LSD1 controls a nuclear checkpoint in Wnt/ß-Catenin signaling to regulate muscle stem cell self-renewal.

The Wnt/ß-Catenin pathway plays a key role in cell fate determination during development and in adult tissue regeneration by stem cells. These processes involve profound gene expression and epigenome remodeling and linking Wnt/ß-Catenin signaling to chromatin modifications has been a challenge over the past decades. Functional studies of the lysine demethylase LSD1/KDM1A converge to indicate that this epigenetic regulator is a key regulator of cell fate, although the extracellular cues controlling LSD1 action remain largely unknown. Here we show that ß-Catenin is a substrate of LSD1. Demethylation by LSD1 prevents ß-Catenin degradation thereby maintaining its nuclear levels. Consistently, in absence of LSD1, ß-Catenin transcriptional activity is reduced in both MuSCs and ESCs. Moreover, inactivation of LSD1 in mouse muscle stem cells and embryonic stem cells shows that LSD1 promotes mitotic spindle orientation via ß-Catenin protein stabilization. Altogether, by inscribing LSD1 and ß-Catenin in the same molecular cascade linking extracellular factors to gene expression, our results provide a mechanistic explanation to the similarity of action of canonical Wnt/ß-Catenin signaling and LSD1 on stem cell fate.

H2A.Z is involved in premature aging and DSB repair initiation in muscle fibers.

Histone variants are key epigenetic players, but their functional and physiological roles remain poorly understood. Here, we show that depletion of the histone variant H2A.Z in mouse skeletal muscle causes oxidative stress, oxidation of proteins, accumulation of DNA damages, and both neuromuscular junction and mitochondria lesions that consequently lead to premature muscle aging and reduced life span. Investigation of the molecular mechanisms involved shows that H2A.Z is required to initiate DNA double strand break repair by recruiting Ku80 at DNA lesions. This is achieved via specific interactions of Ku80 vWA domain with H2A.Z. Taken as a whole, our data reveal that H2A.Z containing nucleosomes act as a molecular platform to bring together the proteins required to initiate and process DNA double strand break repair.

Real life evaluation of AlphaMissense predictions in hematological malignancies.

High-throughput sequencing plays a pivotal role in hematological malignancy diagnostics, but interpreting missense mutations remains challenging. In this study, we used the newly available AlphaMissense database to assess the efficacy of machine learning to predict missense mutation effects and its impact to improve our ability to interpret them. Based on the analysis of 2073 variants from 686 patients analyzed for clinical purpose, we confirmed the very high accuracy of AlphaMissense predictions in a large real-life data set of missense mutations (AUC of ROC curve 0.95), and provided a comprehensive analysis of the discrepancies between AlphaMissense predictions and state of the art clinical interpretation.

Spatial Transcriptomics Reveals Signatures of Histopathological Changes in Muscular Sarcoidosis.

Sarcoidosis is a multisystemic disease characterized by non-caseating granuloma infiltrating various organs. The form with symptomatic muscular involvement is called muscular sarcoidosis. The impact of immune cells composing the granuloma on the skeletal muscle is misunderstood. Here, we investigated the granuloma-skeletal muscle interactions through spatial transcriptomics on two patients affected by muscular sarcoidosis. Five major transcriptomic clusters corresponding to perigranuloma, granuloma, and three successive muscle tissue areas (proximal, intermediate, and distal) around the granuloma were identified. Analyses revealed upregulated pathways in the granuloma corresponding to the activation of T-lymphocytes and monocytes/macrophages cytokines, the upregulation of extracellular matrix signatures, and the induction of the TGF-ß signaling in the perigranuloma. A comparison between the proximal and distal muscles to the granuloma revealed an inverse correlation between the distance to the granuloma and the upregulation of cellular response to interferon-γ/α, TNF-α, IL-1,4,6, fibroblast proliferation, epithelial to mesenchymal cell transition, and the downregulation of muscle gene expression. These data shed light on the intercommunications between granulomas and the muscle tissue and provide pathophysiological mechanisms by showing that granuloma immune cells have a direct impact on proximal muscle tissue by promoting its progressive replacement by fibrosis via the expression of pro-inflammatory and profibrosing signatures. These data could possibly explain the evolution towards a state of disability for some patients.

Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus.

Promyelocytic leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. By using specific approaches, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. Hence, we describe a role of PML NBs as nuclear depot centers to regulate HIRA distribution in the nucleus, dependent both on SP100 and DAXX/H3.3 levels. Upon IFN-I stimulation, PML is required for interferon-stimulated genes (ISGs) transcription and PML NBs become juxtaposed to ISGs loci at late time points of IFN-I treatment. HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of ISGs, well beyond the peak of transcription. Though, HIRA accumulation in PML NBs is dispensable for H3.3 deposition on ISGs. We thus uncover a dual function for PML/PML NBs, as buffering centers modulating the nuclear distribution of HIRA, and as chromosomal hubs regulating ISGs transcription and thus HIRA-mediated H3.3 deposition at ISGs upon inflammatory response.

Homozygous COQ7 mutation: a new cause of potentially treatable distal hereditary motor neuropathy.

Distal hereditary motor neuropathy represents a group of motor inherited neuropathies leading to distal weakness. We report a family of two brothers and a sister affected by distal hereditary motor neuropathy in whom a homozygous variant c.3G>T (p.1Met?) was identified in the COQ7 gene. This gene encodes a protein required for coenzyme Q10 biosynthesis, a component of the respiratory chain in mitochondria. Mutations of COQ7 were previously associated with severe multi-organ disorders characterized by early childhood onset and developmental delay. Using patient blood samples and fibroblasts derived from a skin biopsy, we investigated the pathogenicity of the variant of unknown significance c.3G>T (p.1Met?) in the COQ7 gene and the effect of coenzyme Q10 supplementation in vitro. We showed that this variation leads to a severe decrease in COQ7 protein levels in the patient's fibroblasts, resulting in a decrease in coenzyme Q10 production and in the accumulation of 6-demethoxycoenzyme Q10, the COQ7 substrate. Interestingly, such accumulation was also found in the patient's plasma. Normal coenzyme Q10 and 6-demethoxycoenzyme Q10 levels were restored in vitro by using the coenzyme Q10 precursor 2,4-dihydroxybenzoic acid, thus bypassing the COQ7 requirement. Coenzyme Q10 biosynthesis deficiency is known to impair the mitochondrial respiratory chain. Seahorse experiments showed that the patient's cells mainly rely on glycolysis to maintain sufficient ATP production. Consistently, the replacement of glucose by galactose in the culture medium of these cells reduced their proliferation rate. Interestingly, normal proliferation was restored by coenzyme Q10 supplementation of the culture medium, suggesting a therapeutic avenue for these patients. Altogether, we have identified the first example of recessive distal hereditary motor neuropathy caused by a homozygous variation in the COQ7 gene, which should thus be included in the gene panels used to diagnose peripheral inherited neuropathies. Furthermore, 6-demethoxycoenzyme Q10 accumulation in the blood can be used to confirm the pathogenic nature of the mutation. Finally, supplementation with coenzyme Q10 or derivatives should be considered to prevent the progression of COQ7-related peripheral inherited neuropathy in diagnosed patients.

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons.

Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

Dynamic persistence of UPEC intracellular bacterial communities in a human bladder-chip model of urinary tract infection.

Uropathogenic Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbors of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

Urinary tract infections are one of the most common reasons people need antibiotics. These bacterial infections are typically caused by uropathogenic Escherichia coli (also known as UPEC), which either float freely in the urine and wash away when the bladder empties, or form communities inside cells that the bladder struggles to clear. It is possible that the bacteria living within cells are also more protected from the immune system and antibiotics. But this is hard to study in animal models. To overcome this, Sharma et al. built a 'bladder-chip' which mimics the interface between the blood vessels and the tissue layers of the human bladder. Similar chip devices have also been made for other organs. However, until now, no such model had been developed for the bladder. On the chip created by Sharma et al. is a layer of bladder cells which sit at the bottom of a channel filled with diluted human urine. These cells were infected with UPEC, and then imaged over time to see how the bacteria moved, interacted with the bladder cells, and aggregated together. Immune cells from human blood were then added to a vascular channel underneath the bladder tissue, which is coated with endothelial cells that normally line blood vessels. The immune cells rapidly crossed the endothelial barrier and entered the bladder tissue, and swarmed around sites of infection. In some instances, they released the contents of their cells to form net-like traps to catch the bacteria. But these traps failed to remove the bacteria living inside bladder cells. Antibiotics were then added to the urine flowing over the bladder cells as well as the vascular channel, similar to how drugs would be delivered in live human tissue. Sharma et al. discovered that the antibiotics killed bacteria residing in bladder cells slower than bacteria floating freely in the urine. Furthermore, they found that bacteria living in tightly packed communities within bladder cells were more likely to survive treatment and go on to re-infect other parts of the tissue. Antibiotic resistance is a pressing global challenge, and recurrent urinary tract infections are a significant contributor. The bladder-chip presented here could further our understanding of how these bacterial infections develop in vivo and how good antibiotics are at removing them. This could help researchers identify the best dosing and treatment strategies, as well as provide a platform for rapidly testing new antibiotic drugs and other therapies.

Performances of Targeted RNA Sequencing for the Analysis of Fusion Transcripts, Gene Mutation, and Expression in Hematological Malignancies.

RNA sequencing holds great promise to improve the diagnostic of hematological malignancies, because this technique enables to detect fusion transcripts, to look for somatic mutations in oncogenes, and to capture transcriptomic signatures of nosological entities. However, the analytical performances of targeted RNA sequencing have not been extensively described in diagnostic samples. Using a targeted panel of 1385 cancer-related genes in a series of 100 diagnosis samples and 8 controls, we detected all the already known fusion transcripts and also discovered unknown and/or unsuspected fusion transcripts in 12 samples. Regarding the analysis of transcriptomic profiles, we show that targeted RNA sequencing is performant to discriminate acute lymphoblastic leukemia entities driven by different oncogenic translocations. Additionally, we show that 86% of the mutations identified at the DNA level are also detectable at the messenger RNA (mRNA) level, except for nonsense mutations that are subjected to mRNA decay. We conclude that targeted RNA sequencing might improve the diagnosis of hematological malignancies. Standardization of the preanalytical steps and further refinements of the panel design and of the bioinformatical pipelines will be an important step towards its use in standard diagnostic procedures.

Next-generation sequencing in a series of 80 fetuses with complex cardiac malformations and/or heterotaxy.

Herein, we report the screening of a large panel of genes in a series of 80 fetuses with congenital heart defects (CHDs) and/or heterotaxy and no cytogenetic anomalies. There were 49 males (61%/39%), with a family history in 28 cases (35%) and no parental consanguinity in 77 cases (96%). All fetuses had complex CHD except one who had heterotaxy and midline anomalies while 52 cases (65%) had heterotaxy in addition to CHD. Altogether, 29 cases (36%) had extracardiac and extra-heterotaxy anomalies. A pathogenic variant was found in 10/80 (12.5%) cases with a higher percentage in the heterotaxy group (8/52 cases, 15%) compared with the non-heterotaxy group (2/28 cases, 7%), and in 3 cases with extracardiac and extra-heterotaxy anomalies (3/29, 10%). The inheritance was recessive in six genes (DNAI1, GDF1, MMP21, MYH6, NEK8, and ZIC3) and dominant in two genes (SHH and TAB2). A homozygous pathogenic variant was found in three cases including only one case with known consanguinity. In conclusion, after removing fetuses with cytogenetic anomalies, next-generation sequencing discovered a causal variant in 12.5% of fetal cases with CHD and/or heterotaxy. Genetic counseling for future pregnancies was greatly improved. Surprisingly, unexpected consanguinity accounts for 20% of cases with identified pathogenic variants.

Development of a new expanded next-generation sequencing panel for genetic diseases involved in dyslipidemia.

The aim of this study was to provide an efficient tool: reliable, able to increase the molecular diagnosis performance, to facilitate the detection of copy number variants (CNV), to assess genetic risk scores (wGRS) and to offer the opportunity to explore candidate genes. Custom SeqCap EZ libraries, NextSeq500 sequencing and a homemade pipeline enable the analysis of 311 dyslipidemia-related genes. In the training group (48 DNA from patients with a well-established molecular diagnosis), this next-generation sequencing (NGS) workflow showed an analytical sensitivity >99% (n = 532 variants) without any false negative including a partial deletion of one exon. In the prospective group, from 25 DNA from patients without prior molecular analyses, 18 rare variants were identified in the first intention panel genes, allowing the diagnosis of monogenic dyslipidemia in 11 patients. In six other patients, the analysis of minor genes and wGRS determination provided a hypothesis to explain the dyslipidemia. Remaining data from the whole NGS workflow identified four patients with potentially deleterious variants. This NGS process gives a major opportunity to accede to an enhanced understanding of the genetic of dyslipidemia by simultaneous assessment of multiple genetic determinants.

Involvement of a homozygous exon 6 deletion of LMF1 gene in intermittent severe hypertriglyceridemia.

Severe hypertriglyceridemia (HTG), characterized by triglycerides (TG) permanently over 10 mmol/L, may correspond to familial chylomicronemia syndrome (FCS), a rare disorder. However, hypertriglyceridemic patients more often present multifactorial chylomicronemia syndrome (MCS), characterized by highly variable TG. A few nonsense variants of LMF1 gene were reported in literature in FCS patients. In this study, we described a woman with an intermittent severe HTG. NGS analysis and the sequencing of a long range PCR product revealed a homozygous deletion of 6507 base pairs in LMF1 gene, c.730-1528_898-3417del, removing exon 6, predicted to create an in-frame deletion of 56 amino acids, p.(Thr244_Gln299del). Despite an exon 6 homozygous deletion of LMF1, the patient's highly variable lipid phenotype was suggestive of MCS diagnosis.

Exome sequencing in 57 patients with self-limited focal epilepsies of childhood with typical or atypical presentations suggests novel candidate genes.

OBJECTIVE: Self-limited focal epilepsies of childhood (SFEC) are amongst the best defined and most frequent epilepsy syndromes affecting children with usually normal developmental milestones. They include core syndromes such as Rolandic epilepsy or "Benign" epilepsy with Centro-Temporal Spikes and the benign occipital epilepsies, the early onset Panayiotopoulos syndrome and the late-onset Gastaut type. Atypical forms exist for all of them. Atypical Rolandic epilepsies are conceptualized as belonging to a continuum reaching from the "benign" RE to the severe end of the Landau-Kleffner (LKS) and Continuous Spike-Waves during Sleep syndromes (CSWS). GRIN2A has been shown to cause the epilepsy-aphasia continuum that includes some patients with atypical Rolandic epilepsy with frequent speech disorders, LKS and CSWS. In the present study, we searched novel genes causing SFEC with typical or atypical presentations. METHODS: Exome sequencing was performed in 57 trios. Patients presented with typical or atypical SFEC, negative for GRIN2A pathogenic variant. RESULTS: We found rare candidate variants in 20 patients. Thirteen had occurred de novo and were mostly associated to atypical Rolandic Epilepsy. Two of them could be considered as disease related: a null variant in GRIN2B and a missense variant in CAMK2A. Others were considered good candidates, including a substitution affecting a splice site in CACNG2 and missense variants in genes encoding enzymes involved in chromatin remodeling. SIGNIFICANCE: Our results further illustrate the fact that atypical SFEC are more likely to have Mendelian inheritance than typical SFEC.

H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles.

While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription.

Contribution of rare and predicted pathogenic gene variants to childhood-onset lupus: a large, genetic panel analysis of British and French cohorts.

BACKGROUND: Systemic lupus erythematosus (SLE) is a rare immunological disorder and genetic factors are considered important in its causation. Monogenic lupus has been associated with around 30 genotypes in humans and 60 in mice, while genome-wide association studies have identified more than 90 risk loci. We aimed to analyse the contribution of rare and predicted pathogenic gene variants in a population of unselected cases of childhood-onset SLE. METHODS: For this genetic panel analysis we designed a next-generation sequencing panel comprising 147 genes, including all known lupus-causing genes in humans, and potentially lupus-causing genes identified through GWAS and animal models. We screened 117 probands fulfilling American College of Rheumatology (ACR) criteria for SLE, ascertained through British and French cohorts of childhood-onset SLE, and compared these data with those of 791 ethnically matched controls from the 1000 Genomes Project and 574 controls from the FREX Consortium. FINDINGS: After filtering, mendelian genotypes were confirmed in eight probands, involving variants in C1QA, C1QC, C2, DNASE1L3, and IKZF1. Seven additional patients carried heterozygous variants in complement or type I interferon-associated autosomal recessive genes, with decreased concentrations of the encoded proteins C3 and C9 recorded in two patients. Rare variants that were predicted to be damaging were significantly enriched in the childhood-onset SLE cohort compared with controls; 25% of SLE probands versus 5% of controls were identified to harbour at least one rare, predicted damaging variant (p=2·98 × 10-11). Inborn errors of immunity were estimated to account for 7% of cases of childhood-onset SLE, with defects in innate immunity representing the main monogenic contribution. INTERPRETATION: An accumulation of rare variants that are predicted to be damaging in SLE-associated genes might contribute to disease expression and clinical heterogeneity. FUNDING: European Research Council.

Identification of mobile retrocopies during genetic testing: Consequences for routine diagnosis.

Human retrocopies, that is messenger RNA transcripts benefitting from the long interspersed element 1 machinery for retrotransposition, may have specific consequences for genomic testing. Next genetration sequencing (NGS) techniques allow the detection of such mobile elements but they may be misinterpreted as genomic duplications or be totally overlooked. We report eight observations of retrocopies detected during diagnostic NGS analyses of targeted gene panels, exome, or genome sequencing. For seven cases, while an exons-only copy number gain was called, read alignment inspection revealed a depth of coverage shift at every exon-intron junction where indels were also systematically called. Moreover, aberrant chimeric read pairs spanned entire introns or were paired with another locus for terminal exons. The 8th retrocopy was present in the reference genome and thus showed a normal NGS profile. We emphasize the existence of retrocopies and strategies to accurately detect them at a glance during genetic testing and discuss pitfalls for genetic testing.

The epilepsy phenotypic spectrum associated with a recurrent CUX2 variant.

OBJECTIVE: Cut homeodomain transcription factor CUX2 plays an important role in dendrite branching, spine development, and synapse formation in layer II to III neurons of the cerebral cortex. We identify a recurrent de novo CUX2 p.Glu590Lys as a novel genetic cause for developmental and epileptic encephalopathy (DEE). METHODS: The de novo p.Glu590Lys variant was identified by whole-exome sequencing (n = 5) or targeted gene panel (n = 4). We performed electroclinical and imaging phenotyping on all patients. RESULTS: The cohort comprised 7 males and 2 females. Mean age at study was 13 years (0.5-21.0). Median age at seizure onset was 6 months (2 months to 9 years). Seizure types at onset were myoclonic, atypical absence with myoclonic components, and focal seizures. Epileptiform activity on electroencephalogram was seen in 8 cases: generalized polyspike-wave (6) or multifocal discharges (2). Seizures were drug resistant in 7 or controlled with valproate (2). Six patients had a DEE: myoclonic DEE (3), Lennox-Gastaut syndrome (2), and West syndrome (1). Two had a static encephalopathy and genetic generalized epilepsy, including absence epilepsy in 1. One infant had multifocal epilepsy. Eight had severe cognitive impairment, with autistic features in 6. The p.Glu590Lys variant affects a highly conserved glutamine residue in the CUT domain predicted to interfere with CUX2 binding to DNA targets during neuronal development. INTERPRETATION: Patients with CUX2 p.Glu590Lys display a distinctive phenotypic spectrum, which is predominantly generalized epilepsy, with infantile-onset myoclonic DEE at the severe end and generalized epilepsy with severe static developmental encephalopathy at the milder end of the spectrum. Ann Neurol 2018;83:926-934.

LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription.

MyoD is a master regulator of myogenesis. Chromatin modifications required to trigger MyoD expression are still poorly described. Here, we demonstrate that the histone demethylase LSD1/KDM1a is recruited on the MyoD core enhancer upon muscle differentiation. Depletion of Lsd1 in myoblasts precludes the removal of H3K9 methylation and the recruitment of RNA polymerase II on the core enhancer, thereby preventing transcription of the non-coding enhancer RNA required for MyoD expression (CEeRNA). Consistently, Lsd1 conditional inactivation in muscle progenitor cells during embryogenesis prevented transcription of the CEeRNA and delayed MyoD expression. Our results demonstrate that LSD1 is required for the timely expression of MyoD in limb buds and identify a new biological function for LSD1 by showing that it can activate RNA polymerase II-dependent transcription of enhancers.

Comparison of two next-generation sequencing kits for diagnosis of epileptic disorders with a user-friendly tool for displaying gene coverage, DeCovA.

In recent years, molecular genetics has been playing an increasing role in the diagnostic process of monogenic epilepsies. Knowing the genetic basis of one patient's epilepsy provides accurate genetic counseling and may guide therapeutic options. Genetic diagnosis of epilepsy syndromes has long been based on Sanger sequencing and search for large rearrangements using MLPA or DNA arrays (array-CGH or SNP-array). Recently, next-generation sequencing (NGS) was demonstrated to be a powerful approach to overcome the wide clinical and genetic heterogeneity of epileptic disorders. Coverage is critical for assessing the quality and accuracy of results from NGS. However, it is often a difficult parameter to display in practice. The aim of the study was to compare two library-building methods (Haloplex, Agilent and SeqCap EZ, Roche) for a targeted panel of 41 genes causing monogenic epileptic disorders. We included 24 patients, 20 of whom had known disease-causing mutations. For each patient both libraries were built in parallel and sequenced on an Ion Torrent Personal Genome Machine (PGM). To compare coverage and depth, we developed a simple homemade tool, named DeCovA (Depth and Coverage Analysis). DeCovA displays the sequencing depth of each base and the coverage of target genes for each genomic position. The fraction of each gene covered at different thresholds could be easily estimated. None of the two methods used, namely NextGene and Ion Reporter, were able to identify all the known mutations/CNVs displayed by the 20 patients. Variant detection rate was globally similar for the two techniques and DeCovA showed that failure to detect a mutation was mainly related to insufficient coverage.