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

in Brain: a Journal of Neurology (2023)

Genetic 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 ... [more ▼]

Genetic 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 vs. non-severe disease (P = 2.9e-3, 95% CI: 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/). [less ▲]

Epilepsies of presumed genetic etiology show enrichment of rare variants that occur in the general population

in American Journal of Human Genetics (2023)

Previous studies suggested that severe epilepsies, e.g., developmental and epileptic encephalopathies (DEEs), are mainly caused by ultra-rare de novo genetic variants. For milder disease, rare genetic ... [more ▼]

Previous studies suggested that severe epilepsies, e.g., developmental and epileptic encephalopathies (DEEs), are mainly caused by ultra-rare de novo genetic variants. For milder disease, rare genetic variants could contribute to the phenotype. To determine the importance of rare variants for different epilepsy types, we analyzed a whole-exome sequencing cohort of 9,170 epilepsy-affected individuals and 8,436 control individuals. Here, we separately analyzed three different groups of epilepsies: severe DEEs, genetic generalized epilepsy (GGE), and non-acquired focal epilepsy (NAFE). We required qualifying rare variants (QRVs) to occur in control individuals with an allele count R 1 and a minor allele frequency % 1:1,000, to be predicted as deleterious (CADD R 20), and to have an odds ratio in individuals with epilepsy R 2. We identified genes enriched with QRVs primarily in NAFE (n ¼ 72), followed by GGE (n ¼ 32) and DEE (n ¼ 21). This suggests that rare variants may play a more important role for causality of NAFE than for DEE. Moreover, we found that genes harboring QRVs, e.g., HSGP2, FLNA, or TNC, encode proteins that are involved in structuring the brain extracellular matrix. The present study confirms an involvement of rare variants for NAFE that occur also in the general population, while in DEE and GGE, the contribution of such variants appears more limited. [less ▲]

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

in Brain: a Journal of Neurology (2023), 146(3), 923-934

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 ... [more ▼]

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. [less ▲]

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

E-print/Working paper (2022)

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 ... [more ▼]

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 Format [less ▲]

Sub-genic intolerance, ClinVar, and the epilepsies: A whole-exome sequencing study of 29,165 individuals

in The American Journal of Human Genetics (2021)

Summary Both mild and severe epilepsies are influenced by variants in the same genes, yet an explanation for the resulting phenotypic variation is unknown. As part of the ongoing Epi25 Collaboration, we ... [more ▼]

Summary Both mild and severe epilepsies are influenced by variants in the same genes, yet an explanation for the resulting phenotypic variation is unknown. As part of the ongoing Epi25 Collaboration, we performed a whole-exome sequencing analysis of 13,487 epilepsy-affected individuals and 15,678 control individuals. While prior Epi25 studies focused on gene-based collapsing analyses, we asked how the pattern of variation within genes differs by epilepsy type. Specifically, we compared the genetic architectures of severe developmental and epileptic encephalopathies (DEEs) and two generally less severe epilepsies, genetic generalized epilepsy and non-acquired focal epilepsy (NAFE). Our gene-based rare variant collapsing analysis used geographic ancestry-based clustering that included broader ancestries than previously possible and revealed novel associations. Using the missense intolerance ratio (MTR), we found that variants in DEE-affected individuals are in significantly more intolerant genic sub-regions than those in NAFE-affected individuals. Only previously reported pathogenic variants absent in available genomic datasets showed a significant burden in epilepsy-affected individuals compared with control individuals, and the ultra-rare pathogenic variants associated with DEE were located in more intolerant genic sub-regions than variants associated with non-DEE epilepsies. MTR filtering improved the yield of ultra-rare pathogenic variants in affected individuals compared with control individuals. Finally, analysis of variants in genes without a disease association revealed a significant burden of loss-of-function variants in the genes most intolerant to such variation, indicating additional epilepsy-risk genes yet to be discovered. Taken together, our study suggests that genic and sub-genic intolerance are critical characteristics for interpreting the effects of variation in genes that influence epilepsy. [less ▲]

Predicting functional effects of missense variants in voltage-gated sodium and calcium channels

in Science Translational Medicine (2020), 12(556), 6848

Malfunctions of voltage-gated sodium and calcium channels (encoded by SCNxA and CACNA1x family genes, respectively) have been associated with severe neurologic, psychiatric, cardiac, and other diseases ... [more ▼]

Malfunctions of voltage-gated sodium and calcium channels (encoded by SCNxA and CACNA1x family genes, respectively) have been associated with severe neurologic, psychiatric, cardiac, and other diseases. Altered channel activity is frequently grouped into gain or loss of ion channel function (GOF or LOF, respectively) that often corresponds not only to clinical disease manifestations but also to differences in drug response. Experimental studies of channel function are therefore important, but laborious and usually focus only on a few variants at a time. On the basis of known gene-disease mechanisms of 19 different diseases, we inferred LOF (n = 518) and GOF (n = 309) likely pathogenic variants from the disease phenotypes of variant carriers. By training a machine learning model on sequence- and structure-based features, we predicted LOF or GOF effects [area under the receiver operating characteristics curve (ROC) = 0.85] of likely pathogenic missense variants. Our LOF versus GOF prediction corresponded to molecular LOF versus GOF effects for 87 functionally tested variants in SCN1/2/8A and CACNA1I (ROC = 0.73) and was validated in exome-wide data from 21,703 cases and 128,957 controls. We showed respective regional clustering of inferred LOF and GOF nucleotide variants across the alignment of the entire gene family, suggesting shared pathomechanisms in the SCNxA/CACNA1x family genes. [less ▲]

Predicting Functional Effects of Missense Variants in Voltage-Gated Sodium and Calcium Channels

E-print/Working paper (2019)

Malfunctions of voltage-gated sodium and calcium channels (SCN and CACNA1 genes) have been associated with severe neurologic, psychiatric, cardiac and other diseases. Altered channel activity is ... [more ▼]

Malfunctions of voltage-gated sodium and calcium channels (SCN and CACNA1 genes) have been associated with severe neurologic, psychiatric, cardiac and other diseases. Altered channel activity is frequently grouped into gain or loss of ion channel function (GOF or LOF, respectively) which is not only corresponding to clinical disease manifestations, but also to differences in drug response. Experimental studies of channel function are therefore important, but laborious and usually focus only on a few variants at a time. Based on known gene-disease-mechanisms, we here infer LOF (518 variants) and GOF (309 variants) of likely pathogenic variants from disease phenotypes of variant carriers. We show regional clustering of inferred GOF and LOF variants, respectively, across the alignment of the entire gene family, suggesting shared pathomechanisms in the SCN/CACNA1 genes. By training a machine learning model on sequence- and structure-based features we predict LOF- or GOF- associated disease phenotypes (ROC = 0.85) of likely pathogenic missense variants. We then successfully validate the GOF versus LOF prediction on 87 functionally tested variants in SCN1/2/8A and CACNA1I (ROC = 0.73) and in exome-wide data from > 100.000 cases and controls. Ultimately, functional prediction of missense variants in clinically relevant genes will facilitate precision medicine in clinical practice. [less ▲]

De novo Variants in Neurodevelopmental Disorders with Epilepsy

E-print/Working paper (2018)

Neurodevelopmental disorders (NDD) with epilepsy constitute a complex and heterogeneous phenotypic spectrum of largely unclear genetic architecture. We conducted exome-wide enrichment analyses for protein ... [more ▼]

Neurodevelopmental disorders (NDD) with epilepsy constitute a complex and heterogeneous phenotypic spectrum of largely unclear genetic architecture. We conducted exome-wide enrichment analyses for protein-altering de novo variants (DNV) in 7088 parent-offspring trios with NDD of which 2151 were comorbid with epilepsy. In this cohort, the genetic spectrum of epileptic encephalopathy (EE) and nonspecific NDD with epilepsy were markedly similar. We identified 33 genes significantly enriched for DNV in NDD with epilepsy, of which 27.3 were associated with therapeutic consequences. These 33 DNV-enriched genes were more often associated with synaptic transmission but less with chromatin modification when compared to NDD without epilepsy. On average, only 53 of the DNV-enriched genes were represented on available diagnostic sequencing panels, so our findings should drive significant improvements of genetic testing approaches. [less ▲]

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features

in American Journal of Human Genetics (2017), 100(4), 676-688

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions ... [more ▼]

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease. [less ▲]

Targeted sequencing of 351 candidate genes for epileptic encephalopathy in a large cohort of patients

in Molecular Genetics and Genomic Medicine (2016), 4(5), 568-80

Background Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional ... [more ▼]

Background Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional evidence exists for a definite annotation. Methods To increase the number of validated EE genes, we sequenced 26 known and 351 candidate genes for EE in 360 patients. Variants in 25 genes known to be involved in EE or related phenotypes were followed up in 41 patients. We prioritized the candidate genes, and followed up 31 variants in this prioritized subset of candidate genes. Results Twenty-nine genotypes in known genes for EE (19) or related diseases (10), dominant as well as recessive or X-linked, were classified as likely pathogenic variants. Among those, likely pathogenic de novo variants were found in EE genes that act dominantly, including the recently identified genes EEF1A2, KCNB1 and the X-linked gene IQSEC2. A de novo frameshift variant in candidate gene HNRNPU was the only de novo variant found among the followed-up candidate genes, and the patient's phenotype was similar to a few recent publications. Conclusion Mutations in genes described in OMIM as, for example, intellectual disability gene can lead to phenotypes that get classified as EE in the clinic. We confirmed existing literature reports that de novo loss-of-function HNRNPUmutations lead to severe developmental delay and febrile seizures in the first year of life. [less ▲]