wKinMut: An integrated tool for the analysis and interpretation of mutations in human protein kinases

BACKGROUND: Protein kinases are involved in relevant physiological functions and a broad number of mutations in this superfamily have been reported in the literature to affect protein function and stability. Unfortunately, the exploration of the consequences on the phenotypes of each individual mutation remains a considerable challenge. RESULTS: The wKinMut web-server offers direct prediction of the potential pathogenicity of the mutations from a number of methods, including our recently developed prediction method based on the combination of information from a range of diverse sources, including physicochemical properties and functional annotations from FireDB and Swissprot and kinase-specific characteristics such as the membership to specific kinase groups, the annotation with disease-associated GO terms or the occurrence of the mutation in PFAM domains, and the relevance of the residues in determining kinase subfamily specificity from S3Det. This predictor yields interesting results that compare favourably with other methods in the field when applied to protein kinases. Together with the predictions, wKinMut offers a number of integrated services for the analysis of mutations. These include: the classification of the kinase, information about associations of the kinase with other proteins extracted from iHop, the mapping of the mutations onto PDB structures, pathogenicity records from a number of databases and the classification of mutations in large-scale cancer studies. Importantly, wKinMut is connected with the SNP2L system that extracts mentions of mutations directly from the literature, and therefore increases the possibilities of finding interesting functional information associated to the studied mutations. CONCLUSIONS: wKinMut facilitates the exploration of the information available about individual mutations by integrating prediction approaches with the automatic extraction of information from the literature (text mining) and several state-of-the-art databases. wKinMut has been used during the last year for the analysis of the consequences of mutations in the context of a number of cancer genome projects, including the recent analysis of Chronic Lymphocytic Leukemia cases and is publicly available at http://wkinmut.bioinfo.cnio.es

Rare mendelian forms of obesity and diabetes and their implications for treatment outcomes

Obesity and diabetes are becoming epidemic health issues worldwide. In recent decades, a considerable amount of work has been done to study the pathogenicity underlying those diseases, which has led to valuable insights into the genetic basis, treatment and prevention of obesity and diabetes. Nevertheless, despite our more detailed pathophysiological understanding of the rare forms of diabetes and obesity than of more common polygenic forms, we still know little about their prevalence and implications outside specialised genetics services. In the present work, I have explored the contribution of Mendelian forms of obesity in individuals with severe obesity. Initial segregation analyses of families with an obese proband, led to the identification of an oligogenic mode of inheritance for obesity. This was followed by re-analysis of pre-existing whole exome sequencing data from 91 individuals with extreme obesity, which revealed an additional 21 possible causative variants in known monogenic/syndromic obesity genes and three further cases of oligogenic inheritance. In addition, 11 candidate variants were identified in genes suggested by rodent models of obesity and/or diabetes, but not previously reported in humans. To further expand the analysis, a unique custom genotyping array focusing on, obesity and diabetes mellitus (T2D, and monogenic forms of diabetes) was designed to be applied to a larger number of samples (N=2068). Application of the array led to the identification of a total of 161 potential causative variants in 40 monogenic obesity/syndromic obesity genes, with a putative diagnostic yield of 11%. Initial analysis suggests that having one of these putative Mendelian forms of obesity resulted in no statistical difference in percentage weight loss at 2 years post-surgery and diabetes remission. Our first analysis on obesity indicates that the use of a custom-designed genotyping array for specific rare diseases may be an advantageous first level screening strategy in terms of cost and time. The work presented here also suggests that the true prevalence of Mendelian forms of obesity among bariatric surgery patients is likely to be high - this presents a significant unmet need for genetic analysis and follow-up.  Open Acces

Genomic and Cellular Studies Establish the Pathogenesis and Cellular Mechanisms of Disease-Causing Mutations in Families with Autosomal Recessive Disorders

The majority of the reported genetic disorders in the UAE population are of the autosomal recessive type, which is mainly due to high rates of consanguinity within the UAE national population, and within a significant proportion of other UAE expatriate communities; such as Arabs and Pakistanis. It is estimated that more than 50% of all marriages among Emiratis occur between biologically related couples, with first cousin marriages being the highest. That could be attributed to sociocultural values in the region. Successful management of genetic diseases can be achieved by the implementation of effective preventative programs that could help reduce the number of new cases, and provide early diagnosis to potentially improve disease management. For these desired outcomes to be achieved, it is imperative to identify the molecular causes (i.e. disease-causing genes and mutations) of such disorders. Therefore, the aim of this study is to elucidate the molecular pathology and cellular mechanisms of a group of recessive disorders affecting Emirati and expatriate families in the UAE. Whole exome sequencing, together with homozygosity mapping and segregation analyses, were performed on the recruited families to elucidate the causative genes and mutations. Where necessary, bioinformatics in silico analyses coupled with cellular and other functional studies were performed to confirm pathogenicity and uncover the cellular mechanisms of the studied disease phenotypes. In this dissertation, I report the identification of two novel compound heterozygous mutations in Multiple PDZ domain (MPDZ) gene causing congenital hydrocephalus, and provide experimental evidence on their pathogenesis and mechanisms of action. In addition, I report the identification of a novel mutation in Xylosyltransferase I (XYLT1) gene responsible for Desbuquois dysplasia II (DBQDII), and provide evidence on the involvement of the endoplasmic reticulum (ER) quality control in the cellular mechanism of several DBQDII-causing mutations, including, the newly identified one. Furthermore, I provide preliminary data on candidate genes in two families affected by suspected monogenic intellectual disability syndromes. Overall, this dissertation provides evidence on the pathogenicity of several mutations and associated cellular mechanisms. The outcomes of this project will likely be valuable for implementing effective preventive strategies at least for the extended family members of the affected individuals

Identifying common and rare variants in migraine genetic predisposition

Migraine is an episodic brain disorder that is characterized by recurrent pain. Etiology of migraine is extremely complex; most likely caused by combination of genetic and environmental risk factors. The aim of the thesis is to examine the role of MTHFR polymorphisms rs1801131 and rs1801133 as risk factors for pediatric migraine; also in migraine subtypes – migraine with aura (MA) and without aura (MO). Second part involved exome sequencing of two family trios to discover novel genetic risk factors for migraine. Candidate gene study of MTHFR did not reveal any statistically significant results. Exome sequencing revealed three novel variants that could precipitate migraine. PDLIM5, PRKCE and SCN9A all affect voltage-gated channels. Mutations in those genes could increase neuronal hyperexcitability and neurotransmitter release, which in turn has been associated with pain and visual aura

Is High Resolution Melting Analysis (HRMA) Accurate for Detection of Human Disease-Associated Mutations? A Meta Analysis

BACKGROUND: High Resolution Melting Analysis (HRMA) is becoming the preferred method for mutation detection. However, its accuracy in the individual clinical diagnostic setting is variable. To assess the diagnostic accuracy of HRMA for human mutations in comparison to DNA sequencing in different routine clinical settings, we have conducted a meta-analysis of published reports. METHODOLOGY/PRINCIPAL FINDINGS: Out of 195 publications obtained from the initial search criteria, thirty-four studies assessing the accuracy of HRMA were included in the meta-analysis. We found that HRMA was a highly sensitive test for detecting disease-associated mutations in humans. Overall, the summary sensitivity was 97.5% (95% confidence interval (CI): 96.8-98.5; I(2) = 27.0%). Subgroup analysis showed even higher sensitivity for non-HR-1 instruments (sensitivity 98.7% (95%CI: 97.7-99.3; I(2) = 0.0%)) and an eligible sample size subgroup (sensitivity 99.3% (95%CI: 98.1-99.8; I(2) = 0.0%)). HRMA specificity showed considerable heterogeneity between studies. Sensitivity of the techniques was influenced by sample size and instrument type but by not sample source or dye type. CONCLUSIONS/SIGNIFICANCE: These findings show that HRMA is a highly sensitive, simple and low-cost test to detect human disease-associated mutations, especially for samples with mutations of low incidence. The burden on DNA sequencing could be significantly reduced by the implementation of HRMA, but it should be recognized that its sensitivity varies according to the number of samples with/without mutations, and positive results require DNA sequencing for confirmation

Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing's disease.

The CABLES1 cell cycle regulator participates in the adrenal-pituitary negative feedback, and its expression is reduced in corticotropinomas, pituitary tumors with a largely unexplained genetic basis. We investigated the presence of CABLES1 mutations/copy number variations (CNVs) and their associated clinical, histopathological and molecular features in patients with Cushing's disease (CD). Samples from 146 pediatric (118 germline DNA only/28 germline and tumor DNA) and 35 adult (tumor DNA) CD patients were screened for CABLES1 mutations. CNVs were assessed in 116 pediatric CD patients (87 germline DNA only/29 germline and tumor DNA). Four potentially pathogenic missense variants in CABLES1 were identified, two in young adults (c.532G > A, p.E178K and c.718C > T, p.L240F) and two in children (c.935G > A, p.G312D and c.1388A > G, and p.D463G) with CD; no CNVs were found. The four variants affected residues within or close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein and impaired its ability to block cell growth in a mouse corticotropinoma cell line (AtT20/D16v-F2). The four patients had macroadenomas. We provide evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene. Its function might link two of the main molecular mechanisms altered in corticotropinomas: the cyclin-dependent kinase/cyclin group of cell cycle regulators and the epidermal growth factor receptor signaling pathway. Further studies are needed to assess the prevalence of CABLES1 mutations among patients with other types of pituitary adenomas and to elucidate the pituitary-specific functions of this gene

The association between ATM D1853N polymorphism and breast cancer susceptibility: a meta-analysis

BACKGROUND: Emerging evidence suggests that ataxia telangiectasia-mutated (ATM) is involved in numerous damage repair signaling pathways and cell-cycle checkpoints. Heterozygous carriers of ATM-mutations have an increased risk for the development of breast cancer. The purpose of this study is to evaluate the association between ATM exon39 5557G > A (D1853N, rs1801516) polymorphism and breast cancer susceptibility with the use of a meta-analysis. METHODS: By searching PubMed and Embase databases, a total of 9 epidemiological studies with 4,191 cases and 3,780 controls were identified. Crude odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) for ATM D1853N polymorphism and breast cancer risk were calculated using fixed- or random-effects model based on the degree of heterogeneity among studies. RESULTS: No significant association between the ATM D1853N polymorphism and breast cancer risk was observed in overall analysis (GA versus GG: OR = 1.18; 95% CI, 0.90-1.53; AA versus GG: OR = 0.77; 95% CI, 0.58-1.03; dominant model: OR = 1.16; 95% CI, 0.89-1.51; and recessive model: OR = 0.78; 95% CI, 0.59-1.04, respectively). CONCLUSION: Our results indicate that ATM D1853N polymorphism is not a risk factor for developing breast cancer

Fifteen new risk loci for coronary artery disease highlight arterial-wall-specific mechanisms

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP-CAD associations (P < 5 × 10(-8), in fixed-effects meta-analysis) from 15 genomic regions, including SNPs in or near genes involved in cellular adhesion, leukocyte migration and atherosclerosis (PECAM1, rs1867624), coagulation and inflammation (PROCR, rs867186 (p.Ser219Gly)) and vascular smooth muscle cell differentiation (LMOD1, rs2820315). Correlation of these regions with cell-type-specific gene expression and plasma protein levels sheds light on potential disease mechanisms