Clonal hematopoiesis as a pitfall in germline variant interpretation in the context of Mendelian disorders.

Clonal hematopoiesis because of somatic mutations in hematopoietic stem/progenitor cells is an age-related phenomenon and commonly observed when sequencing blood DNA in elderly individuals. Several genes that are implicated in clonal hematopoiesis are also associated with Mendelian disorders when mutated in the germline, potentially leading to variant misinterpretation. We performed a literature search to identify genes associated with age-related clonal hematopoiesis followed by an OMIM query to identify the subset of genes in which germline variants are associated with Mendelian disorders. We retrospectively screened for diagnostic cases in which the presence of age-related clonal hematopoiesis confounded exome sequencing data interpretation. We found 58 genes in which somatic mutations are implicated in clonal hematopoiesis, while germline variants in the same genes are associated with Mendelian (mostly neurodevelopmental) disorders. Using five selected cases of individuals with suspected monogenic disorders, we illustrate how clonal hematopoiesis in either variant databases or exome sequencing datasets poses a pitfall, potentially leading to variant misclassification and erroneous conclusions regarding gene-disease associations.

Bi-allelic Truncating Mutations in TANGO2 Cause Infancy-Onset Recurrent Metabolic Crises with Encephalocardiomyopathy.

Molecular diagnosis of mitochondrial disorders is challenging because of extreme clinical and genetic heterogeneity. By exome sequencing, we identified three different bi-allelic truncating mutations in TANGO2 in three unrelated individuals with infancy-onset episodic metabolic crises characterized by encephalopathy, hypoglycemia, rhabdomyolysis, arrhythmias, and laboratory findings suggestive of a defect in mitochondrial fatty acid oxidation. Over the course of the disease, all individuals developed global brain atrophy with cognitive impairment and pyramidal signs. TANGO2 (transport and Golgi organization 2) encodes a protein with a putative function in redistribution of Golgi membranes into the endoplasmic reticulum in Drosophila and a mitochondrial localization has been confirmed in mice. Investigation of palmitate-dependent respiration in mutant fibroblasts showed evidence of a functional defect in mitochondrial ß-oxidation. Our results establish TANGO2 deficiency as a clinically recognizable cause of pediatric disease with multi-organ involvement.

Mitochondrial DNA mutation analysis from exome sequencing-A more holistic approach in diagnostics of suspected mitochondrial disease.

Diagnostics for suspected mitochondrial disease (MD) can be challenging and necessitate invasive procedures like muscle biopsy. This is due to the extremely broad genetic and phenotypic spectrum, disease genes on both nuclear and mitochondrial DNA (mtDNA), and the tissue specificity of mtDNA variants. Exome sequencing (ES) has revolutionized the diagnostics for MD. However, the nuclear and mtDNA are investigated with separate tests, increasing costs and duration of diagnostics. The full potential of ES is often not exploited as the additional analysis of "off-target reads" deriving from the mtDNA can be used to analyze both genomes. We performed mtDNA analysis by ES of 2111 cases in a clinical setting. We further assessed the recall rate and precision as well as the estimation of heteroplasmy by ES data by comparison with targeted mtDNA next generation sequencing in 49 cases. ES identified known pathogenic mtDNA point mutations in 38 individuals, increasing the diagnostic yield by nearly 2%. Analysis of mtDNA variants by ES had a high recall rate (96.2 ± 5.6%) and an excellent precision (99.5 ± 2.2%) when compared to the gold standard of targeted mtDNA next generation sequencing. ES estimated heteroplasmy levels with an average difference of 6.6 ± 3.8%, sufficient for clinical decision making. Taken together, the mtDNA analysis from ES is of sufficient quality for clinical diagnostics. We therefore propose ES, investigating both nuclear and mtDNA, as first line test in individuals with suspected MD. One should be aware, that a negative result does not exclude MD and necessitates further test (in additional tissues).

Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island.

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 that is prevalent on the island of Réunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a 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 open new treatment opportunities.

Eight Novel Mutations Confirm the Role of AAGAB in Punctate Palmoplantar Keratoderma Type 1 (Buschke-Fischer-Brauer) and Show Broad Phenotypic Variability.

Punctate palmoplantar keratoderma (PPKP1; Buschke-Fischer-Brauer) is a rare autosomal dominant inherited skin disease characterized by multiple hyperkeratotic papules involving the palms and soles. Mutations have been found at 2 loci, on chromosomes 15q22-15q24 and 8q24.13-8q24.21. We recently identified mutations in 3 families, in the AAGAB gene on 15q, which encodes the alpha- and gamma-adaptin-binding protein p34. The current study examined 14 additional families, comprising a total of 26 affected individuals and identified 8 novel mutations in 9 families. In one family a mutation that was present only in the affected individuals was found, and in 4 other families, previously reported mutations were found (1, 2). These results confirm the role of AAGAB in PPKP1. Our findings suggest that there is no correlation with age, but with mechanical factors. No additional obvious genotype-phenotype correlation was observed, even when comparing different types of mutations. Rather, identical genotypes presented a very broad interfamilial and intrafamilial variability of phenotypes.

Genome-wide association study of restless legs syndrome identifies common variants in three genomic regions.

Restless legs syndrome (RLS) is a frequent neurological disorder characterized by an imperative urge to move the legs during night, unpleasant sensation in the lower limbs, disturbed sleep and increased cardiovascular morbidity. In a genome-wide association study we found highly significant associations between RLS and intronic variants in the homeobox gene MEIS1, the BTBD9 gene encoding a BTB(POZ) domain as well as variants in a third locus containing the genes encoding mitogen-activated protein kinase MAP2K5 and the transcription factor LBXCOR1 on chromosomes 2p, 6p and 15q, respectively. Two independent replications confirmed these association signals. Each genetic variant was associated with a more than 50% increase in risk for RLS, with the combined allelic variants conferring more than half of the risk. MEIS1 has been implicated in limb development, raising the possibility that RLS has components of a developmental disorder.

Nonsense mutations in AAGAB cause punctate palmoplantar keratoderma type Buschke-Fischer-Brauer.

Punctate palmoplantar keratodermas (PPKPs) are rare autosomal-dominant inherited skin diseases that are characterized by multiple hyperkeratotic plaques distributed on the palms and soles. To date, two different loci in chromosomal regions 15q22-15q24 and 8q24.13-8q24.21 have been reported. Pathogenic mutations, however, have yet to be identified. In order to elucidate the genetic cause of PPKP type Buschke-Fischer-Brauer (PPKP1), we performed exome sequencing in five affected individuals from three families, and we identified in chromosomal region 15q22.33-q23 two heterozygous nonsense mutations-c.370C>T (p.Arg124(∗)) and c.481C>T (p.Arg161(∗))-in AAGAB in all affected individuals. Using immunoblot analysis, we showed that both mutations result in premature termination of translation and truncated protein products. Analyses of mRNA of affected individuals revealed that the disease allele is either not detectable or only detectable at low levels. To assess the consequences of the mutations in skin, we performed immunofluorescence analyses. Notably, the amount of granular staining in the keratinocytes of affected individuals was lower in the cytoplasm but higher around the nucleus than it was in the keratinocytes of control individuals. AAGAB encodes the alpha-and gamma-adaptin-binding protein p34 and might play a role in membrane traffic as a chaperone. The identification of mutations, along with the results from additional studies, defines the genetic basis of PPKP1 and provides evidence that AAGAB plays an important role in skin integrity.

Stimulation of soluble guanylyl cyclase (sGC) by riociguat attenuates heart failure and pathological cardiac remodelling.

BACKGROUND AND PURPOSE: Heart failure is associated with an impaired NO-soluble guanylyl cyclase (sGC)-cGMP pathway and its augmentation is thought to be beneficial for its therapy. We hypothesized that stimulation of sGC by the sGC stimulator riociguat prevents pathological cardiac remodelling and heart failure in response to chronic pressure overload. EXPERIMENTAL APPROACH: Transverse aortic constriction or sham surgery was performed in C57BL/6N mice. After 3 weeks of transverse aortic constriction when heart failure was established, animals receive either riociguat or its vehicle for 5 additional weeks. Cardiac function was evaluated weekly by echocardiography. Eight weeks after surgery, histological analyses were performed to evaluate remodelling and the transcriptome of the left ventricles (LVs) was analysed by RNA sequencing. Cell culture experiments were used for mechanistically studies. KEY RESULTS: Transverse aortic constriction resulted in a continuous decrease of LV ejection fraction and an increase in LV mass until week 3. Five weeks of riociguat treatment resulted in an improved LV ejection fraction and a decrease in the ratio of left ventricular mass to total body weight (LVM/BW), myocardial fibrosis and myocyte cross-sectional area. RNA sequencing revealed that riociguat reduced the expression of myocardial stress and remodelling genes (e.g. Nppa, Nppb, Myh7 and collagen) and attenuated the activation of biological pathways associated with cardiac hypertrophy and heart failure. Riociguat reversed pathological stress response in cultivated myocytes and fibroblasts. CONCLUSION AND IMPLICATIONS: Stimulation of the sGC reverses transverse aortic constriction-induced heart failure and remodelling, which is associated with improved myocardial gene expression. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Human skin-resident host T cells can persist long term after allogeneic stem cell transplantation and maintain recirculation potential.

Tissue-resident memory T cells (TRM) have recently emerged as crucial cellular players for host defense in a wide variety of tissues and barrier sites. Insights into the maintenance and regulatory checkpoints of human TRM cells remain scarce, especially due to the difficulties associated with tracking T cells through time and space in humans. We therefore sought to identify and characterize skin-resident T cells in humans defined by their long-term in situ lodgment. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) preceded by myeloablative chemotherapy unmasked long-term sequestration of host T cell subsets in human skin despite complete donor T cell chimerism in the blood. Single-cell chimerism analysis paired with single-cell transcriptional profiling comprehensively characterized these bona fide long-term skin-resident T cells and revealed differential tissue maintenance for distinct T cell subsets, specific TRM cell markers such as galectin-3, but also tissue exit potential with retention of the transcriptomic TRM cell identity. Analysis of 26 allo-HSCT patients revealed profound interindividual variation in the tissue maintenance of host skin T cells. The long-term persistence of host skin T cells in a subset of these patients did not correlate with the development of chronic GvHD. Our data exemplify the power of exploiting a clinical situation as a proof of concept for the existence of bona fide human skin TRM cells and reveal long-term persistence of host T cells in a peripheral tissue but not in the circulation or bone marrow in a subset of allo-HSCT patients.

Riociguat attenuates the changes in left ventricular proteome and microRNA profile after experimental aortic stenosis in mice.

BACKGROUND AND PURPOSE: Development and progression of heart failure involve endothelial and myocardial dysfunction as well as a dysregulation of the NO-sGC-cGMP signalling pathway. Recently, we reported that the sGC stimulator riociguat has beneficial effects on cardiac remodelling and progression of heart failure in response to chronic pressure overload. Here, we examined if these beneficial effects of riociguat were also reflected in alterations of the myocardial proteome and microRNA profiles. EXPERIMENTAL APPROACH: Male C57BL/6N mice underwent transverse aortic constriction (TAC) and sham-operated mice served as controls. TAC and sham animals were randomised and treated with either riociguat or vehicle for 5 weeks, starting 3 weeks after surgery, when cardiac hypertrophy was established. Afterwards, we performed mass spectrometric proteome analyses and microRNA sequencing of proteins and RNAs, respectively, isolated from left ventricles (LVs). KEY RESULTS: TAC-induced changes of the LV proteome were significantly reduced by treatment with riociguat. Bioinformatics analyses revealed that riociguat improved TAC-induced cardiovascular disease-related pathways, metabolism and energy production, for example, reversed alterations in the levels of myosin heavy chain 7, cardiac phospholamban and ankyrin repeat domain-containing protein 1. Riociguat also attenuated TAC-induced changes of microRNA levels in the LV. CONCLUSION AND IMPLICATIONS: The sGC stimulator riociguat exerted beneficial effects on cardiac structure and function during pressure overload, which was accompanied by a reversal of TAC-induced changes of the cardiac proteome and microRNA profile. Our data support the potential of riociguat as a novel therapeutic agent for heart failure.

Genome-wide scan identifies CDH13 as a novel susceptibility locus contributing to blood pressure determination in two European populations.

Hypertension is a complex disease that affects a large proportion of adult population. Although approximately half of the inter-individual variance in blood pressure (BP) level is heritable, identification of genes responsible for its regulation has remained challenging. Genome-wide association study (GWAS) is a novel approach to search for genetic variants contributing to complex diseases. We conducted GWAS for three BP traits [systolic and diastolic blood pressure (SBP and DBP); hypertension (HYP)] in the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) S3 cohort (n = 1644) recruited from general population in Southern Germany. GWAS with 395,912 single nucleotide polymorphisms (SNPs) identified an association between BP traits and a common variant rs11646213 (T/A) upstream of the CDH13 gene at 16q23.3. The initial associations with HYP and DBP were confirmed in two other European population-based cohorts: KORA S4 (Germans) and HYPEST (Estonians). The associations between rs11646213 and three BP traits were replicated in combined analyses (dominant model: DBP, P = 5.55 x 10(-5), effect -1.40 mmHg; SBP, P = 0.007, effect -1.56 mmHg; HYP, P = 5.30 x 10(-8), OR = 0.67). Carriers of the minor allele A had a decreased risk of hypertension. A non-significant trend for association was also detected with severe family based hypertension in the BRIGHT sample (British). The novel susceptibility locus, CDH13, encodes for an adhesion glycoprotein T-cadherin, a regulator of vascular wall remodeling and angiogenesis. Its function is compatible with the BP biology and may improve the understanding of the pathogenesis of hypertension.

The rare and the common: An Austrian DRPLA family harboring the European haplotype.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a CAG trinucleotide repeat expansion disorder with an autosomal-dominant mode of inheritance and very low prevalence in Europe. We herein report the clinical characteristics of the first Austrian DRPLA family. Genetic analysis revealed the presence of a common European haplotype, suggesting a founder mutation in Europe.

Congenital heart disease risk loci identified by genome-wide association study in European patients.

Genetic factors undoubtedly affect the development of congenital heart disease (CHD) but still remain ill defined. We sought to identify genetic risk factors associated with CHD and to accomplish a functional analysis of SNP-carrying genes. We performed a genome-wide association study (GWAS) of 4034 White patients with CHD and 8486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified 4 highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence the expression of WNT3, and the variant rs870142 related to septal defects is proposed to influence the expression of MSX1. We analyzed the expression of all 4 genes during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNA-Seq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3, and MSX1 play an essential functional role in heart development at the embryonic and newborn stages.

Evaluation of the interaction between LRRK2 and PARK16 loci in determining risk of Parkinson's disease: analysis of a large multicenter study.

A recent study MacLeod et al. has shown that an interaction between variants at the LRRK2 and PARK16 loci influences risk of development of Parkinson's disease (PD). Our study examines the proposed interaction between LRRK2 and PARK16 variants in modifying PD risk using a large multicenter series of PD patients (7715) and controls (8261) from sites participating in the Genetic Epidemiology of Parkinson's Disease Consortium. Our data does not support a strong direct interaction between LRRK2 and PARK16 variants; however, given the role of retromer and lysosomal pathways in PD, further studies are warranted.

Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine.

BACKGROUND: Familial hemiplegic migraine is an autosomal dominant severe subtype of migraine with aura characterised by some degree of hemiparesis during the attacks. So far, mutations in two genes regulating ion translocation-CACNA1A and ATP1A2-have been identified in pedigrees with this disease. METHODS: To identify additional genes for familial hemiplegic migraine, we did a genome-wide linkage analysis of two disease pedigrees without mutations in CACNA1A and ATP1A2. Ion channel genes in the candidate interval were analysed for mutations, and the functional consequences of the recorded sequence alteration were determined. FINDINGS: We identified a novel locus for familial hemiplegic migraine on chromosome 2q24. Sequencing of candidate genes in this region revealed a heterozygous missense mutation (Gln1489Lys) in the neuronal voltage-gated sodium channel gene SCN1A, mutations of which have been associated with epilepsy. This same mutation was present in three families with familial hemiplegic migraine. It results in a charge-altering aminoacid exchange in the so-called hinged-lid domain of the protein, which is critical for fast inactivation of the channel. Whole-cell recordings in transiently transfected tsA201 cells expressing the highly homologous SCN5A sodium channel showed that the mutation induces a two-fold to four-fold accelerated recovery from fast inactivation without altering any of the other channel parameters investigated. INTERPRETATION: Dysfunction of the neuronal sodium channel SCN1A can cause familial hemiplegic migraine. Our findings have implications for the understanding of migraine aura. Moreover, our study reinforces the molecular links between migraine and epilepsy, two common paroxysmal disorders.

Genomic heterogeneity of osteosarcoma - shift from single candidates to functional modules.

Osteosarcoma (OS), a bone tumor, exhibit a complex karyotype. On the genomic level a highly variable degree of alterations in nearly all chromosomal regions and between individual tumors is observable. This hampers the identification of common drivers in OS biology. To identify the common molecular mechanisms involved in the maintenance of OS, we follow the hypothesis that all the copy number-associated differences between the patients are intercepted on the level of the functional modules. The implementation is based on a network approach utilizing copy number associated genes in OS, paired expression data and protein interaction data. The resulting functional modules of tightly connected genes were interpreted regarding their biological functions in OS and their potential prognostic significance. We identified an osteosarcoma network assembling well-known and lesser-known candidates. The derived network shows a significant connectivity and modularity suggesting that the genes affected by the heterogeneous genetic alterations share the same biological context. The network modules participate in several critical aspects of cancer biology like DNA damage response, cell growth, and cell motility which is in line with the hypothesis of specifically deregulated but functional modules in cancer. Further, we could deduce genes with possible prognostic significance in OS for further investigation (e.g. EZR, CDKN2A, MAP3K5). Several of those module genes were located on chromosome 6q. The given systems biological approach provides evidence that heterogeneity on the genomic and expression level is ordered by the biological system on the level of the functional modules. Different genomic aberrations are pointing to the same cellular network vicinity to form vital, but already neoplastically altered, functional modules maintaining OS. This observation, exemplarily now shown for OS, has been under discussion already for a longer time, but often in a hypothetical manner, and can here be exemplified for OS.

Rare variants in ß-Amyloid precursor protein (APP) and Parkinson's disease.

Many individuals with Parkinson's disease (PD) develop cognitive deficits, and a phenotypic and molecular overlap between neurodegenerative diseases exists. We investigated the contribution of rare variants in seven genes of known relevance to dementias (ß-amyloid precursor protein (APP), PSEN1/2, MAPT (microtubule-associated protein tau), fused in sarcoma (FUS), granulin (GRN) and TAR DNA-binding protein 43 (TDP-43)) to PD and PD plus dementia (PD+D) in a discovery sample of 376 individuals with PD and followed by the genotyping of 25 out of the 27 identified variants with a minor allele frequency <5% in 975 individuals with PD, 93 cases with Lewy body disease on neuropathological examination, 613 individuals with Alzheimer's disease (AD), 182 cases with frontotemporal dementia and 1014 general population controls. Variants identified in APP were functionally followed up by Aß mass spectrometry in transiently transfected HEK293 cells. PD+D cases harbored more rare variants across all the seven genes than PD individuals without dementia, and rare variants in APP were more common in PD cases overall than in either the AD cases or controls. When additional controls from publically available databases were added, one rare variant in APP (c.1795G>A(p.(E599K))) was significantly associated with the PD phenotype but was not found in either the PD cases or controls of an independent replication sample. One of the identified rare variants (c.2125G>A (p.(G709S))) shifted the Aß spectrum from Aß40 to Aß39 and Aß37. Although the precise mechanism remains to be elucidated, our data suggest a possible role for APP in modifying the PD phenotype as well as a general contribution of genetic factors to the development of dementia in individuals with PD.

Large-scale assessment of polyglutamine repeat expansions in Parkinson disease.

OBJECTIVES: We aim to clarify the pathogenic role of intermediate size repeat expansions of SCA2, SCA3, SCA6, and SCA17 as risk factors for idiopathic Parkinson disease (PD). METHODS: We invited researchers from the Genetic Epidemiology of Parkinson's Disease Consortium to participate in the study. There were 12,346 cases and 8,164 controls genotyped, for a total of 4 repeats within the SCA2, SCA3, SCA6, and SCA17 genes. Fixed- and random-effects models were used to estimate the summary risk estimates for the genes. We investigated between-study heterogeneity and heterogeneity between different ethnic populations. RESULTS: We did not observe any definite pathogenic repeat expansions for SCA2, SCA3, SCA6, and SCA17 genes in patients with idiopathic PD from Caucasian and Asian populations. Furthermore, overall analysis did not reveal any significant association between intermediate repeats and PD. The effect estimates (odds ratio) ranged from 0.93 to 1.01 in the overall cohort for the SCA2, SCA3, SCA6, and SCA17 loci. CONCLUSIONS: Our study did not support a major role for definite pathogenic repeat expansions in SCA2, SCA3, SCA6, and SCA17 genes for idiopathic PD. Thus, results of this large study do not support diagnostic screening of SCA2, SCA3, SCA6, and SCA17 gene repeats in the common idiopathic form of PD. Likewise, this largest multicentered study performed to date excludes the role of intermediate repeats of these genes as a risk factor for PD.

A gene locus responsible for the familial hair shaft abnormality pili annulati maps to chromosome 12q24.32-24.33.

Pili annulati, a rare hair shaft abnormality with a characteristic shiny appearance due to alternating light and dark bands of the hair, is assumed to be inherited in an autosomal dominant mode with high penetrance. A locus for pili annulati has not been found yet. We identified one large and four small European kindreds with pili annulati and conducted a genomewide linkage analysis using 382 microsatellite markers. A multipoint logarithm of the odds (LOD) score of 3.19 was demonstrated between D12S1659 and D12S1723 on the telomeric part of the long arm of chromosome 12. Subsequent finemapping in a region of 20 cM gave a maximum multipoint LOD score of 3.24 at D12S1723 under the assumption of homogeneity and a LOD score of 3.57 around D12S343 under the assumption of heterogeneity, both exceed the statistical thresholds necessary to conclude linkage. Most of this LOD score came from the largest family, which reached a maximum LOD score of 3.81. The maximum two-point LOD score for all families was 3.97 at D12S1609. Definite recombination events narrowed the region of shared haplotype in the affected individuals to an 8 Mb region between the marker D12S324 and the telomeric end of the long arm of chromosome 12.