Bioinformatic approaches to determine pathogenicity and function of clinical genetic variants across ion channels and neurodevelopmental disorder associated genes

Clinical genetic testing for rare monogenic diseases has the scope of identifying the disease-causing variants. Identification of the molecular etiology of the disease can already today improve clinical care and is essential for the administration of precision medicines that are currently in development for many disorders. However, distinguishing pathogenic variants from benign genetic variants remains a challenge – in particular for missense variants where a single amino acid is substituted. The effects of a pathogenic variant on the protein function, for example, whether it causes a gain (GoF) or a loss (LoF) of the protein function, is most of the time not understood since most genetic variants are ultra-rare and have not been molecularly tested. In particular, for genes associated with severe developmental disorders, first-generation symptomatic treatments offer often only limited relief. Consequently, the development and application of targeted treatments that promise improvement is urgently needed. Identifying the disease-causing pathogenic and predicting their function is crucial as targeted therapies can only be administered to patients with classified pathogenic variants whose functional effects are known to avoid adverse treatment outcomes. In this dissertation, I present bioinformatic approaches to enhance the assessment of variant pathogenicity and understanding of the functional effects of genetic variants. The developed approaches were applied on an exome-wide scale using public datasets and for selected disorders for which I had expert-curated clinical-genetic data available from collaborators. The major focus of this thesis is on genes implicated in neurodevelopmental disorders and diseases associated with ion channel dysfunction for which collaboration with other research groups enabled the aggregation of required genetic, clinical, and functional datasets to develop and test the bioinformatic approaches. In the first study (Bruenger and Ivaniuk et al., in preparation for submission to Genetics in Medicine), we developed a novel approach to extend the application of current variant interpretation guidelines as proposed by the American College of Medical Genetics and Genomics (ACMG). Currently, a major limitation of interpreting the pathogenicity of variants with the ACMG guidelines presents the rare applicability of some of the proposed evidence criteria. We evaluated the potential of incorporating individual pathogenic variants observed in paralogous genes to extend the applicability of two criteria of the guidelines. Our results demonstrated that pathogenic variants in evolutionarily conserved paralogous genes can serve as evidence for a variant's pathogenicity and thus extend the current criteria's applicability by more than four times. We further explored whether the selection of the paralogous pathogenic variants can be improved by incorporating phenotype information. We assembled a clinically well-defined cohort of patients with variants in voltage-gated sodium channels (VGSC) and identified phenotype correlations among paralogous genes based on the shared variant properties. By integrating these phenotype correlations into our proposed extension of the ACMG criteria, we demonstrated an enhanced ability to provide evidence for the pathogenicity of genetic variants in VGSC-encoding genes. In the second study (Brunklaus, Feng, and Bruenger et al., Brain, 2022), we examined whether experimentally obtained functional effects of variants in one VGSC encoding gene could predict function in conserved variants in paralogous genes with high sequence similarity. We aggregated 437 in-vitro functionally tested variants from an intensive literature search and found that the functional effect across conserved variants in paralogous genes was conserved in 94% of cases. Our findings represent the first GoF versus LoF topological map of VGSC proteins, which could guide precision therapy as functionally tested variants are rare across VGSC. We integrated our findings into a publicly accessible webtool (http://SCN-viewer.broadinstitute.org) to facilitate functional variant interpretation across VGSC. In the third study (Bruenger et al., Brain, 2022), we systematically identified biological properties associated with variant pathogenicity across all major voltage and ligand-gated ion-channel families. We discovered and independently replicated that several pore residue properties and proximity to the pore axis were significantly enriched for pathogenic variants compared to population variants across all ion channels. Using a newly developed structural framework, we provide quantitative evidence that variants at the pore showed the strongest pathogenic variant enrichment. Moreover, we found that a hydrophobic pore environment was most strongly associated with variant pathogenicity. Finally, we showed that the identified biological properties correlated with in-vitro functional readouts from 679 variants and clinical phenotypes in 1,422 patients with neurodevelopmental disorders which were collected through collaboration with other research groups. In summary, we identified biological properties associated with ion-channel malfunction and show that these are correlated with in vitro functional readouts and clinical phenotypes in patients with neurodevelopmental disorders. Our results suggest that clinical decision support algorithms that predict variant pathogenicity and function are feasible in the future. In the fourth study (Iqbal and Bruenger et al., Brain, 2022), we developed a novel consensus approach that combines evolutionary and population-based genomic scores to identify 3D essential sites (Essential3D) on protein structures encoded by genes associated with neurodevelopmental disorders (NDDs). NDDs encompass severe clinical conditions caused by pathogenic variants in different genes. However, many of those genes were just recently associated with NDDs and are not well studied. We identified 14,377 Essential3D sites on protein structures encoded by 189 genes and found that these sites were eight-fold enriched for pathogenic versus population controls in an independent cohort of over 360,000 patient and population variants. The Essential3D sites offer insights into molecular mechanisms of protein function, such as key protein-protein interaction sites. The provided annotations are available at https://es-ndd.broadinstitute.org and will guide clinical variant interpretation. In summary, within these major studies in my Ph.D., we aggregated genetic, clinical, and functional datasets and developed bioinformatic approaches to enhance the assessment of variant pathogenicity and improve understanding of the functional effects of genetic variants on protein function. The advances made during my Ph.D. research demonstrate the power of integrating multiple data sources to study novel genetic variants and their implication for rare monogenic diseases. Our approaches specifically improve variant function and pathogenicity assessment in genes implicated in several severe diseases for which currently applied first-generation therapies cannot adequately lower the disease burden. Thus, our results contribute to a new era in precision medicine, where personalized treatments and improved clinical care become increasingly accessible to patients. Finally, the annotations developed in these can serve as a foundation for further studies, including the application of machine learning methods to predict variant pathogenicity and protein functional effects more accurately

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis

Epilepsy; Genetics; Neurodevelopmental disorderEpilèpsia; Genètica; Trastorn del neurodesenvolupamentEpilepsia; Genética; Trastorno del neurodesarrolloGenetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10−3, 95% confidence interval: 1.5–15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).D.L.’s work was supported by funds from the Dravet Syndrome Foundation (grant number, 272016), the BMBF (Treat-ION grant, 01GM1907), National Institute of Neurological Disorders and Stroke (Channelopathy-Associated Epilepsy Research Center, 5-U54-NS108874). E.P-P. is supported by Chilean National Agency for Investigation and Development, ANID Fondecyt grant 1221464 and the FamilieSCN2A foundation 2020 Action Potential Grant. P.M. received support by the BMBF (Treat-Ion2, 01GM2210B) and the Fonds National de la Recherche Luxembourg in Luxembourg (Research Unit FOR-2715, FNR grant NTER/DFG/21/16394868 MechEPI2)

Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases

CNV-ClinViewer: Enhancing the clinical interpretation of large copy-number variants online

Purpose Large copy number variants (CNVs) can cause a heterogeneous spectrum of rare and severe disorders. However, most CNVs are benign and are part of natural variation in human genomes. CNV pathogenicity classification, genotype-phenotype analyses, and therapeutic target identification are challenging and time-consuming tasks that require the integration and analysis of information from multiple scattered sources by experts. Methods We developed a web-application combining >250,000 patient and population CNVs together with a large set of biomedical annotations and provide tools for CNV classification based on ACMG/ClinGen guidelines and gene-set enrichment analyses. Results Here, we introduce the CNV-ClinViewer (https://cnv-ClinViewer.broadinstitute.org), an open-source web-application for clinical evaluation and visual exploration of CNVs. The application enables real-time interactive exploration of large CNV datasets in a user-friendly designed interface. Conclusion Overall, this resource facilitates semi-automated clinical CNV interpretation and genomic loci exploration and, in combination with clinical judgment, enables clinicians and researchers to formulate novel hypotheses and guide their decision-making process. Subsequently, the CNV-ClinViewer enhances for clinical investigators patient care and for basic scientists translational genomic research

Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design

Severe communication delays are independent of seizure burden and persist despite contemporary treatments in SCN1A + Dravet syndrome: Insights from the ENVISION natural history study

Objective: Dravet syndrome (DS) is a developmental and epileptic encephalopathy characterized by high seizure burden, treatment‐resistant epilepsy, and developmental stagnation. Family members rate communication deficits among the most impactful disease manifestations. We evaluated seizure burden and language/communication development in children with DS. Methods: ENVISION was a prospective, observational study evaluating children with DS associated with SCN1A pathogenic variants (SCN1A+ DS) enrolled at age ≤5 years. Seizure burden and antiseizure medications were assessed every 3 months and communication and language every 6 months with the Bayley Scales of Infant and Toddler Development 3rd edition and the parent‐reported Vineland Adaptive Behavior Scales 3rd edition. We report data from the first year of observation, including analyses stratified by age at Baseline: 0:6–2:0 years:months (Y:M; youngest), 2:1–3:6 Y:M (middle), and 3:7–5:0 Y:M (oldest). Results: Between December 2020 and March 2023, 58 children with DS enrolled at 16 sites internationally. Median follow‐up was 17.5 months (range = .0–24.0), with 54 of 58 (93.1%) followed for at least 6 months and 51 of 58 (87.9%) for 12 months. Monthly countable seizure frequency (MCSF) increased with age (median [minimum–maximum] = 1.0 in the youngest [1.0–70.0] and middle [1.0–242.0] age groups and 4.5 [.0–2647.0] in the oldest age group), and remained high, despite use of currently approved antiseizure medications. Language/communication delays were observed early, and developmental stagnation occurred after age 2 years with both instruments. In predictive modeling, chronologic age was the only significant covariate of seizure frequency (effect size = .52, p = .024). MCSF, number of antiseizure medications, age at first seizure, and convulsive status epilepticus were not predictors of language/communication raw scores. Significance: In infants and young children with SCN1A+ DS, language/communication delay and stagnation were independent of seizure burden. Our findings emphasize that the optimal therapeutic window to prevent language/communication delay is before 3 years of age

Pathogenic paralogous variants can be used to apply the ACMG PS1 and PM5 variant interpretation criteria 2023.08.22.23294353

Purpose The majority of missense variants in clinical genetic tests are classified as variants of uncertain significance. Broadening the evidence of the PS1 and PM5 criteria has the potential to increase conclusive variant interpretation. Methods We hypothesized that incorporation of pathogenic missense variants in conserved residues across paralogous genes can increase the number of variants where ACMG PS1/PM5 criteria can be applied. We mapped over 2.5 million pathogenic and general population variants from ClinVar, HGMD, and gnomAD databases onto 9,990 genes and aligned these by gene families. Subsequently, we developed a novel framework to extend PS1/PM5 by incorporating pathogenic paralogous variants annotations (para-PS1/PM5). Results We demonstrate that para-PS1/PM5 criteria increase the number of classifiable amino acids 3.6-fold compared to PS1 and PM5. Across all gene families with at least two disease-associated genes, the calculated likelihood ratios suggest moderate evidence for pathogenicity. Moreover, for 36 genes, the extended para-PS1/PM5 criteria reach strong evidence level. Conclusion We show that single pathogenic paralogous variants incorporation at paralogous protein positions increases the applicability of the PS1 and PM5 criteria, likely leading to a reduction of variants of uncertain significance across many monogenic disorders. Future iterations of the ACMG guidelines may consider para-PS1 and para-PM5

Genome-wide identification and phenotypic characterization of seizure-associated copy number variations in 741,075 individuals.

Copy number variants (CNV) are established risk factors for neurodevelopmental disorders with seizures or epilepsy. With the hypothesis that seizure disorders share genetic risk factors, we pooled CNV data from 10,590 individuals with seizure disorders, 16,109 individuals with clinically validated epilepsy, and 492,324 population controls and identified 25 genome-wide significant loci, 22 of which are novel for seizure disorders, such as deletions at 1p36.33, 1q44, 2p21-p16.3, 3q29, 8p23.3-p23.2, 9p24.3, 10q26.3, 15q11.2, 15q12-q13.1, 16p12.2, 17q21.31, duplications at 2q13, 9q34.3, 16p13.3, 17q12, 19p13.3, 20q13.33, and reciprocal CNVs at 16p11.2, and 22q11.21. Using genetic data from additional 248,751 individuals with 23 neuropsychiatric phenotypes, we explored the pleiotropy of these 25 loci. Finally, in a subset of individuals with epilepsy and detailed clinical data available, we performed phenome-wide association analyses between individual CNVs and clinical annotations categorized through the Human Phenotype Ontology (HPO). For six CNVs, we identified 19 significant associations with specific HPO terms and generated, for all CNVs, phenotype signatures across 17 clinical categories relevant for epileptologists. This is the most comprehensive investigation of CNVs in epilepsy and related seizure disorders, with potential implications for clinical practice

Conserved patterns across ion channels correlate with variant pathogenicity and clinical phenotypes 2022.03.23.485339

Clinically identified genetic variants in ion channels can be benign or cause disease by increasing or decreasing the protein function. Consequently, therapeutic decision-making is challenging without molecular testing of each variant. Our biophysical knowledge of ion channel structures and function is just emerging, and it is currently not well understood which amino acid residues cause disease when mutated.We sought to systematically identify biological properties associated with variant pathogenicity across all major voltage and ligand-gated ion channel families. We collected and curated 3,049 pathogenic variants from hundreds of neurodevelopmental and other disorders and 12,546 population variants for 30 ion channel or channel subunits for which a high-quality protein structure was available. Using a wide range of bioinformatics approaches, we computed 163 structural features and tested them for pathogenic variant enrichment. We developed a novel 3D spatial distance scoring approach that enables comparisons of pathogenic and population variant distribution across protein structures.We discovered and independently replicated that several pore residue properties and proximity to the pore axis were most significantly enriched for pathogenic variants compared to population variants. Using our novel 3D scoring approach, we showed that the strongest pathogenic variant enrichment was observed for pore-lining residues and alpha-helix residues within 5 A distance from the pore axis center and not involved in gating. Within the subset of residues located at the pore, the hydrophobicity of the pore was the feature most strongly associated with variant pathogenicity. We also found an association between the identified properties and both clinical phenotypes and fucntional in vitro assays for voltage-gated sodium channels (SCN1A, SCN2A, SCN8A) and N-methyl-D-aspartate (NMDA) receptor (GRIN1, GRIN2A, GRIN2B) encoding genes. In an independent expert-curated dataset of 1,422 neurodevelopmental disorder pathogenic patient variants, and 679 electrophysiological experiments that pore axis distance is associated with seizure age of onset and cognitive performance as well as differential gain vs. loss-of-channel function.In summary, we identified biological properties associated with ion-channel malfunction and show that these are correlated with in vitro functional read-outs and clinical phenotypes in patients with neurodevelopmental disorders. Our results suggest that clinical decision support algorithms that predict variant pathogenicity and function are feasible in the future.Competing Interest StatementThe authors have declared no competing interest.DSSPDictionary of Protein Secondary StructuregnomADGenome aggregation DatabaseGoFGain of functionGRIN genesGRIN1, GRIN2A. GRIN2BHGMDHuman Gene Mutation DatabaseNMDA receptorN-methyl-D-aspartate receptorGABA receptorGamma-aminobutyric acid receptorLoFLoss of functionSCN genesSCN1A, SCN2A, SCN8AVCFVariant Call Forma

Conserved patterns across ion channels correlate with variant pathogenicity and clinical phenotypes

Clinically identified genetic variants in ion channels can be benign or cause disease by increasing or decreasing the protein function. Consequently, therapeutic decision-making is challenging without molecular testing of each variant. Our biophysical knowledge of ion channel structures and function is just emerging, and it is currently not well understood which amino acid residues cause disease when mutated.We sought to systematically identify biological properties associated with variant pathogenicity across all major voltage and ligand-gated ion channel families. We collected and curated 3,049 pathogenic variants from hundreds of neurodevelopmental and other disorders and 12,546 population variants for 30 ion channel or channel subunits for which a high-quality protein structure was available. Using a wide range of bioinformatics approaches, we computed 163 structural features and tested them for pathogenic variant enrichment. We developed a novel 3D spatial distance scoring approach that enables comparisons of pathogenic and population variant distribution across protein structures.We discovered and independently replicated that several pore residue properties and proximity to the pore axis were most significantly enriched for pathogenic variants compared to population variants. Using our 3D scoring approach, we showed that the strongest pathogenic variant enrichment was observed for pore-lining residues and alpha-helix residues within 5Å distance from the pore axis center and not involved in gating. Within the subset of residues located at the pore, the hydrophobicity of the pore was the feature most strongly associated with variant pathogenicity. We also found an association between the identified properties and both clinical phenotypes and functional in vitro assays for voltage-gated sodium channels (SCN1A, SCN2A, SCN8A) and N-methyl-D-aspartate (NMDA) receptor (GRIN1, GRIN2A, GRIN2B) encoding genes. In an independent expert-curated dataset of 1,422 neurodevelopmental disorder pathogenic patient variants and 679 electrophysiological experiments, we show that pore axis distance is associated with seizure age of onset and cognitive performance as well as differential gain vs. loss-of-channel function.In summary, we identified biological properties associated with ion-channel malfunction and show that these are correlated with in vitro functional read-outs and clinical phenotypes in patients with neurodevelopmental disorders. Our results suggest that clinical decision support algorithms that predict variant pathogenicity and function are feasible in the future