Functional Characterization of Rare RAB12 Variants and Their Role in Musician's and Other Dystonias

Mutations in RAB (member of the Ras superfamily) genes are increasingly recognized as cause of a variety of disorders including neurological conditions. While musician's dystonia (MD) and writer's dystonia (WD) are task-specific movement disorders, other dystonias persistently affect postures as in cervical dystonia. Little is known about the underlying etiology. Next-generation sequencing revealed a rare missense variant (c.586A> G; p.Ile196Val) in RAB12 in two of three MD/WD families. Next, we tested 916 additional dystonia patients; 512 Parkinson's disease patients; and 461 healthy controls for RAB12 variants and identified 10 additional carriers of rare missense changes among dystonia patients (1.1%) but only one carrier in non-dystonic individuals (0.1%; p = 0.005). The detected variants among index patients comprised p.Ile196Val (n = 6); p.Ala174Thr (n = 3); p.Gly13Asp; p.Ala148Thr; and p.Arg181Gln in patients with MD; cervical dystonia; or WD. Two relatives of MD patients with WD also carried p.Ile196Val. The two variants identified in MD patients (p.Ile196Val; p.Gly13Asp) were characterized on endogenous levels in patient-derived fibroblasts and in two RAB12-overexpressing cell models. The ability to hydrolyze guanosine triphosphate (GTP), so called GTPase activity, was increased in mutants compared to wildtype. Furthermore, subcellular distribution of RAB12 in mutants was altered in fibroblasts. Soluble Transferrin receptor 1 levels were reduced in the blood of all three tested p.Ile196Val carriers. In conclusion, we demonstrate an enrichment of missense changes among dystonia patients. Functional characterization revealed altered enzyme activity and lysosomal distribution in mutants suggesting a contribution of RAB12 variants to MD and other dystonias

Comparison of Physical Properties of Untreated and Heat Treated Beech and Hornbeam

Istraživanjem fizikalnih svojstava toplinski obrađene bukovine i grabovine utvrđeno je da je njihova srednja vrijednost manja i signifikantno se razlikuje od srednjih vrijednosti fizikalnih svojstava neobrađene bukovine i grabovine. Srednja vrijednost gustoće u apsolutno suhom stanju toplinski obrađene bukovine manja je za 8,5 % od neobrađene, a za grabovinu je ona manja za 7,5 %. Smanjenje srednjih vrijednosti maksimalnih utezanja toplinski obrađene bukovine i grabovine u odnosu prema neobrađenoj još je veće. Maksimalno radijalno utezanje toplinski obrađene bukovine manje je za 7 %, maksimalno tangencijalno utezanje za 23,5 %, a maksimalno volumno utezanje za 19,3 % od istih fizikalnih svojstava neobrađene bukovine. Toplinski obrađena grabovina ima srednju vrijednost maksimalnoga radijalnog utezanja za 123 %, maksimalnoga tangencijalnog utezanja za 86 % i maksimalnoga volumnog utezanja za 99,5 % manju od istih fizikalnih svojstava neobrađene grabovine. Takvim smanjenjem maksimalnih utezanja u radijalnome i tangencijalnom smjeru toplinskom obradom grabovina postaje znatno prihvatljivija za izradu proizvoda za koje je važna dimenzionalna stabilnost.The investigation of physical properties of heat treated beech wood and hornbeam wood found that their average value is lower and significantly different from average values of physical properties of untreated beech wood and hornbeam wood. The average value of density in absolutely dry condition of heat treated beech wood is smaller by 8.5% from the untreated, and the hornbeam wood is smaller by 7.5%. Reduction of average values of maximum shrinkage of heat treated beech wood and hornbeam wood is even bigger in relation to the untreated wood. Maximum radial shrinkage of heat treated beech wood is smaller by 7%, maximum tangential shrinkage by 23.5% and maximum volumetric shrinkage by 19.3% compared to the same physical properties of untreated beech wood. Heat treated hornbeam wood has an average value of maximum radial shrinkage smaller by 123%, maximum tangential shrinkage by 86% and maximum volume shrinkage by 99.5% compared to the same physical properties of untreated hornbeam wood. With such reduction in the maximum shrinkage in radial and tangential direction using heat treatment, hornbeam becomes particulary suitable for making products where dimensional stability is important

Genomic correlates of glatiramer acetate adverse cardiovascular effects lead to a novel locus mediating coronary risk

Glatiramer acetate is used therapeutically in multiple sclerosis but also known for adverse effects including elevated coronary artery disease (CAD) risk. The mechanisms underlying the cardiovascular side effects of the medication are unclear. Here, we made use of the chromosomal variation in the genes that are known to be affected by glatiramer treatment. Focusing on genes and gene products reported by drug-gene interaction database to interact with glatiramer acetate we explored a large meta-analysis on CAD genome-wide association studies aiming firstly, to investigate whether variants in these genes also affect cardiovascular risk and secondly, to identify new CAD risk genes. We traced association signals in a 200-kb region around genomic positions of genes interacting with glatiramer in up to 60 801 CAD cases and 123 504 controls. We validated the identified association in additional 21 934 CAD cases and 76 087 controls. We identified three new CAD risk alleles within the TGFB1 region on chromosome 19 that independently affect CAD risk. The lead SNP rs12459996 was genome-wide significantly associated with CAD in the extended meta-analysis (odds ratio 1.09, p = 1.58×10-12). The other two SNPs at the locus were not in linkage disequilibrium with the lead SNP and by a conditional analysis showed p-values of 4.05 × 10-10 and 2.21 × 10-6. Thus, studying genes reported to interact with glatiramer acetate we identified genetic variants that concordantly with the drug increase the risk of CAD. Of these, TGFB1 displayed signal for association. Indeed, the gene has been associated with CAD previously in both in vivo and in vitro studies. Here we establish genome-wide significant association with CAD in large human samples.This work was supported by grants from the Fondation Leducq (CADgenomics: Understanding CAD Genes, 12CVD02), the German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept (e:AtheroSysMed, grant 01ZX1313A-2014 and SysInflame, grant 01ZX1306A), and the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement no HEALTH-F2-2013-601456 (CVgenes-at-target). Further grants were received from the DFG as part of the Sonderforschungsbereich CRC 1123 (B2). T.K. was supported by a DZHK Rotation Grant. I.B. was supported by the Deutsche Forschungsgemeinschaft (DFG) cluster of excellence ‘Inflammation at Interfaces’. F.W.A. is supported by a Dekker scholarship-Junior Staff Member 2014T001 - Netherlands Heart Foundation and UCL Hospitals NIHR Biomedical Research Centre

Genomewide association analysis of coronary artery disease

Background - Modern genotyping platforms permit a systematic search for inherited components of complex diseases. We performed a joint analysis of two genomewide association studies of coronary artery disease. Methods - We first identified chromosomal loci that were strongly associated with coronary artery disease in the Wellcome Trust Case Control Consortium (WTCCC) study (which involved 1926 case subjects with coronary artery disease and 2938 controls) and looked for replication in the German MI [Myocardial Infarction] Family Study (which involved 875 case subjects with myocardial infarction and 1644 controls). Data on other single-nucleotide polymorphisms (SNPs) that were significantly associated with coronary artery disease in either study (P<0.001) were then combined to identify additional loci with a high probability of true association. Genotyping in both studies was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix). Results - Of thousands of chromosomal loci studied, the same locus had the strongest association with coronary artery disease in both the WTCCC and the German studies: chromosome 9p21.3 (SNP, rs1333049) (P=1.80x10–14 and P=3.40x10–6, respectively). Overall, the WTCCC study revealed nine loci that were strongly associated with coronary artery disease (P80%) of a true association: chromosomes 1p13.3 (rs599839), 1q41 (rs17465637), 10q11.21 (rs501120), and 15q22.33 (rs17228212). Conclusions - We identified several genetic loci that, individually and in aggregate, substantially affect the risk of development of coronary artery disease

Whole-exome sequencing in an extended family with myocardial infarction unmasks familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal-dominant disease leading to markedly elevated low-density lipoprotein (LDL) cholesterol levels and increased risk for premature myocardial infarction (MI). Mutation carriers display variable LDL cholesterol levels, which may obscure the diagnosis. We examined by whole-exome sequencing a family in which multiple myocardial infarctions occurred at a young age with unclear etiology.Whole-exome sequencing of three affected family members, validation of the identified variant with Sanger-sequencing, and subsequent co-segregation analysis in the family.The index patient (LDL cholesterol 188 mg/dL) was referred for molecular-genetic investigations. He had coronary artery bypass graft (CABG) at the age of 59 years; 12 out of 15 1st, 2nd and 3rd degree relatives were affected with coronary artery disease (CAD) and/or premature myocardial infarction (MI). We sequenced the whole-exome of the patient and two cousins with premature MI. After filtering, we were left with a potentially disease causing variant in the LDL receptor (LDLR) gene, which we validated by Sanger-sequencing (nucleotide substitution in the acceptor splice-site of exon 10, c.1359-1G > A). Sequencing of all family members available for genetic analysis revealed co-segregation of the variant with CAD (LOD 3.0) and increased LDLC (>190 mg/dL), following correction for statin treatment (LOD 4.3). Interestingly, mutation carriers presented with highly variable corrected (183-354 mg/dL) and on-treatment LDL levels (116-274 mg/dL) such that the diagnosis of FH in this family was made only after the molecular-genetic analysis.Even in families with unusual clustering of CAD FH remains to be underdiagnosed, which underscores the need for implementation of systematic screening programs. Whole-exome sequencing may facilitate identification of disease-causing variants in families with unclear etiology of MI and enable preventive treatment of mutation carriers in a more timely fashion

Genomic correlates of glatiramer acetate adverse cardiovascular effects lead to a novel locus mediating coronary risk.

Glatiramer acetate is used therapeutically in multiple sclerosis but also known for adverse effects including elevated coronary artery disease (CAD) risk. The mechanisms underlying the cardiovascular side effects of the medication are unclear. Here, we made use of the chromosomal variation in the genes that are known to be affected by glatiramer treatment. Focusing on genes and gene products reported by drug-gene interaction database to interact with glatiramer acetate we explored a large meta-analysis on CAD genome-wide association studies aiming firstly, to investigate whether variants in these genes also affect cardiovascular risk and secondly, to identify new CAD risk genes. We traced association signals in a 200-kb region around genomic positions of genes interacting with glatiramer in up to 60 801 CAD cases and 123 504 controls. We validated the identified association in additional 21 934 CAD cases and 76 087 controls. We identified three new CAD risk alleles within the TGFB1 region on chromosome 19 that independently affect CAD risk. The lead SNP rs12459996 was genome-wide significantly associated with CAD in the extended meta-analysis (odds ratio 1.09, p = 1.58×10-12). The other two SNPs at the locus were not in linkage disequilibrium with the lead SNP and by a conditional analysis showed p-values of 4.05 × 10-10 and 2.21 × 10-6. Thus, studying genes reported to interact with glatiramer acetate we identified genetic variants that concordantly with the drug increase the risk of CAD. Of these, TGFB1 displayed signal for association. Indeed, the gene has been associated with CAD previously in both in vivo and in vitro studies. Here we establish genome-wide significant association with CAD in large human samples

A genomic exploration identifies mechanisms that may explain adverse cardiovascular effects of COX-2 inhibitors.

Cyclooxygenase-2 inhibitors (coxibs) are characterized by multiple molecular off-target effects and increased coronary artery disease (CAD) risk. Here, we systematically explored common variants of genes representing molecular targets of coxibs for association with CAD. Given a broad spectrum of pleiotropic effects of coxibs, our intention was to narrow potential mechanisms affecting CAD risk as we hypothesized that the affected genes may also display genomic signals of coronary disease risk. A Drug Gene Interaction Database search identified 47 gene products to be affected by coxibs. We traced association signals in 200-kb regions surrounding these genes in 84,813 CAD cases and 202,543 controls. Based on a threshold of 1 × 10(Bonferroni correction for 3131 haplotype blocks), four gene loci yielded significant associations. The lead SNPs were rs7270354 (MMP9), rs4888383 (BCAR1), rs6905288 (VEGFA1), and rs556321 (CACNA1E). By additional genotyping, rs7270354 at MMP9 and rs4888383 at BCAR1 also reached the established GWAS threshold for genome-wide significance. The findings demonstrate overlap of genes affected by coxibs and those mediating CAD risk and points to further mechanisms, which are potentially responsible for coxib-associated CAD risk. The novel approach furthermore suggests that genetic studies may be useful to explore the clinical relevance of off-target drug effects

Systematic analysis of variants related to familial hypercholesterolemia in families with premature myocardial infarction.

Familial hypercholesterolemia (FH) is an oligogenic disorder characterized by markedly elevated low-density lipoprotein cholesterol (LDLC) levels. Variants in four genes have been reported to cause the classical autosomal-dominant form of the disease. FH is largely under-diagnosed in European countries. As FH increases the risk for coronary artery disease (CAD) and myocardial infarction (MI), it might be specifically overlooked in the large number of such patients. Here, we systematically examined the frequency of potential FH-causing variants by exome sequencing in 255 German patients with premature MI and a positive family history for CAD. We further performed co-segregation analyses in an average of 5.5 family members per MI patient. In total, we identified 11 potential disease-causing variants that co-segregate within the families, that is, 5% of patients with premature MI and positive CAD family history had FH. Eight variants were previously reported as disease-causing and three are novel (LDLR.c.811G>A p.(V271I)), PCSK9.c.610G>A (p.(D204N)) and STAP1.c.139A>G (p.(T47A))). Co-segregation analyses identified multiple additional family members carrying one of these FH variants and the clinical phenotype of either FH (n=2) or FH and premature CAD (n=15). However, exome sequencing also revealed that some variants in FH genes, which have been reported to cause FH, do not co-segregate with FH. The data reveal that a large proportion of FH patients escape the diagnosis, even when they have premature MI. Hence, systematic molecular-genetic screening for FH in such patients may reveal a substantial number of cases and thereby allow a timely LDLC-lowering in both FH/MI patients as well as their variant-carrying family members

Prediction of Causal Candidate Genes in Coronary Artery Disease Loci.

Genome-wide association studies have to date identified 159 significant and suggestive loci for coronary artery disease (CAD). We now report comprehensive bioinformatics analyses of sequence variation in these loci to predict candidate causal genes.All annotated genes in the loci were evaluated with respect to protein-coding single-nucleotide polymorphism and gene expression parameters. The latter included expression quantitative trait loci, tissue specificity, and miRNA binding. High priority candidate genes were further identified based on literature searches and our experimental data. We conclude that the great majority of causal variations affecting CAD risk occur in noncoding regions, with 41% affecting gene expression robustly versus 6% leading to amino acid changes. Many of these genes differed from the traditionally annotated genes, which was usually based on proximity to the lead single-nucleotide polymorphism. Indeed, we obtained evidence that genetic variants at CAD loci affect 98 genes which had not been linked to CAD previously.Our results substantially revise the list of likely candidates for CAD and suggest that genome-wide association studies efforts in other diseases may benefit from similar bioinformatics analyses