Across a variety of Mendelian disorders, ∼50-75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome ...sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.
Cardiac macrophages (cMPs) are increasingly recognized as important regulators of myocardial homeostasis and disease, yet the role of noncoding RNA in these cells is largely unknown. Small RNA ...sequencing of the entire miRNomes of the major cardiac cell fractions revealed microRNA-21 (miR-21) as the single highest expressed microRNA in cMPs, both in health and disease (25% and 43% of all microRNA reads, respectively). MiR-21 has been previously reported as a key microRNA driving tissue fibrosis. Here, we aimed to determine the function of macrophage miR-21 on myocardial homeostasis and disease-associated remodeling.
Macrophage-specific ablation of miR-21 in mice driven by Cx3cr1-Cre was used to determine the function of miR-21 in this cell type. As a disease model, mice were subjected to pressure overload for 6 and 28 days. Cardiac function was assessed in vivo by echocardiography, followed by histological analyses and single-cell sequencing. Cocultures of macrophages and cardiac fibroblasts were used to study macrophage-to-fibroblast signaling.
Mice with macrophage-specific genetic deletion of miR-21 were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload of the left ventricle. Single-cell sequencing of pressure-overloaded hearts from these mice revealed that miR-21 in macrophages is essential for their polarization toward a M1-like phenotype. Systematic quantification of intercellular communication mediated by ligand-receptor interactions across all cell types revealed that miR-21 primarily determined macrophage-fibroblast communication, promoting the transition from quiescent fibroblasts to myofibroblasts. Polarization of isolated macrophages in vitro toward a proinflammatory (M1-like) phenotype activated myofibroblast transdifferentiation of cardiac fibroblasts in a paracrine manner and was dependent on miR-21 in cMPs.
Our data indicate a critical role of cMPs in pressure overload-induced cardiac fibrosis and dysfunction and reveal macrophage miR-21 as a key molecule for the profibrotic role of cMPs.
Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing ...adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na(+)/K(+) ATPase α subunit) and ATP2B3 (encoding a Ca(2+) ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Summary Background The genetic cause of intellectual disability in most patients is unclear because of the absence of morphological clues, information about the position of such genes, and suitable ...screening methods. Our aim was to identify de-novo variants in individuals with sporadic non-syndromic intellectual disability. Methods In this study, we enrolled children with intellectual disability and their parents from ten centres in Germany and Switzerland. We compared exome sequences between patients and their parents to identify de-novo variants. 20 children and their parents from the KORA Augsburg Diabetes Family Study were investigated as controls. Findings We enrolled 51 participants from the German Mental Retardation Network. 45 (88%) participants in the case group and 14 (70%) in the control group had de-novo variants. We identified 87 de-novo variants in the case group, with an exomic mutation rate of 1·71 per individual per generation. In the control group we identified 24 de-novo variants, which is 1·2 events per individual per generation. More participants in the case group had loss-of-function variants than in the control group (20/51 vs 2/20; p=0·022), suggesting their contribution to disease development. 16 patients carried de-novo variants in known intellectual disability genes with three recurrently mutated genes ( STXBP1, SYNGAP1 , and SCN2A ). We deemed at least six loss-of-function mutations in six novel genes to be disease causing. We also identified several missense alterations with potential pathogenicity. Interpretation After exclusion of copy-number variants, de-novo point mutations and small indels are associated with severe, sporadic non-syndromic intellectual disability, accounting for 45–55% of patients with high locus heterogeneity. Autosomal recessive inheritance seems to contribute little in the outbred population investigated. The large number of de-novo variants in known intellectual disability genes is only partially attributable to known non-specific phenotypes. Several patients did not meet the expected syndromic manifestation, suggesting a strong bias in present clinical syndrome descriptions. Funding German Ministry of Education and Research, European Commission 7th Framework Program, and Swiss National Science Foundation.
Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG, POLG2 and C10orf2) or the biosynthesis pathways of ...deoxyribonucleoside 5'-triphosphates for mtDNA synthesis. However, in many of these disorders, the underlying genetic defect has yet to be discovered. Here, we identify homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characterized by external ophthalmoplegia, emaciation and respiratory failure. Muscle biopsies showed mtDNA depletion and multiple mtDNA deletions. C20orf72, hereafter MGME1 (mitochondrial genome maintenance exonuclease 1), encodes a mitochondrial RecB-type exonuclease belonging to the PD-(D/E)XK nuclease superfamily. We show that MGME1 cleaves single-stranded DNA and processes DNA flap substrates. Fibroblasts from affected individuals do not repopulate after chemically induced mtDNA depletion. They also accumulate intermediates of stalled replication and show increased levels of 7S DNA, as do MGME1-depleted cells. Thus, we show that MGME1-mediated mtDNA processing is essential for mitochondrial genome maintenance.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Metabolomic profiling and the integration of whole-genome genetic association data has proven to be a powerful tool to comprehensively explore gene regulatory networks and to investigate the effects ...of genetic variation at the molecular level. Serum metabolite concentrations allow a direct readout of biological processes, and association of specific metabolomic signatures with complex diseases such as Alzheimer's disease and cardiovascular and metabolic disorders has been shown. There are well-known correlations between sex and the incidence, prevalence, age of onset, symptoms, and severity of a disease, as well as the reaction to drugs. However, most of the studies published so far did not consider the role of sexual dimorphism and did not analyse their data stratified by gender. This study investigated sex-specific differences of serum metabolite concentrations and their underlying genetic determination. For discovery and replication we used more than 3,300 independent individuals from KORA F3 and F4 with metabolite measurements of 131 metabolites, including amino acids, phosphatidylcholines, sphingomyelins, acylcarnitines, and C6-sugars. A linear regression approach revealed significant concentration differences between males and females for 102 out of 131 metabolites (p-values<3.8×10(-4); Bonferroni-corrected threshold). Sex-specific genome-wide association studies (GWAS) showed genome-wide significant differences in beta-estimates for SNPs in the CPS1 locus (carbamoyl-phosphate synthase 1, significance level: p<3.8×10(-10); Bonferroni-corrected threshold) for glycine. We showed that the metabolite profiles of males and females are significantly different and, furthermore, that specific genetic variants in metabolism-related genes depict sexual dimorphism. Our study provides new important insights into sex-specific differences of cell regulatory processes and underscores that studies should consider sex-specific effects in design and interpretation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Serum metabolite concentrations provide a direct readout of biological processes in the human body, and they are associated with disorders such as cardiovascular and metabolic diseases. We present a ...genome-wide association study (GWAS) of 163 metabolic traits measured in human blood from 1,809 participants from the KORA population, with replication in 422 participants of the TwinsUK cohort. For eight out of nine replicated loci (FADS1, ELOVL2, ACADS, ACADM, ACADL, SPTLC3, ETFDH and SLC16A9), the genetic variant is located in or near genes encoding enzymes or solute carriers whose functions match the associating metabolic traits. In our study, the use of metabolite concentration ratios as proxies for enzymatic reaction rates reduced the variance and yielded robust statistical associations with P values ranging from 3 × 10−24 to 6.5 × 10−179. These loci explained 5.6%-36.3% of the observed variance in metabolite concentrations. For several loci, associations with clinically relevant parameters have been reported previously.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of ...pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder which is prevalent on the Island of Réunion and whose molecular basis is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and 2 families from other countries. Functional consequences of the mutated alleles were assessed by growth and methionine incorporation assays in yeast. Enzyme activity was attenuated in liquid medium without methionine, but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly opens new treatment opportunities.
The rapidly evolving field of metabolomics aims at a comprehensive measurement of ideally all endogenous metabolites in a cell or body fluid. It thereby provides a functional readout of the ...physiological state of the human body. Genetic variants that associate with changes in the homeostasis of key lipids, carbohydrates, or amino acids are not only expected to display much larger effect sizes due to their direct involvement in metabolite conversion modification, but should also provide access to the biochemical context of such variations, in particular when enzyme coding genes are concerned. To test this hypothesis, we conducted what is, to the best of our knowledge, the first GWA study with metabolomics based on the quantitative measurement of 363 metabolites in serum of 284 male participants of the KORA study. We found associations of frequent single nucleotide polymorphisms (SNPs) with considerable differences in the metabolic homeostasis of the human body, explaining up to 12% of the observed variance. Using ratios of certain metabolite concentrations as a proxy for enzymatic activity, up to 28% of the variance can be explained (p-values 10(-16) to 10(-21)). We identified four genetic variants in genes coding for enzymes (FADS1, LIPC, SCAD, MCAD) where the corresponding metabolic phenotype (metabotype) clearly matches the biochemical pathways in which these enzymes are active. Our results suggest that common genetic polymorphisms induce major differentiations in the metabolic make-up of the human population. This may lead to a novel approach to personalized health care based on a combination of genotyping and metabolic characterization. These genetically determined metabotypes may subscribe the risk for a certain medical phenotype, the response to a given drug treatment, or the reaction to a nutritional intervention or environmental challenge.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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