aims of this study were to clarify the role of the FKBP5 gene in depression and anxiety disorders through a case–control study and an association study with personality traits using the Temperament and Character Inventory (TCI) in healthy subjects.

Combined DOCK8 and CLEC7A mutations causing immunodeficiency in 3 brothers with diarrhea, eczema, and infections

To the Editor: We report four male brothers from a consanguineous Italian family with an immunodeficiency syndrome characterized by severe eczema, milk and egg allergies, recurrent infections, intractable diarrhea, failure to thrive and, in two of the three, lymphoma. One of them (II-2) died at age 12 months of cardiovascular failure due to intractable diarrhea and the other three were evaluated as part of this study (Table 1, Figure 1A). The proband (II-1) presented shortly after birth with severe eczema and intractable diarrhea, without gastrointestinal infection. In the first year of life, he had multiple allergies to milk, soy, and egg proteins. He had repeated episodes of pneumonia leading to bilateral bronchiectasis at 16 years of age. At age 25 years, he developed lymphoma involving the tonsil, lung and spleen. This was defined as a high grade large cell B cell lymphoma (EBV-negative), and was successfully treated with immune-chemotherapy. At age 27, he had Pseudomonas aeruginosa sepsis. At age 28, he was successfully transplanted with hematopoietic stem cells (HSC) from a matched unrelated donor (MUD). Likewise, a younger sibling (II-3) presented at 6 months of age with allergy to milk proteins and intractable diarrhea, despite a milk protein-free diet. He had two episodes of pneumonia and several upper respiratory tract infections. At age 15 years, he developed an anaplastic peripheral T cell lymphoma, involving skin, lymph nodes, liver and spleen. It proved refractory to chemotherapy. The patient died of infection while receiving high-dose methotrexate and cytarabine. Sibling II-4 was fed with an extensively hydrolyzed formula, but presented with non-infectious diarrhea as an infant. He developed respiratory infections and otitis. At age 13 he was successfully transplanted with HSC from a MUD. Figure 1 (A)Family tree. The proband is indicated by arrow. (B) Scatter plot of absolute counts of T-, B- and NK- cells (◆ II-1, ● II-3, ■ II-4) compared to 20 age-matched HD (○)( mean±SD). (C)FOXP3 expression in CD4 of ... Table 1 Clinical Features of Patients Immunological studies in the three siblings surviving infancy showed severe T cell lymphopenia (160-1140 cells/ l), with normal relative percentage of B and NK cells (Figure 1B). Immunedysregulation, Polyendocrinopathy, Enteropathy, X-linked syndrome (IPEX) (OMIM #304790), was suspected 1, 2, but FOXP3 sequencing did not reveal any mutation in the coding region except a previously reported, silent single nucleotide polymorphism (SNP) at serine 181 (543C>T, rs2232367 in the NCBI dbSNP) in all of the affected siblings. A previous study had suggested this variant to be associated with IPEX syndrome3. However, analysis of 100 chromosomes from healthy subjects showed the allele frequency of rs2232367 to be 5% in the Italian population. Sequencing of reverse transcribed mRNA did not reveal any splicing variant of FOXP3 (data not shown) and expression of FOXP3 protein was normal in CD25+/CD4+ T cells in the proband (Figure 1C). Next, we examined T-regulatory functional properties of CD4+CD25hi+ regulatory cells (Tregs) and CD4+CD25-effector cells from two of the siblings surviving infancy (the proband II-1 and sibling II-4). Tregs isolated by magnetic bead cell sorting, in parallel with those of a healthy donor, and stimulated with anti-CD3 mAb in the presence of allogeneic antigen presenting cells strongly suppressed both the proliferation and IFN-gamma secretion of co-cultured autologous effector T cells at a ratio [Responder: suppressor] of 2:1 (Figure 1D). The proliferative capacity of Treg cells from the proband was also examined, revealing them to be anergic. Altogether, these genetic and functional data ruled out the diagnosis of IPEX syndrome due to FOXP3 mutation. Genome-wide genotyping of SNPs in the three siblings and their mother with the Affymetrix 6.0 microarray and analysis with GenCol4 revealed regions with identical genotype, including chromosomes 9p24 and 12p13 (Supp. figures 1A and 3), suggesting an autosomal recessive disorder. Exome enrichment (37.7 million nucleotides, Agilent SureSelect v.1) and next generation sequencing (NGS) was performed on the proband, as previously described5. Mean enrichment of the exome was 44-fold. NGS yielded 171 million sequences each 130 nucleotides long. ~95% of sequences aligned to the reference human genome uniquely, covering each exonic nucleotide ~135 times. ~1.45% of target nucleotides were not sequenced, while ~88.3% had at least 20-fold sequence coverage. A bioinformatic decision tree was used to identify nucleotide variants in the aligned sequences5. Homozygous variants were detected in two genes in the proband (Supp. figures 1B, 1C): The first was a known nonsense mutation in the pattern recognition receptor gene (CLEC7A, p.Tyr238X, rs16910526), that signals via CARD9 (Supp. figure 2C). This mutation has been reported to cause familial chronic mucocutaneous candidiasis (CANDF4, OMIM#613108)7.The second was a novel homozygous frameshift variant in exon 27 of the DOCK8 gene, c.3193delA. DOCK8 mutations cause autosomal recessive hyper-IgE syndrome (MIM#243700), characterized by T lymphopenia, eosinophilia, elevated IgE, eczema, recurrent infections and severe food allergies 8, 9. Sanger sequencing confirmed homozygosity for both the DOCK8 and CLEC7A variants in the three siblings and heterozygosity in their parents. The DOCK8 mutation is predicted to result in a premature stop codon 17 residues downstream of the deletion (Ser1065AlafsX17). However, Sanger sequencing of DOCK8 mRNA revealed a small number of transcripts that read through the premature stop codon (Supp.figure 2A and data not shown). Indeed, immunoblots of T cells from the three patients showed low level DOCK8 protein expression (Supp. figure 2B), suggesting residual protein synthesis by alternative splicing. Exome sequencing, when combined with confirmatory testing, has previously shown efficacy for molecular elucidation of Mendelian diseases. Here, we describe another application, namely the identification of potentially causative mutations in two or more loci in inherited diseases. Patients with DOCK8 mutations display skin infections, pneumonia, elevated serum IgE, but do not usually have diarrhea. The latter is typically associated with IPEX syndrome. The phenotypic severity and enteropathy despite residual DOCK8 protein expression may reflect the combined defects in two genes acting in parallel immunologic pathways. Interestingly, this unusual phenotype partially resembles IPEX syndrome. The prevalence of mutations in two genes in apparently Mendelian disorders is not known. Such interactions would not be discovered by conventional, serial, univariate molecular testing since the DOCK8 result would be interpreted as a definitive diagnosis, and further molecular evidence would not be sought. We suggest that exome sequencing be considered in patients with atypical presentations of Mendelian diseases for examination of possible underpinning mutations in more than one locus.

Copy number variants in attention-deficit hyperactive disorder: identification of the 15q13 deletion and its functional role.

Objectives Evidence has supported a role for rare copy number variants in the etiology of attention-deficit hyperactivity disorder (ADHD), in particular, the region 15q13, which is also a hot spot for several neuropsychiatric disorders. This region spans several genes, but their role and the biological implications remain unclear. Methods We carried out, for the first time, an analysis of the 15q13 region in an Italian cohort of 117 ADHD patients and 77 controls using the MLPA method, confirmed by a genome single-nucleotide polymorphism array. In addition, we probed for downstream effects of the 15q13 deletions on gene expression by carrying out a transcriptomic analysis in blood. Results We found 15q13 deletions in two ADHD patients and identified 129 genes as significantly dysregulated in the blood of the two ADHD patients carrying 15q13 deletions compared with ADHD patients without 15q13 deletions. As expected, genes in the deleted region (KLF13, MTMR10) were downregulated in the two patients with deletions. Moreover, a pathway analysis identified apoptosis, oxidation reduction, and immune response as the mechanisms that were altered most significantly in the ADHD patients with 15q13 deletions. Interestingly, we showed that deletions in KLF13 and CHRNA7 influenced the expression of genes belonging to the same immune/inflammatory and oxidative stress signaling pathways. Conclusion Our findings are consistent with the presence of 15q13 deletions in Italian ADHD patients. More interestingly, we show that pathways related to immune/inflammatory response and oxidative stress signaling are affected by the deletion of KFL13 and CHRNA7. Because the phenotypic effects of 15q13 are pleiotropic, our findings suggest that there are shared biologic pathways among multiple neuropsychiatric conditions.

Analysis of complete mitochondrial genomes of patients with schizophrenia and bipolar disorder.

The present study aims at investigating the association between common and rare variants of mitochondrial DNA (mtDNA), and increased risk of schizophrenia (SZ) and bipolar disorder (BPD) in a cohort of patients originating from the same Italian population. The distribution of the major European mtDNA haplogroups was determined in 89 patients and their frequencies did not significantly differ from those observed in the Italian population. Moreover, 27 patients with high probability of having inherited the disease from the maternal side were selected for whole mitochondrial genome sequencing to investigate the possible presence of causative point mutations. Overall, 213 known variants and 2 novel changes were identified, but none of them was predicted to have functional effects. Hence, none of the sequence changes we found in our sample could explain the maternal component of SZ and BPD predisposition.

Association study and mutational screening of SYNGR1 as a candidate susceptibility gene for schizophrenia.

Objective Synaptogyrin 1 (SYNGR1) is a transmembrane protein of neurotransmitter-containing vesicle. Recently, suggestive association between SYNGR1 intragenic polymorphisms and schizophrenia has been reported in the Indian population. Furthermore, some rare nucleotide changes with a potential pathogenic effect have been found in Indian and Chinese schizophrenia patients. In this study, we have performed an association study and a resequencing analysis in an Italian sample. Methods Eight polymorphisms of the SYNGR1 gene were typed in a case–control sample consisting of 274 patients and 335 controls. In parallel, a mutational screening covering all SYNGR1 exons was conducted. Results Evidence of association has been found for rs715505 (P = 0.028), a marker already reported to be associated with the disease. Resequencing analysis revealed two novel polymorphisms and several rare variants (13 of 16 as new variants), some of which might have relevance for gene expression and function. Conclusion The results of our association study support a contribution of SYNGR1 to schizophrenia susceptibility. In addition, the resequencing analysis evidenced mutations with a potential functional role at the mRNA and/or protein level. Of particular interest is the p.isoc:S26G missense mutation identified in six patients (0.011) and three controls (0.004) which might be involved in the elimination of a potential protein kinase C phosphorylation site.

The GRM7 gene, early response to risperidone, and schizophrenia: a genome-wide association study and a confirmatory pharmacogenetic analysis

The search for biomarkers of response to antipsychotic medications is hindered by difficulties inherent in the topic or related to persistent methodological difficulties, such as high rates of anticipated discontinuation and consequent distortions in the imputation of missing data. Because early response to antipsychotics represents a sufficiently reliable index of the subsequent treatment response in patients with schizophrenia, we undertook a real-world, genome-wide association study (GWAS) with the aim of identifying genetic predictors of response to risperidone after 2 weeks in 86 patients with schizophrenia. Limited to the associations reaching significance in the GWAS, confirmatory analysis relative to risperidone response over 9 months was also designed involving 97 patients (European only) enroled in the CATIE (Clinical Antipsychotic Trials of Intervention Effectiveness) genetic substudy. The GWAS revealed a significant association (false discovery rate 0.02) of the single-nucleotide polymorphism rs2133450 inside the GRM7 gene with Emsley’s positive domain derived from the positive and negative syndrome scale (PANSS). Patients with the rs2133450 CC genotype presented poorer improvement in the positive domain over 2 weeks, with odds ratios of 12.68 (95% CI, 3.51–45.76) and 6.95 (95% confidence interval (CI), 2.37–20.37) compared with patients with the AA and AC genotypes, respectively. Compared with A homozygotes, rs2133450 C homozygotes enroled in the CATIE-derived confirmatory analysis showed less improvement in Emsley’s positive, excited and depression domains, positive and general PANSS subtypes, and total PANSS after 9 months of treatment with risperidone. The original GWAS and the CATIE-derived confirmatory analysis support the proposal that the rs2133450 may have translational relevance as a predictor of response to risperidone.

Proteasome system dysregulation and treatment resistance mechanisms in major depressive disorder

Several studies have demonstrated that allelic variants related to inflammation and the immune system may increase the risk for major depressive disorder (MDD) and reduce patient responsiveness to antidepressant treatment. Proteasomes are fundamental complexes that contribute to the regulation of T-cell function. Only one study has shown a putative role of proteasomal PSMA7, PSMD9 and PSMD13 genes in the susceptibility to an antidepressant response, and sparse data are available regarding the potential alterations in proteasome expression in psychiatric disorders such as MDD. The aim of this study was to clarify the role of these genes in the mechanisms underlying the response/resistance to MDD treatment. We performed a case-control association study on 621 MDD patients, of whom 390 were classified as treatment-resistant depression (TRD), and we collected peripheral blood cells and fibroblasts for mRNA expression analyses. The analyses showed that subjects carrying the homozygous GG genotype of PSMD13 rs3817629 had a twofold greater risk of developing TRD and exhibited a lower PSMD13 mRNA level in fibroblasts than subjects carrying the A allele. In addition, we found a positive association between PSMD9 rs1043307 and the presence of anxiety disorders in comorbidity with MDD, although this result was not significant following correction for multiple comparisons. In conclusion, by confirming the involvement of PSMD13 in the MDD treatment response, our data corroborate the hypothesis that the dysregulation of the complex responsible for the degradation of intracellular proteins and potentially controlling autoimmunity- and immune tolerance–related processes may be involved in several phenotypes, including the TRD.

Exome sequencing in schizophrenic patients with high levels of homozygosity identifies novel and extremely rare mutations in the GABA/glutamatergic pathways

Inbreeding is a known risk factor for recessive Mendelian diseases and previous studies have suggested that it could also play a role in complex disorders, such as psychiatric diseases. Recent inbreeding results in the presence of long runs of homozygosity (ROHs) along the genome, which are also defined as autozygosity regions. Genetic variants in these regions have two alleles that are identical by descent, thus increasing the odds of bearing rare recessive deleterious mutations due to a homozygous state. A recent study showed a suggestive enrichment of long ROHs in schizophrenic patients, suggesting that recent inbreeding could play a role in the disease. To better understand the impact of autozygosity on schizophrenia risk, we selected, from a cohort of 180 Italian patients, seven subjects with extremely high numbers of large ROHs that were likely due to recent inbreeding and characterized the mutational landscape within their ROHs using Whole Exome Sequencing and, gene set enrichment analysis. We identified a significant overlap (17%; empirical p-value = 0.0171) between genes inside ROHs affected by low frequency functional homozygous variants (107 genes) and the group of most promising candidate genes mutated in schizophrenia. Moreover, in four patients, we identified novel and extremely rare damaging mutations in the genes involved in neurodevelopment (MEGF8) and in GABA/glutamatergic synaptic transmission (GAD1, FMN1, ANO2). These results provide insights into the contribution of rare recessive mutations and inbreeding as risk factors for schizophrenia. ROHs that are likely due to recent inbreeding harbor a combination of predisposing low-frequency variants and extremely rare variants that have a high impact on pivotal biological pathways implicated in the disease. In addition, this study confirms that focusing on patients with high levels of homozygosity could be a useful prioritization strategy for discovering new high-impact mutations in genetically complex disorders.

Genome-wide analysis of RNA sites consistently edited in human blood reveals interactions with mRNA processing genes and suggests correlations with biological and drug-related variables.

Background A-to-I RNA editing is a co-/post-transcriptional modification catalyzed by ADAR enzymes, that deaminate Adenosines (A) into Inosines (I). Most of known editing events are located within inverted ALU repeats, but they also occur in coding sequences and may alter the function of encoded proteins. RNA editing contributes to generate transcriptomic diversity and it is found altered in cancer, autoimmune and neurological disorders. However, little is known about how editing process could be influenced by genetic variations, biological and environmental variables. Results We analyzed RNA editing levels in human blood using RNA-seq data from 459 healthy individuals and identified 2,079 sites consistently edited in this tissue, that we considered the most biologically relevant editing sites. As expected, analysis of gene expression revealed that ADAR is the major contributor to editing on these sites, explaining ~13% of observed variability. After removing ADAR effect, we found significant associations for 1,122 genes, mainly involved in RNA processing. These genes were significantly enriched in genes encoding proteins interacting with ADARs, including 276 potential ADARs interactors and 9 ADARs direct partners. In addition, association analysis on 28 biological and drugs intake variables revealed several factors potentially influencing RNA editing in blood, including sex, age, BMI, drugs and medications. Finally, we identified genetic loci associated to editing levels, including known ADAR eQTLs and a small region on chromosome 7, containing LOC730338 lincRNA gene. Conclusions Our data provides a detailed picture of the most relevant RNA editing events and their variability in human blood, giving interesting insights on the mechanisms behind this post-transcriptional modification and its regulation in this tissue.

A novel homozygous mutation in GAD1 gene described in a schizophrenic patient impairs activity and dimerization of GAD67 enzyme

Recently, by whole exome sequencing of schizophrenia (SCZ) patients, we identified a subject that was homozygous for a novel missense substitution (c.391 A > G) in the glutamate acid decarboxylase 1 (GAD1) gene. GAD1 encodes for GAD67 enzyme, catalyzing the production of gamma-aminobutyric acid (GABA) from L-glutamic acid. Here, we studied the impact of this mutation on GAD67 activity, dimerization and subcellular localization. Biochemical assay revealed that c.391 A > G reduces GAD67 enzymatic activity by ~30%, probably due to the impaired homodimerization of homozygous mutants as highlighted by proximity ligation assays. The mutational screening of 120 genes of the “GABAergic system” in a cohort of 4,225 SCZ cases and 5,834 controls (dbGaP: phs000473.v1.p2), did not identify other cases that were homozygous for ultra-rare variants in GAD1, but highlighted an increased frequency of cases that were homozygous for rare variants in genes of the GABA system (SCZ: 0.14% vs. Controls: 0.00%; p-value = 0.0055). In conclusion, this study demonstrates the functional impact of c.391 A > G variant and its biological effect makes it a good candidate as risk variant for SCZ. This study also supports an involvement of ultra-rare variants in GABAergic genes in the etiopathogenesis of SCZ.