Gene family information facilitates variant interpretation and identification of disease-associated genes in neurodevelopmental disorders

in Genome Medicine (2020), 12(28),

Background: Classifying pathogenicity of missense variants represents a major challenge in clinical practice during the diagnoses of rare and genetic heterogeneous neurodevelopmental disorders (NDDs ... [more ▼]

Background: Classifying pathogenicity of missense variants represents a major challenge in clinical practice during the diagnoses of rare and genetic heterogeneous neurodevelopmental disorders (NDDs). While orthologous gene conservation is commonly employed in variant annotation, approximately 80% of known disease-associated genes belong to gene families. The use of gene family information for disease gene discovery and variant interpretation has not yet been investigated on genome-wide scale. We empirically evaluate whether paralog conserved or non-conserved sites in human gene families are important in NDDs. Methods: Gene family information was collected from Ensembl. Paralog conserved sites were defined based on paralog sequence alignments. 10,068 NDD patients and 2,078 controls were statistically evaluated for de novo variant burden in gene families. Results: We demonstrate that disease-associated missense variants are enriched at paralog conserved sites across all disease groups and inheritance models tested. We developed a gene family de novo enrichment framework that identified 43 exome-wide enriched gene families including 98 de novo variant carrying genes in NDD patients of which 28 represent novel candidate genes for NDD which are brain expressed and under evolutionary constraint. Conclusion: This study represents the first method to incorporate gene-family information into a statistical framework to interpret variant data for NDDs and to discover newly NDD -associated genes. [less ▲]

Assessment of genetic variant burden in epilepsy-associated brain lesions

in European Journal of Human Genetics (2019)

It is challenging to estimate genetic variant burden across different subtypes of epilepsy. Herein, we used a comparative approach to assess the diagnostic yield and genotype-phenotype correlations in the ... [more ▼]

It is challenging to estimate genetic variant burden across different subtypes of epilepsy. Herein, we used a comparative approach to assess the diagnostic yield and genotype-phenotype correlations in the four most common brain lesions in patients with drug-resistant focal epilepsy. Targeted sequencing analysis was performed for a panel of 161 genes with a mean coverage of > 400x. Lesional tissue was histopathologically reviewed and dissected from hippocampal sclerosis (n=15), ganglioglioma (n=16), dysembryoplastic neuroepithelial tumors (n=8) and ocal cortical dysplasia type II (n=15). Peripheral blood (n=12) or surgical tissue samples histopathologically classified as lesion-free (n=42) were available for comparison. Variants were classified as pathogenic or likely pathogenic according to American College of Medical Genetics and Genomics guidelines. Overall, we identified pathogenic and likely pathogenic variants in 25.9% of patients with a mean coverage of 383x. The highest number of pathogenic/ likely pathogenic variants was observed in patients with ganglioglioma (43.75%; all somatic) and dysembryoplastic neuroepithelial tumors (37.5%; all somatic), and in 20% of cases with focal cortical dysplasia type II (13.33% somatic, 6.67% germline). Pathogenic/likely pathogenic positive genes were disorder-specific and BRAF V600E the only recurrent pathogenic variant. This study represents a reference for diagnostic yield across the four most common lesion entities in patients with drug-resistant focal epilepsy. The observed large variability in variant burden by epileptic lesion type calls for whole exome sequencing of histopathologically well characterized tissue in a diagnostic setting and in research to discover novel disease-associated genes. [less ▲]

Identification of pathogenic variant enriched regions across genes and gene families

E-print/Working paper (2019)

Missense variant interpretation is challenging. Essential regions for protein function are conserved among gene family members, and genetic variants within these regions are potentially more likely to ... [more ▼]

Missense variant interpretation is challenging. Essential regions for protein function are conserved among gene family members, and genetic variants within these regions are potentially more likely to confer risk to disease. Here, we generated 2,871 gene family protein sequence alignments involving 9,990 genes and performed missense variant burden analyses to identify novel essential protein regions. We mapped 2,219,811 variants from the general population into these alignments and compared their distribution with 65,034 missense variants from patients. With this gene family approach, we identified 398 regions enriched for patient variants spanning 33,887 amino acids in 1,058 genes. As a comparison, testing the same genes individually we identified less patient variant enriched regions involving only 2,167 amino acids and 180 genes. Next, we selected de novo variants from 6,753 patients with neurodevelopmental disorders and 1,911 unaffected siblings, and observed a 5.56-fold enrichment of patient variants in our identified regions (95% C.I. =2.76-Inf, p-value = 6.66×10−8). Using an independent ClinVar variant set, we found missense variants inside the identified regions are 111-fold more likely to be classified as pathogenic in comparison to benign classification (OR = 111.48, 95% C.I = 68.09-195.58, p-value < 2.2e−16). All patient variant enriched regions identified (PERs) are available online through a user-friendly platform for interactive data mining, visualization and download at http://per.broadinstitute.org. In summary, our gene family burden analysis approach identified novel patient variant enriched regions in protein sequences. This annotation can empower variant interpretation. [less ▲]

Guideline-based and bioinformatic reassessment of lesion-associated gene and variant pathogenicity in focal human epilepsies

in Epilepsia (2018)

Objective: Increasing availability of surgically resected brain tissue from patients with focal epilepsy and Focal Cortical Dysplasia (FCD) or low-grade glio-neuronal tumors has fostered large-scale ... [more ▼]

Objective: Increasing availability of surgically resected brain tissue from patients with focal epilepsy and Focal Cortical Dysplasia (FCD) or low-grade glio-neuronal tumors has fostered large-scale genetic examination. However, assessment of pathogenicity of germline and somatic variants remains difficult. Here, we present a state of the art evaluation of reported genes and variants associated with epileptic brain lesions. Methods: We critically re-evaluated the pathogenicity for all neuropathology-associated variants reported to date in PubMed and ClinVar databases including 101 neuropathology-associated missense variants encompassing 11 disease-related genes. We assessed gene variant tolerance and classified all identified missense variants according to guidelines from the American College of Medical Genetics and Genomics (ACMG). We further extended the bioinformatic variant prediction by introducing a novel gene-specific deleteriousness ranking for prediction scores. Results: Application of ACMG guidelines and in silico gene variant tolerance analysis classified only seven out of 11 genes to be likely disease-associated according to the reported a disease mechanism, while 61 (60.4%) of 101 variants of those genes were classified as of uncertain significance (VUS), 37 (36.6%) as being likely pathogenic (LP) and 3 (3%) as being pathogenic (P). Significance: We concluded that the majority of neuropathology-associated variants reported to date do not have enough evidence to be classified as pathogenic. Interpretation of lesion-associated variants remains challenging and application of current ACMG guidelines is recommended for interpretation and prediction. [less ▲]

Rare coding variants in genes encoding GABAA receptors in genetic generalised epilepsies: an exome-based case-control study

in Lancet Neurology (2018), 17(8), 699-708

Background Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We ... [more ▼]

Background Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We aimed to investigate the burden of rare genetic variants in genetic generalised epilepsy. Methods For this exome-based case-control study, we used three different genetic generalised epilepsy case cohorts and three independent control cohorts, all of European descent. Cases included in the study were clinically evaluated for genetic generalised epilepsy. Whole-exome sequencing was done for the discovery case cohort, a validation case cohort, and two independent control cohorts. The replication case cohort underwent targeted next-generation sequencing of the 19 known genes encoding subunits of GABAA receptors and was compared to the respective GABAA receptor variants of a third independent control cohort. Functional investigations were done with automated two-microelectrode voltage clamping in Xenopus laevis oocytes. Findings Statistical comparison of 152 familial index cases with genetic generalised epilepsy in the discovery cohort to 549 ethnically matched controls suggested an enrichment of rare missense (Nonsyn) variants in the ensemble of 19 genes encoding GABAA receptors in cases (odds ratio [OR] 2·40 [95% CI 1·41–4·10]; pNonsyn=0·0014, adjusted pNonsyn=0·019). Enrichment for these genes was validated in a whole-exome sequencing cohort of 357 sporadic and familial genetic generalised epilepsy cases and 1485 independent controls (OR 1·46 [95% CI 1·05–2·03]; pNonsyn=0·0081, adjusted pNonsyn=0·016). Comparison of genes encoding GABAA receptors in the independent replication cohort of 583 familial and sporadic genetic generalised epilepsy index cases, based on candidate-gene panel sequencing, with a third independent control cohort of 635 controls confirmed the overall enrichment of rare missense variants for 15 GABAA receptor genes in cases compared with controls (OR 1·46 [95% CI 1·02–2·08]; pNonsyn=0·013, adjusted pNonsyn=0·027). Functional studies for two selected genes (GABRB2 and GABRA5) showed significant loss-of-function effects with reduced current amplitudes in four of seven tested variants compared with wild-type receptors. Interpretation Functionally relevant variants in genes encoding GABAA receptor subunits constitute a significant risk factor for genetic generalised epilepsy. Examination of the role of specific gene groups and pathways can disentangle the complex genetic architecture of genetic generalised epilepsy. [less ▲]

Discovery and pathogenicity assessment of neuropathology-associated gene variants

in Epilepsia (2017, December 08), 58(Suppl.5), 174

Germline and brain-specific somatic variants have been reported as an underlying cause in patients with epilepsy-associated neuropathologies, including focal cortical dysplasias (FCDs) and long-term ... [more ▼]

Germline and brain-specific somatic variants have been reported as an underlying cause in patients with epilepsy-associated neuropathologies, including focal cortical dysplasias (FCDs) and long-term epilepsy associated tumors (LEAT). However, evaluation of identified neuropathology associated variants in genetic screens is complex since not all observed variants contribute to the etiology of neuropathologies not even in genuinely disease-associated genes. Here, we critically reevaluated the pathogenicity of 12 previously published disease-related genes and of 79 neuropathology-associated missense variants listed in the PubMed and ClinVar databases. We (1) assessed the evolutionary gene constraint using the pLI and the missense z score, (2) used the latest American College of Medical Genetics and Genomics (ACMG) guidelines, and (3) performed bioinformatic variant pathogenicity prediction analyses using PolyPhen-2, CADD and GERP. Constraint analysis classified only seven out of 12 genes to be likely disease-associated. Furthermore, 78 (89%) of 88 neuropathology-associated missense variants were classified as being of unknown significance (VUS) and only 10 (11%) as being likely pathogenic (LPII). Pathogenicity prediction yielded a discrimination between LPII variants and a discrimination for VUS compared with rare variant scores from individuals present in the Genome Aggregation Database (gnomAD). In summary, our results demonstrate that interpretation of variants associated with neuropathologies is complex while the application of current ACMG guidelines including bioinformatic pathogenicity prediction can help improving variant evaluation. Furthermore, we will augment this set of literature-identified variants at the conference by results from our variant screen using self-generated deep sequencing data in >150 candidate genes in >50 patients not yet analyzed. [less ▲]

Reassessment Of Lesion-Associated Gene And Variant Pathogenicity In Focal Human Epilepsies

E-print/Working paper (2017)

Purpose: Increasing availability of surgically resected brain tissue from Focal Cortical Dysplasia and low-grade epilepsy-associated tumor patients fostered large-scale genetic examination. However ... [more ▼]

Purpose: Increasing availability of surgically resected brain tissue from Focal Cortical Dysplasia and low-grade epilepsy-associated tumor patients fostered large-scale genetic examination. However, assessment of germline and somatic variant pathogenicity remains difficult. Methods: Here, we critically reevaluated the pathogenicity for all neuropathology-associated variants reported to date in the PubMed and ClinVar databases, including 12 disease-related genes and 88 neuropathology-associated missense variants. We (1) assessed evolutionary gene constraint using the pLI and missense z scores, (2) applied guidelines by the American College of Medical Genetics and Genomics (ACMG), and (3) predicted pathogenicity by using PolyPhen-2, CADD, and GERP. Results: Constraint analysis classified only seven out of 12 genes to be likely disease-associated, while 35 (40\%) of those 88 variants were classified as being variants of unknown significance (VUS) and 53 (60\%) as being likely pathogenic (LPII). Pathogenicity prediction yielded discrimination between neuropathology-associated variants (LPII and VUS) and rare variant scores obtained from individuals present in the Genome Aggregation Database (gnomAD). Conclusion: We conclude that several VUS are likely disease-associated and will be reclassified by future molecular evidence. In summary, interpretation of lesion-associated gene variants remains complex while the application of current ACMG guidelines including bioinformatic pathogenicity prediction will help improving interpretation and prediction. [less ▲]

A systematic comparison of two new releases of exome sequencing products: the aim of use determines the choice of product.

in Biological chemistry (2016), 397(8), 791-801

We received early access to the newest releases of exome sequencing products, namely Agilent SureSelect v6 (Agilent, Santa Clara, CA, USA) and NimbleGen MedExome (Roche NimbleGen, Basel, Switzerland), and ... [more ▼]

We received early access to the newest releases of exome sequencing products, namely Agilent SureSelect v6 (Agilent, Santa Clara, CA, USA) and NimbleGen MedExome (Roche NimbleGen, Basel, Switzerland), and we conducted whole exome sequencing (WES) of several DNA samples with each of these products in order to assess their performance. Here, we provide a detailed evaluation of the original, normalized (with respect to the different target sizes), and trimmed data sets and compare them in terms of the amount of duplicates, the reads on target, and the enrichment evenness. In addition to these general statistics, we performed a detailed analysis of the frequently mutated and newly described genes found in 'The Deciphering Developmental Disorders Study' published very recently (Fitzgerald, T.W., Gerety, S.S., Jones, W.D., van Kogelenberg, M., King, D.A., McRae, J., Morley, K.I., Parthiban, V., Al-Turki, S., Ambridge, K., et al. (2015). Large-scale discovery of novel genetic causes of developmental disorders. Nature 519, 223-228.). In our comparison, the Agilent v6 exome performs better than the NimbleGen's MedExome both in terms of efficiency and evenness of coverage distribution. With its larger target size, it is also more comprehensive, and therefore the better choice in research projects that aim to identify novel disease-associated genes. In contrast, if the exomes are mainly used in a diagnostic setting, we see advantages for the new NimbleGen MedExome. We find a superior coverage here in those genes of high clinical relevance that likely allows for a better detection of relevant, disease-causing mutations. [less ▲]

CHD2 variants are a risk factor for photosensitivity in epilepsy

in Brain: a Journal of Neurology (2015)

Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes

in Nature Genetics (2014), 46(12), 1327-32

Febrile seizures affect 2–4% of all children1 and have a strong genetic component2. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2)3, 4, 5 have been identified that cause febrile ... [more ▼]

Febrile seizures affect 2–4% of all children1 and have a strong genetic component2. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2)3, 4, 5 have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B6, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees7, 8 identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes. [less ▲]