Global characterization of copy number variants in epilepsy patients from whole genome sequencing.

Epilepsy will affect nearly 3% of people at some point during their lifetime. Previous copy number variants (CNVs) studies of epilepsy have used array-based technology and were restricted to the detection of large or exonic events. In contrast, whole-genome sequencing (WGS) has the potential to more comprehensively profile CNVs but existing analytic methods suffer from limited accuracy. We show that this is in part due to the non-uniformity of read coverage, even after intra-sample normalization. To improve on this, we developed PopSV, an algorithm that uses multiple samples to control for technical variation and enables the robust detection of CNVs. Using WGS and PopSV, we performed a comprehensive characterization of CNVs in 198 individuals affected with epilepsy and 301 controls. For both large and small variants, we found an enrichment of rare exonic events in epilepsy patients, especially in genes with predicted loss-of-function intolerance. Notably, this genome-wide survey also revealed an enrichment of rare non-coding CNVs near previously known epilepsy genes. This enrichment was strongest for non-coding CNVs located within 100 Kbp of an epilepsy gene and in regions associated with changes in the gene expression, such as expression QTLs or DNase I hypersensitive sites. Finally, we report on 21 potentially damaging events that could be associated with known or new candidate epilepsy genes. Our results suggest that comprehensive sequence-based profiling of CNVs could help explain a larger fraction of epilepsy cases.

Testing association of rare genetic variants with resistance to three common antiseizure medications.

OBJECTIVE: Drug resistance is a major concern in the treatment of individuals with epilepsy. No genetic markers for resistance to individual antiseizure medication (ASM) have yet been identified. We aimed to identify the role of rare genetic variants in drug resistance for three common ASMs: levetiracetam (LEV), lamotrigine (LTG), and valproic acid (VPA). METHODS: A cohort of 1622 individuals of European descent with epilepsy was deeply phenotyped and underwent whole exome sequencing (WES), comprising 575 taking LEV, 826 LTG, and 782 VPA. We performed gene- and gene set-based collapsing analyses comparing responders and nonresponders to the three drugs to determine the burden of different categories of rare genetic variants. RESULTS: We observed a marginally significant enrichment of rare missense, truncating, and splice region variants in individuals who were resistant to VPA compared to VPA responders for genes involved in VPA pharmacokinetics. We also found a borderline significant enrichment of truncating and splice region variants in the synaptic vesicle glycoprotein (SV2) gene family in nonresponders compared to responders to LEV. We did not see any significant enrichment using a gene-based approach. SIGNIFICANCE: In our pharmacogenetic study, we identified a slightly increased burden of damaging variants in gene groups related to drug kinetics or targeting in individuals presenting with drug resistance to VPA or LEV. Such variants could thus determine a genetic contribution to drug resistance.

Human copy number variants are enriched in regions of low mappability.

Copy number variants (CNVs) are known to affect a large portion of the human genome and have been implicated in many diseases. Although whole-genome sequencing (WGS) can help identify CNVs, most analytical methods suffer from limited sensitivity and specificity, especially in regions of low mappability. To address this, we use PopSV, a CNV caller that relies on multiple samples to control for technical variation. We demonstrate that our calls are stable across different types of repeat-rich regions and validate the accuracy of our predictions using orthogonal approaches. Applying PopSV to 640 human genomes, we find that low-mappability regions are approximately 5 times more likely to harbor germline CNVs, in stark contrast to the nearly uniform distribution observed for somatic CNVs in 95 cancer genomes. In addition to known enrichments in segmental duplication and near centromeres and telomeres, we also report that CNVs are enriched in specific types of satellite and in some of the most recent families of transposable elements. Finally, using this comprehensive approach, we identify 3455 regions with recurrent CNVs that were missing from existing catalogs. In particular, we identify 347 genes with a novel exonic CNV in low-mappability regions, including 29 genes previously associated with disease.

Impact of Paternal Age at Conception on Human Health.

BACKGROUND: The effect of maternal age at conception on various aspects of offspring health is well documented and often discussed. We seldom hear about the paternal age effect on offspring health, although the link is now almost as solid as with maternal age. The causes behind this, however, are drastically different between males and females. CONTENT: In this review article, we will first examine documented physiological changes linked to paternal age effect. We will start with all morphological aspects of the testis that have been shown to be altered with aging. We will then move on to all the parameters of spermatogenesis that are linked with paternal age at conception. The biggest part of this review will focus on genetic changes associated with paternal age effects. Several studies that have established a strong link between paternal age at conception and the rate of de novo mutations will be reviewed. We will next discuss paternal age effects associated with telomere length and try to better understand the seemingly contradictory results. Finally, severe diseases that affect brain functions and normal development have been associated with older paternal age at conception. In this context, we will discuss the cases of autism spectrum disorder and schizophrenia, as well as several childhood cancers. SUMMARY: In many Western civilizations, the age at which parents have their first child has increased substantially in recent decades. It is important to summarize major health issues associated with an increased paternal age at conception to better model public health systems.

Exome sequencing of sporadic childhood-onset schizophrenia suggests the contribution of X-linked genes in males.

Childhood-onset schizophrenia (COS) is a rare and severe form of schizophrenia, defined as having an onset before the age of 13. The male COS cases have a slightly younger age of onset than female cases. They also present with a higher rate of comorbid developmental disorders. These sex differences are not explained by the frequency of chromosomal abnormalities, and the contribution of other forms of genetic variations remains unestablished. Using a whole-exome sequencing approach, we examined 12 COS trios where the unaffected parents had an affected male child. The sequencing data enabled us to test if the hemizygous variants, transmitted from the unaffected carrying mother, could mediate the phenotype (X-linked recessive inheritance model). Our results revealed that affected children have a significantly greater number of X-linked rare variants than their unaffected fathers. The variants identified in the male probands were mostly found in genes previously linked to other neuropsychiatric diseases like autism, intellectual disability, and epilepsy, including LUZP4, PCDH19, RPS6KA3, and OPHN1. The level of expression of the genes was assessed at different developmental periods in normal brain using the BrainSpan database. This approach revealed that some of them were expressed earlier in males than in females, consistent with the younger age of onset in male COS. In conclusion, this article suggests that X-linked genes might play a role in the pathophysiology of COS. Candidate genes detailed here could explain the higher level of comorbidities and the earlier age of onset observed in a subset of the male COS cases.

Deleterious mutations in the essential mRNA metabolism factor, hGle1, in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons. Causative mutations in the global RNA-processing proteins TDP-43 and FUS among others, as well as their aggregation in ALS patients, have identified defects in RNA metabolism as an important feature in this disease. Lethal congenital contracture syndrome 1 and lethal arthrogryposis with anterior horn cell disease are autosomal recessive fetal motor neuron diseases that are caused by mutations in another global RNA-processing protein, hGle1. In this study, we carried out the first screening of GLE1 in ALS patients (173 familial and 760 sporadic) and identified 2 deleterious mutations (1 splice site and 1 nonsense mutation) and 1 missense mutation. Functional analysis of the deleterious mutants revealed them to be unable to rescue motor neuron pathology in zebrafish morphants lacking Gle1. Furthermore, in HeLa cells, both mutations caused a depletion of hGle1 at the nuclear pore where it carries out an essential role in nuclear export of mRNA. These results suggest a haploinsufficiency mechanism and point to a causative role for GLE1 mutations in ALS patients. This further supports the involvement of global defects in RNA metabolism in ALS.

Genome-wide association study in essential tremor identifies three new loci.

We conducted a genome-wide association study of essential tremor, a common movement disorder characterized mainly by a postural and kinetic tremor of the upper extremities. Twin and family history studies show a high heritability for essential tremor. The molecular genetic determinants of essential tremor are unknown. We included 2807 patients and 6441 controls of European descent in our two-stage genome-wide association study. The 59 most significantly disease-associated markers of the discovery stage were genotyped in the replication stage. After Bonferroni correction two markers, one (rs10937625) located in the serine/threonine kinase STK32B and one (rs17590046) in the transcriptional coactivator PPARGC1A were associated with essential tremor. Three markers (rs12764057, rs10822974, rs7903491) in the cell-adhesion molecule CTNNA3 were significant in the combined analysis of both stages. The expression of STK32B was increased in the cerebellar cortex of patients and expression quantitative trait loci database mining showed association between the protective minor allele of rs10937625 and reduced expression in cerebellar cortex. We found no expression differences related to disease status or marker genotype for the other two genes. Replication of two lead single nucleotide polymorphisms of previous small genome-wide association studies (rs3794087 in SLC1A2, rs9652490 in LINGO1) did not confirm the association with essential tremor.

Partitioning the heritability of Tourette syndrome and obsessive compulsive disorder reveals differences in genetic architecture.

The direct estimation of heritability from genome-wide common variant data as implemented in the program Genome-wide Complex Trait Analysis (GCTA) has provided a means to quantify heritability attributable to all interrogated variants. We have quantified the variance in liability to disease explained by all SNPs for two phenotypically-related neurobehavioral disorders, obsessive-compulsive disorder (OCD) and Tourette Syndrome (TS), using GCTA. Our analysis yielded a heritability point estimate of 0.58 (se = 0.09, p = 5.64e-12) for TS, and 0.37 (se = 0.07, p = 1.5e-07) for OCD. In addition, we conducted multiple genomic partitioning analyses to identify genomic elements that concentrate this heritability. We examined genomic architectures of TS and OCD by chromosome, MAF bin, and functional annotations. In addition, we assessed heritability for early onset and adult onset OCD. Among other notable results, we found that SNPs with a minor allele frequency of less than 5% accounted for 21% of the TS heritability and 0% of the OCD heritability. Additionally, we identified a significant contribution to TS and OCD heritability by variants significantly associated with gene expression in two regions of the brain (parietal cortex and cerebellum) for which we had available expression quantitative trait loci (eQTLs). Finally we analyzed the genetic correlation between TS and OCD, revealing a genetic correlation of 0.41 (se = 0.15, p = 0.002). These results are very close to previous heritability estimates for TS and OCD based on twin and family studies, suggesting that very little, if any, heritability is truly missing (i.e., unassayed) from TS and OCD GWAS studies of common variation. The results also indicate that there is some genetic overlap between these two phenotypically-related neuropsychiatric disorders, but suggest that the two disorders have distinct genetic architectures.

Exome sequencing identifies FUS mutations as a cause of essential tremor.

Essential tremor (ET) is a common neurodegenerative disorder that is characterized by a postural or motion tremor. Despite a strong genetic basis, a gene with rare pathogenic mutations that cause ET has not yet been reported. We used exome sequencing to implement a simple approach to control for misdiagnosis of ET, as well as phenocopies involving sporadic and senile ET cases. We studied a large ET-affected family and identified a FUS p.Gln290(∗) mutation as the cause of ET in this family. Further screening of 270 ET cases identified two additional rare missense FUS variants. Functional considerations suggest that the pathogenic effects of ET-specific FUS mutations are different from the effects observed when FUS is mutated in amyotrophic lateral sclerosis cases; we have shown that the ET FUS nonsense mutation is degraded by the nonsense-mediated-decay pathway, whereas amyotrophic lateral sclerosis FUS mutant transcripts are not.

Deciphering the genetic structure of the Quebec founder population using genealogies.

Using genealogy to study the demographic history of a population makes it possible to overcome the models and assumptions often used in population genetics. The Quebec founder population is one of the few populations in the world having access to the complete genealogy of the last 400 years. The goal of this study is to follow the evolution of the Quebec population structure over time from the beginning of European colonization until the present day. To do so, we calculated the kinship coefficients of all ancestors' pairs in the ascending genealogy of 665 subjects from eight regional and ethnocultural groups per 25-year period. We show that the Quebec population structure appeared progressively in the St. Lawrence valley as early as 1750 with the distinction of the Saguenay and Gaspesian groups. At that time, the ancestors of two groups, the Sagueneans and the Acadians from the Gaspé Peninsula, experienced a marked increase in kinship and inbreeding levels which have shaped the structure and led to the contemporary population structure. Interestingly, this structure arose before the colonization of the Saguenay region and at the very beginning of the Gaspé Peninsula settlement. The resulting regional founder effects in these groups led to differences in the present-day identity-by-descent sharing, the Gaspé and North Shore groups sharing more large segments and the Sagueneans more short segments. This is also reflected by the distribution of the number of most recent common ancestors at different generations and their genetic contribution to the studied subjects.

Investigation of rare variants in LRP1, KPNA1, ALS2CL and ZNF480 genes in schizophrenia patients reflects genetic heterogeneity of the disease.

BACKGROUND: Schizophrenia is a severe psychiatric disease characterized by a high heritability and a complex genetic architecture. Recent reports based on exome sequencing analyses have highlighted a significant increase of potentially deleterious de novo mutations in different genes in individuals with schizophrenia. FINDINGS: This report presents the mutation screening results of four candidate genes for which such de novo mutations were previously reported (LRP1, KPNA1, ALS2CL and ZNF480). We have not identified any excess of rare variants in the additional SCZ cases we have screened. CONCLUSIONS: This supports the notion that de novo mutations in these four genes are extremely rare in schizophrenia and further highlights the high degree of genetic heterogeneity of this disease.

Multivariate extension of penalized regression on summary statistics to construct polygenic risk scores for correlated traits.

Genetic correlations between human traits and disorders such as schizophrenia (SZ) and bipolar disorder (BD) diagnoses are well established. Improved prediction of individual traits has been obtained by combining predictors of multiple genetically correlated traits derived from summary statistics produced by genome-wide association studies, compared with single trait predictors. We extend this idea to penalized regression on summary statistics in Multivariate Lassosum, expressing regression coefficients for the multiple traits on single nucleotide polymorphisms (SNPs) as correlated random effects, similarly to multi-trait summary statistic best linear unbiased predictors (MT-SBLUPs). We also allow the SNP contributions to genetic covariance and heritability to depend on genomic annotations. We conducted simulations with two dichotomous traits having polygenic architecture similar to SZ and BD, using genotypes from 29,330 subjects from the CARTaGENE cohort. Multivariate Lassosum produced polygenic risk scores (PRSs) more strongly correlated with the true genetic risk predictor and had better discrimination power between affected and non-affected subjects than previously published sparse multi-trait (PANPRS) and univariate (Lassosum, sparse LDpred2, and the standard clumping and thresholding) methods in most simulation settings. Application of Multivariate Lassosum to predict SZ, BD, and related psychiatric traits in the Eastern Quebec SZ and BD kindred study revealed associations with every trait stronger than those obtained with univariate sparse PRSs, particularly when heritability and genetic covariance depended on genomic annotations. Multivariate Lassosum thus appears promising to improve prediction of genetically correlated traits with summary statistics for a selected subset of SNPs.

Unraveling the role of non-coding rare variants in epilepsy.

The discovery of new variants has leveled off in recent years in epilepsy studies, despite the use of very large cohorts. Consequently, most of the heritability is still unexplained. Rare non-coding variants have been largely ignored in studies on epilepsy, although non-coding single nucleotide variants can have a significant impact on gene expression. We had access to whole genome sequencing (WGS) from 247 epilepsy patients and 377 controls. To assess the functional impact of non-coding variants, ExPecto, a deep learning algorithm was used to predict expression change in brain tissues. We compared the burden of rare non-coding deleterious variants between cases and controls. Rare non-coding highly deleterious variants were significantly enriched in Genetic Generalized Epilepsy (GGE), but not in Non-Acquired Focal Epilepsy (NAFE) or all epilepsy cases when compared with controls. In this study we showed that rare non-coding deleterious variants are associated with epilepsy, specifically with GGE. Larger WGS epilepsy cohort will be needed to investigate those effects at a greater resolution. Nevertheless, we demonstrated the importance of studying non-coding regions in epilepsy, a disease where new discoveries are scarce.

Restless legs syndrome-associated MEIS1 risk variant influences iron homeostasis.

Restless legs syndrome (RLS) is a frequent sleep disorder that is linked to disturbed iron homeostasis. Genetic studies identified MEIS1 as an RLS-predisposing gene, where the RLS risk haplotype is associated with decreased MEIS1 mRNA and protein expression. We show here that RNA interference treatment of the MEIS1 worm orthologue increases ferritin expression in Caenorhabditis elegans and that the RLS-associated haplotype leads to increased expression of ferritin and DMT1 in RLS brain tissues. Additionally, human cells cultured under iron-deficient conditions show reduced MEIS1 expression. Our data establish a link between the RLS MEIS1 gene and iron metabolism.

Schizophrenia genetics: putting all the pieces together.

Schizophrenia is a major mental disorder characterized by a deep disruption of the thinking process and of emotional response. For many decades, genetics studies have yielded little success in identifying genetic factors responsible for the disease. However, with the recent breakthroughs in genome analysis technologies, the field of the genetics of schizophrenia has progressed a lot in the last years. Both common and rare variants have been successfully associated with the disease and a particular emphasis has been made on rare copy number variations. Recently, a new paradigm linking de novo mutations to the genetic mechanism of schizophrenia has been unravelled. The aim of this review is to discuss the most important genetic studies made in the field to give a general perspective of where to go in the future.

Assessment of burden and segregation profiles of CNVs in patients with epilepsy.

OBJECTIVE: Microdeletions are associated with different forms of epilepsy but show incomplete penetrance, which is not well understood. We aimed to assess whether unmasked variants or double CNVs could explain incomplete penetrance. METHODS: We analyzed copy number variants (CNVs) in 603 patients with four different subgroups of epilepsy and 945 controls. CNVs were called from genotypes and validated on whole-genome (WGS) or whole-exome sequences (WES). CNV burden difference between patients and controls was obtained by fitting a logistic regression. CNV burden was assessed for small and large (>1 Mb) deletions and duplications and for deletions overlapping different gene sets. RESULTS: Large deletions were enriched in genetic generalized epilepsies (GGE) compared to controls. We also found enrichment of deletions in epilepsy genes and hotspots for GGE. We did not find truncating or functional variants that could have been unmasked by the deletions. We observed a double CNV hit in two patients. One patient also carried a de novo deletion in the 22q11.2 hotspot. INTERPRETATION: We could corroborate previous findings of an enrichment of large microdeletions and deletions in epilepsy genes in GGE. We could also replicate that microdeletions show incomplete penetrance. However, we could not validate the hypothesis of unmasked variants nor the hypothesis of double CNVs to explain the incomplete penetrance. We found a de novo CNV on 22q11.2 that could be of interest. We also observed GGE families carrying a deletion on 15q13.3 hotspot that could be investigated in the Quebec founder population.

Association of Essential Tremor With Novel Risk Loci: A Genome-Wide Association Study and Meta-analysis.

Importance: Essential tremor (ET) is one of the most common movement disorders, affecting 5% of the general population older than 65 years. Common variants are thought to contribute toward susceptibility to ET, but no variants have been robustly identified. Objective: To identify common genetic factors associated with risk of ET. Design, Setting, and Participants: Case-control genome-wide association study. Inverse-variance meta-analysis was used to combine cohorts. Multicenter samples collected from European populations were collected from January 2010 to September 2019 as part of an ongoing study. Included patients were clinically diagnosed with or reported having ET. Control individuals were not diagnosed with or reported to have ET. Of 485 250 individuals, data for 483 054 passed data quality control and were used. Main Outcomes and Measures: Genotypes of common variants associated with risk of ET. Results: Of the 483 054 individuals included, there were 7177 with ET (3693 [51.46%] female; mean [SD] age, 62.66 [15.12] years), and 475 877 control individuals (253 785 [53.33%] female; mean [SD] age, 56.40 [17.6] years). Five independent genome-wide significant loci and were identified and were associated with approximately 18% of ET heritability. Functional analyses found significant enrichment in the cerebellar hemisphere, cerebellum, and axonogenesis pathways. Genetic correlation (r), which measures the degree of genetic overlap, revealed significant common variant overlap with Parkinson disease (r, 0.28; P = 2.38 × 10-8) and depression (r, 0.12; P = 9.78 × 10-4). A separate fine-mapping of transcriptome-wide association hits identified genes such as BACE2, LRRN2, DHRS13, and LINC00323 in disease-relevant brain regions, such as the cerebellum. Conclusions and Relevance: The results of this genome-wide association study suggest that a portion of ET heritability can be explained by common genetic variation and can help identify new common genetic risk factors for ET.

MEIS1 intronic risk haplotype associated with restless legs syndrome affects its mRNA and protein expression levels.

Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible urge to move the legs at night, which is often accompanied by unpleasant sensations. A recent genomewide association study identified an association between RLS and intronic markers from the MEIS1 gene. Comparative genomic analysis indicates that MEIS1 is the only gene encompassed in this evolutionarily conserved chromosomal segment, i.e. a conservation synteny block, from mammals to fish. We carried out a series of experiments to delineate the role of MEIS1 in RLS pathogenesis and the underlying genetic mechanism. We sequenced all 13 MEIS1 exons and their splice junctions in 285 RLS probands with confirmed clinical diagnosis and did not identify any causative coding or exon-intron junction mutations. We found no evidence of structural variation or disease-associated haplotype differential splicing. However, sequencing of conserved regions of MEIS1 introns 8 and 9 identified a novel single nucleotide polymorphism (C13B_2) significantly associated with RLS (allelic association, P = 1.81E-07). We detected a significant decrease in MEIS1 mRNA expression by quantitative real-time polymerase chain reaction in lymphoblastoid cell lines (LCLs) and brain tissues from RLS patients homozygous for the intronic RLS risk haplotype, compared with those homozygous for the non-risk haplotype. Finally, we found significantly decreased MEIS1 protein levels in the same batch of LCLs and brain tissues from the homozygous carriers of the risk haplotype, compared with the homozygous non-carriers. Therefore, these data suggest that reduced expression of the MEIS1 gene, possibly through intronic cis-regulatory element(s), predisposes to RLS.

Assessing the role of rare genetic variants in drug-resistant, non-lesional focal epilepsy.

OBJECTIVE: Resistance to antiseizure medications (ASMs) is one of the major concerns in the treatment of epilepsy. Despite the increasing number of ASMs available, the proportion of individuals with drug-resistant epilepsy remains unchanged. In this study, we aimed to investigate the role of rare genetic variants in ASM resistance. METHODS: We performed exome sequencing of 1,128 individuals with non-familial non-acquired focal epilepsy (NAFE) (762 non-responders, 366 responders) and were provided with 1,734 healthy controls. We undertook replication in a cohort of 350 individuals with NAFE (165 non-responders, 185 responders). We performed gene-based and gene-set-based kernel association tests to investigate potential enrichment of rare variants in relation to drug response status and to risk for NAFE. RESULTS: We found no gene or gene set that reached genome-wide significance. Yet, we identified several prospective candidate genes - among them DEPDC5, which showed a potential association with resistance to ASMs. We found some evidence for an enrichment of truncating variants in dominant familial NAFE genes in our cohort of non-familial NAFE and in association with drug-resistant NAFE. INTERPRETATION: Our study identifies potential candidate genes for ASM resistance. Our results corroborate the role of rare variants for non-familial NAFE and imply their involvement in drug-resistant epilepsy. Future large-scale genetic research studies are needed to substantiate these findings.

Polygenic risk scores of several subtypes of epilepsies in a founder population.

OBJECTIVE: Polygenic risk scores (PRSs) are used to quantify the cumulative effects of a number of genetic variants, which may individually have a very small effect on susceptibility to a disease; we used PRSs to better understand the genetic contribution to common epilepsy and its subtypes. METHODS: We first replicated previous single associations using 373 unrelated patients. We then calculated PRSs in the same French Canadian patients with epilepsy divided into 7 epilepsy subtypes and population-based controls. We fitted a logistic mixed model to calculate the variance explained by the PRS using pseudo-R2 statistics. RESULTS: We show that the PRS explains more of the variance in idiopathic generalized epilepsy than in patients with nonacquired focal epilepsy. We also demonstrate that the variance explained is different within each epilepsy subtype. CONCLUSIONS: Globally, we support the notion that PRSs provide a reliable measure to rightfully estimate the contribution of genetic factors to the pathophysiologic mechanism of epilepsies, but further studies are needed on PRSs before they can be used clinically.