Understanding mutational effects in digenic diseases.

To further our understanding of the complexity and genetic heterogeneity of rare diseases, it has become essential to shed light on how combinations of variants in different genes are responsible for a disease phenotype. With the appearance of a resource on digenic diseases, it has become possible to evaluate how digenic combinations differ in terms of the phenotypes they produce. All instances in this resource were assigned to two classes of digenic effects, annotated as true digenic and composite classes. Whereas in the true digenic class variants in both genes are required for developing the disease, in the composite class, a variant in one gene is sufficient to produce the phenotype, but an additional variant in a second gene impacts the disease phenotype or alters the age of onset. We show that a combination of variant, gene and higher-level features can differentiate between these two classes with high accuracy. Moreover, we show via the analysis of three digenic disorders that a digenic effect decision profile, extracted from the predictive model, motivates why an instance was assigned to either of the two classes. Together, our results show that digenic disease data generates novel insights, providing a glimpse into the oligogenic realm.

DIDA: A curated and annotated digenic diseases database.

DIDA (DIgenic diseases DAtabase) is a novel database that provides for the first time detailed information on genes and associated genetic variants involved in digenic diseases, the simplest form of oligogenic inheritance. The database is accessible via http://dida.ibsquare.be and currently includes 213 digenic combinations involved in 44 different digenic diseases. These combinations are composed of 364 distinct variants, which are distributed over 136 distinct genes. The web interface provides browsing and search functionalities, as well as documentation and help pages, general database statistics and references to the original publications from which the data have been collected. The possibility to submit novel digenic data to DIDA is also provided. Creating this new repository was essential as current databases do not allow one to retrieve detailed records regarding digenic combinations. Genes, variants, diseases and digenic combinations in DIDA are annotated with manually curated information and information mined from other online resources. Next to providing a unique resource for the development of new analysis methods, DIDA gives clinical and molecular geneticists a tool to find the most comprehensive information on the digenic nature of their diseases of interest.

Prolonged Right Ventricular Ejection Delay in Brugada Syndrome Depends on the Type of SCN5A Variant　- Electromechanical Coupling Through Tissue Velocity Imaging as a Bridge Between Genotyping and Phenotyping.

BACKGROUND: Patients with Brugada syndrome (BrS) and a history of syncope or sustained ventricular arrhythmia have longer right ventricular ejection delays (RVEDs) than asymptomatic BrS patients. Different types ofSCN5Avariants leading to different reductions in sodium current (INa) may have different effects on conduction delay, and consequently on electromechanical coupling (i.e., RVED). Thus, we investigated the genotype-phenotype relationship by measuring RVED to establish whether BrS patients carrying more severeSCN5Avariants leading to premature protein truncation (T) and presumably 100%INareduction have a longer RVED than patients carrying missense variants (M) with different degrees ofINareduction.Methods and Results:There were 34 BrS patients (mean [±SD] age 43.3±12.9 years; 52.9% male) carrying anSCN5Avariant and 66 non-carriers in this cross-sectional study. Patients carrying aSCN5Avariant were divided into T-carriers (n=13) and M-carriers (n=21). Using tissue velocity imaging, RVED and left ventricular ejection delay (LVED) were measured as the time from QRS onset to the onset of the systolic ejection wave at the end of the isovolumetric contraction. T-carriers had longer RVEDs than M-carriers (139.3±15.1 vs. 124.8±11.9 ms, respectively; P=0.008) and non-carriers (127.7±17.3 ms, P=0.027). There were no differences in LVED among groups. CONCLUSIONS: Using the simple, non-invasive echocardiographic parameter RVED revealed a more pronounced 'electromechanical' delay in BrS patients carrying T variants ofSCN5A.

Convert your favorite protein modeling program into a mutation predictor: "MODICT".

BACKGROUND: Predict whether a mutation is deleterious based on the custom 3D model of a protein. RESULTS: We have developed MODICT, a mutation prediction tool which is based on per residue RMSD (root mean square deviation) values of superimposed 3D protein models. Our mathematical algorithm was tested for 42 described mutations in multiple genes including renin (REN), beta-tubulin (TUBB2B), biotinidase (BTD), sphingomyelin phosphodiesterase-1 (SMPD1), phenylalanine hydroxylase (PAH) and medium chain Acyl-Coa dehydrogenase (ACADM). Moreover, MODICT scores corresponded to experimentally verified residual enzyme activities in mutated biotinidase, phenylalanine hydroxylase and medium chain Acyl-CoA dehydrogenase. Several commercially available prediction algorithms were tested and results were compared. The MODICT PERL package and the manual can be downloaded from https://github.com/IbrahimTanyalcin/MODICT . CONCLUSIONS: We show here that MODICT is capable tool for mutation effect prediction at the protein level, using superimposed 3D protein models instead of sequence based algorithms used by POLYPHEN and SIFT.

Contribution of Cardiac Sodium Channel ß-Subunit Variants to Brugada Syndrome.

BACKGROUND: Brugada syndrome (BrS) is an inheritable cardiac disease associated with syncope, malignant ventricular arrhythmias and sudden cardiac death. The largest proportion of mutations in BrS is found in the SCN5A gene encoding the α-subunit of cardiac sodium channels (Nav1.5). Causal SCN5A mutations are present in 18-30% of BrS patients. The additional genetic diagnostic yield of variants in cardiac sodium channel ß-subunits in BrS patients was explored and functional studies on 3 novel candidate variants were performed. METHODS AND RESULTS: TheSCN1B-SCN4B genes were screened, which encode the 5 sodium channel ß-subunits, in a SCN5A negative BrS population (n=74). Five novel variants were detected; in silico pathogenicity prediction classified 4 variants as possibly disease causing. Three variants were selected for functional study. These variants caused only limited alterations of Nav1.5 function. Next generation sequencing of a panel of 88 arrhythmia genes could not identify other major causal mutations. CONCLUSIONS: It was hypothesized that the studied variants are not the primary cause of BrS in these patients. However, because small functional effects of these ß-subunit variants can be discriminated, they might contribute to the BrS phenotype and be considered a risk factor. The existence of these risk factors can give an explanation to the reduced penetrance and variable expressivity seen in this syndrome. We therefore recommend including the SCN1-4B genes in a next generation sequencing-based gene panel.

An exome-wide study of renal operational tolerance.

Background: Renal operational tolerance is a rare and beneficial state of prolonged renal allograft function in the absence of immunosuppression. The underlying mechanisms are unknown. We hypothesized that tolerance might be driven by inherited protein coding genetic variants with large effect, at least in some patients. Methods: We set up a European survey of over 218,000 renal transplant recipients and collected DNAs from 40 transplant recipients who maintained good allograft function without immunosuppression for at least 1 year. We performed an exome-wide association study comparing the distribution of moderate to high impact variants in 36 tolerant patients, selected for genetic homogeneity using principal component analysis, and 192 controls, using an optimal sequence-kernel association test adjusted for small samples. Results: We identified rare variants of HOMER2 (3/36, FDR 0.0387), IQCH (5/36, FDR 0.0362), and LCN2 (3/36, FDR 0.102) in 10 tolerant patients vs. 0 controls. One patient carried a variant in both HOMER2 and LCN2. Furthermore, the three genes showed an identical variant in two patients each. The three genes are expressed at the primary cilium, a key structure in immune responses. Conclusion: Rare protein coding variants are associated with operational tolerance in a sizable portion of patients. Our findings have important implications for a better understanding of immune tolerance in transplantation and other fields of medicine.ClinicalTrials.gov, identifier: NCT05124444.

Novel inactivating follicle-stimulating hormone receptor mutations in a patient with premature ovarian insufficiency identified by next-generation sequencing gene panel analysis.

OBJECTIVE: To find the genetic etiology of premature ovarian insufficiency (POI) in a patient with primary amenorrhea and hypergonadotropic hypogonadism. DESIGN: Case report. SETTING: University hospital. PATIENTS: A Belgian woman aged 32 years with POI at the age of 17, her parents, and her sister whose POI was diagnosed at age 29. INTERVENTIONS: Analysis of a panel of 31 genes implicated in POI (POIGP) using next-generation sequencing (NGS), Sanger sequencing, and in vitro functional study. MAIN OUTCOME MEASURES: Gene variants, family mutational segregation, and in vitro functional impact of the mutant proteins. RESULTS: The analysis of the gene panel using NGS identified the presence of two novel follicle-stimulating hormone receptor (FSHR) missense mutations at a compound heterozygous state in the affected patient: c.646 G>A, p.Gly216Arg, and c.1313C>T, p.Thr438Ile. Sanger sequencing showed the presence of each mutation at heterozygous state in the patient's parents and at heterozygous compound state in the affected sister. Both substituted amino acids (Gly216 and Thr438) were conserved in FSHR of several vertebrate species as well as in other glycoproteins receptors (TSHR and LHCGHR), suggesting a potentially important role in glycoprotein receptor function. An in vitro functional study showed similar results for both variants with more than 90% reduction of their cell surface expression and a 55% reduction of their FSH-induced cyclic adenosine 3':5' monophosphate (cAMP) production compared with the wild-type FSHR. CONCLUSIONS: The analysis of a gene panel of 31 genes implicated in POI allowed us to identify two novel partially inactivating mutations of FSHR that are likely responsible for the POI phenotype of the proband and of her affected sister.

Detection of Heteroplasmic Variants in the Mitochondrial Genome through Massive Parallel Sequencing.

Detecting heteroplasmies in the mitochondrial DNA (mtDNA) has been a challenge for many years. In the past, Sanger sequencing was the main option to perform this analysis, however, this method could not detect low frequency heteroplasmies. Massive Parallel Sequencing (MPS) provides the opportunity to study the mtDNA in depth, but a controlled pipeline is necessary to reliably retrieve and quantify the low frequency variants. It has been shown that differences in methods can significantly affect the number and frequency of the retrieved variants. In this protocol, we present a method involving both wet lab and bioinformatics that allows identifying and quantifying single nucleotide variants in the full mtDNA sequence, down to a heteroplasmic load of 1.5%. For this, we set up a PCR-based amplification of the mtDNA, followed by MPS using Illumina chemistry, and variant calling with two different algorithms, mtDNA server and Mutect. The PCR amplification is used to enrich the mitochondrial fraction, while the bioinformatic processing with two algorithms is used to discriminate the true heteroplasmies from background noise. The protocol described here allows for deep sequencing of the mitochondrial DNA in bulk DNA samples as well as single cells (both large cells such as human oocytes, and small-sized single cells such as human embryonic stem cells) with minor modifications to the protocol.

Accurate and comprehensive analysis of single nucleotide variants and large deletions of the human mitochondrial genome in DNA and single cells.

Massive parallel sequencing (MPS) can accurately quantify mitochondrial DNA (mtDNA) single nucleotide variants (SNVs), but no MPS methods are currently validated to simultaneously and accurately establish the breakpoints and frequency of large deletions at low heteroplasmic loads. Here we present the thorough validation of an MPS protocol to quantify the load of very low frequency, large mtDNA deletions in bulk DNA and single cells, along with SNV calling by standard methods. We used a set of well-characterized DNA samples, DNA mixes and single cells to thoroughly control the study. We developed a custom script for the detection of mtDNA rearrangements that proved to be more accurate in detecting and quantifying deletions than pre-existing tools. We also show that PCR conditions and primersets must be carefully chosen to avoid biases in the retrieved variants and an increase in background noise, and established a lower detection limit of 0.5% heteroplasmic load for large deletions, and 1.5 and 2% for SNVs, for bulk DNA and single cells, respectively. Finally, the analysis of different single cells provided novel insights into mtDNA cellular mosaicism.

SCN4A variants and Brugada syndrome: phenotypic and genotypic overlap between cardiac and skeletal muscle sodium channelopathies.

SCN5A mutations involving the α-subunit of the cardiac voltage-gated muscle sodium channel (NaV1.5) result in different cardiac channelopathies with an autosomal-dominant inheritance such as Brugada syndrome. On the other hand, mutations in SCN4A encoding the α-subunit of the skeletal voltage-gated sodium channel (NaV1.4) cause non-dystrophic myotonia and/or periodic paralysis. In this study, we investigated whether cardiac arrhythmias or channelopathies such as Brugada syndrome can be part of the clinical phenotype associated with SCN4A variants and whether patients with Brugada syndrome present with non-dystrophic myotonia or periodic paralysis and related gene mutations. We therefore screened seven families with different SCN4A variants and non-dystrophic myotonia phenotypes for Brugada syndrome and performed a neurological, neurophysiological and genetic work-up in 107 Brugada families. In the families with an SCN4A-associated non-dystrophic myotonia, three patients had a clinical diagnosis of Brugada syndrome, whereas we found a remarkably high prevalence of myotonic features involving different genes in the families with Brugada syndrome. One Brugada family carried an SCN4A variant that is predicted to probably affect function, one family suffered from a not genetically confirmed non-dystrophic myotonia, one family was diagnosed with myotonic dystrophy (DMPK gene) and one family had a Thomsen disease myotonia congenita (CLCN1 variant that affects function). Our findings and data suggest a possible involvement of SCN4A variants in the pathophysiological mechanism underlying the development of a spontaneous or drug-induced type 1 electrocardiographic pattern and the occurrence of malignant arrhythmias in some patients with Brugada syndrome.

Signatures of selection in the three-spined stickleback along a small-scale brackish water - freshwater transition zone.

Local adaptation is often obvious when gene flow is impeded, such as observed at large spatial scales and across strong ecological contrasts. However, it becomes less certain at small scales such as between adjacent populations or across weak ecological contrasts, when gene flow is strong. While studies on genomic adaptation tend to focus on the former, less is known about the genomic targets of natural selection in the latter situation. In this study, we investigate genomic adaptation in populations of the three-spined stickleback Gasterosteus aculeatus L. across a small-scale ecological transition with salinities ranging from brackish to fresh. Adaptation to salinity has been repeatedly demonstrated in this species. A genome scan based on 87 microsatellite markers revealed only few signatures of selection, likely owing to the constraints that homogenizing gene flow puts on adaptive divergence. However, the detected loci appear repeatedly as targets of selection in similar studies of genomic adaptation in the three-spined stickleback. We conclude that the signature of genomic selection in the face of strong gene flow is weak, yet detectable. We argue that the range of studies of genomic divergence should be extended to include more systems characterized by limited geographical and ecological isolation, which is often a realistic setting in nature.

Prolonged right ventricular ejection delay identifies high risk patients and gender differences in Brugada syndrome.

BACKGROUND AND OBJECTIVES: Right ventricular (RV) conduction delay has been suggested as an underlying pathophysiological mechanism in Brugada syndrome (BS). In this cross-sectional study we non-invasively assessed the value of echocardiographic markers reflecting ventricular ejection delay to further assess electromechanical abnormalities in BS and to identify patients at risk for life-threatening arrhythmic events. Furthermore, we sought to assess differences in ejection delays between genders because male BS patients demonstrate a more malignant clinical phenotype. METHODS: 124 BS patients (57.3% males) and 62 controls (CTR) (48.4% males) were included. Using Tissue Velocity Imaging, the ejection delay, determined as the time from QRS onset to the onset of the sustained systolic contraction, was measured for both RV free wall (RVED) and lateral LV wall (LVED). From these parameters, the interventricular ejection delay between both walls (IVED) was calculated. RESULTS: BS patients had longer RVEDs and IVEDs compared to the CTR. BS patients with a previous history of syncope or spontaneous ventricular arrhythmia showed the longest RVEDs and IVEDs. Male BS patients demonstrated longer RVEDs and IVEDs than females. Male BS patients with malignant events had the longest delays. No significant differences regarding LVED were observed between BS patients and CTR. CONCLUSIONS: We demonstrated that a previous history of malignant events was associated with longer RVEDs. Our findings supported the RV conduction delay mechanism behind BS and demonstrated for the first time that the predominant malignant male Brugada phenotype might also be the result of a more delayed RV conduction in males.