A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Facteurs antitryspiques de la graine de soja : évaluation de la variabilité génotypique dans une collection de référence ; effet du semis précoce et de la réduction de l’irrigation

La graine de soja, source exceptionnelle de protéines et d’acides gras polyinsaturés, contient de nombreux facteurs antinutritionnels (FAN) qui rendent impossible sa consommation sans traitement préalable. Parmi ces FAN, les facteurs antitrypsiques (FAT) réduisent fortement la croissance des monogastriques en perturbant l’assimilation des protéines et le fonctionnement pancréatique. Les FAT sont de deux types chez le soja : les inhibiteurs de Kunitz (KTI), et les inhibiteurs de Bowman-Birk (BBI). L’introduction d’un allèle KTI nul réduit l’activité antitrypsique (AAT) mais les BBI restants produisent encore une AAT trop élevée pour que la graine soit consommée crue. Les conditions environnementales peuvent aussi fortement influencer l’AAT. L’objectif de ce travail est d’explorer la variabilité dans une collection variétale et d’analyser l’effet d’une modification des pratiques culturales sur l’AAT en utilisant une méthode enzymatique sur microplaque, plus rapide et moins couteuse. L’erreur relative obtenue est de 7 %. Les variétés de la collection ont des AAT de 23 à 60 TIU.mg-1. Ces AAT ne sont pas corrélées avec la teneur en protéines. Certaines variétés sont probablement porteuses de la mutation KTI nulle, tout en présentant de très fortes teneurs en protéines. L’avancement de la date de semis induit une légère diminution de l’AAT, ce qui montre qu’un tel changement de pratique, tout en préservant la teneur en protéine, serait compatible avec l’amélioration de la qualité de la graine.Soybean seed is a highly edible source of proteins and polyinsaturated fatty acids. However, it contains many antinutritional components (FAN) that pose restriction to the consumption of raw soybean. Among them, trypsin inhibitors (FAT) strongly reduce the growth of monogastric animals by the reduction of protein digestibility and the induction of pancreatic disorders. Two kinds of FAT are found in soybean seed: Kunitz inhibitors (KTI) and Bowman-Birk inhibitors (BBI). The trypsin inhibition activity (AAT) can be reduced by the use of a KTI null allele in breeding programs but the activity due to the BBI peptides remains too high to allow the use of raw soybean in animal feeding. Environmental growth conditions can also influence the seed AAT. The aim of this work was the evaluation of genotypic variability in a core collection and the study of the effect of a modification of the agronomic practices on the AAT of the seed by the use of a faster and cheaper microanalysis. The relative error was 7%. The AAT of the sampled accessions of the core collection was between 23 and 60 TIU.mg(-1). The correlation between AAT and protein content was not significant. Some accessions with a high protein content are probably homozygous for the KTI null allele. Very early sowing induces a slight but significant decrease of the AAT. Thus, such a change in agronomic practices can both preserve the high protein content and the seed nutritional quality

Facteurs antitryspiques de la graine de soja : évaluation de la variabilité génotypique dans une collection de référence ; effet du semis précoce et de la réduction de l’irrigation

La graine de soja, source exceptionnelle de protéines et d’acides gras polyinsaturés, contient de nombreux facteurs antinutritionnels (FAN) qui rendent impossible sa consommation sans traitement préalable. Parmi ces FAN, les facteurs antitrypsiques (FAT) réduisent fortement la croissance des monogastriques en perturbant l’assimilation des protéines et le fonctionnement pancréatique. Les FAT sont de deux types chez le soja : les inhibiteurs de Kunitz (KTI), et les inhibiteurs de Bowman-Birk (BBI). L’introduction d’un allèle KTI nul réduit l’activité antitrypsique (AAT) mais les BBI restants produisent encore une AAT trop élevée pour que la graine soit consommée crue. Les conditions environnementales peuvent aussi fortement influencer l’AAT. L’objectif de ce travail est d’explorer la variabilité dans une collection variétale et d’analyser l’effet d’une modification des pratiques culturales sur l’AAT en utilisant une méthode enzymatique sur microplaque, plus rapide et moins couteuse. L’erreur relative obtenue est de 7 %. Les variétés de la collection ont des AAT de 23 à 60 TIU.mg-1. Ces AAT ne sont pas corrélées avec la teneur en protéines. Certaines variétés sont probablement porteuses de la mutation KTI nulle, tout en présentant de très fortes teneurs en protéines. L’avancement de la date de semis induit une légère diminution de l’AAT, ce qui montre qu’un tel changement de pratique, tout en préservant la teneur en protéine, serait compatible avec l’amélioration de la qualité de la graine

Functional and genetic analyses of ZYG11B

Abstract Background The Oculo‐Auriculo‐Vertebral Spectrum (OAVS) or Goldenhar Syndrome is an embryonic developmental disorder characterized by hemifacial microsomia associated with auricular, ocular and vertebral malformations. The clinical heterogeneity of this spectrum and its incomplete penetrance limited the molecular diagnosis. In this study, we describe a novel causative gene, ZYG11B. Methods A sporadic case of OAVS was analyzed by whole exome sequencing in trio strategy. The identified candidate gene, ZYG11B, was screened in 143 patients by next generation sequencing. Overexpression and immunofluorescence of wild‐type and mutated ZYG11B forms were performed in Hela cells. Moreover, morpholinos were used for transient knockdown of its homologue in zebrafish embryo. Results A nonsense de novo heterozygous variant in ZYG11B, (NM_024646, c.1609G>T, p.Glu537*) was identified in a single OAVS patient. This variant leads in vitro to a truncated protein whose subcellular localization is altered. Transient knockdown of the zebrafish homologue gene confirmed its role in craniofacial cartilages architecture and in notochord development. Moreover, ZYG11B expression regulates a cartilage master regulator, SOX6, and is regulated by Retinoic Acid, a known developmental toxic molecule leading to clinical features of OAVS. Conclusion Based on genetic, cellular and animal model data, we proposed ZYG11B as a novel rare causative gene for OAVS

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

International audienceCRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations

International audienceCRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nucleaseinduced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on-and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies

Psychosocial Impact of Predictive Genetic Testing in Hereditary Heart Diseases: The PREDICT Study

International audiencePredictive genetic testing (PGT) is offered to asymptomatic relatives at risk of hereditary heart disease, but the impact of result disclosure has been little studied. We evaluated the psychosocial impacts of PGT in hereditary heart disease, using self-report questionnaires (including the State-Trait Anxiety Inventory) in 517 adults, administered three times to the prospective cohort (PCo: n = 264) and once to the retrospective cohort (RCo: n = 253). The main motivations for undergoing PGT were "to remove doubt" and "for their children". The level of anxiety increased between pre-test and result appointments (p <0.0001), returned to baseline after the result (PCo), and was moderately elevated at 4.4 years (RCo). Subjects with a history of depression or with high baseline anxiety were more likely to develop anxiety after PGT result (p = 0.004 and p <0.0001, respectively), whatever it was. Unfavourable changes in professional and/or family life were observed in 12.4% (PCo) and 18.7% (RCo) of subjects. Few regrets about PGT were expressed (0.8% RCo, 2.3% PCo). Medical benefit was not the main motivation, which emphasises the role of pre/post-test counselling. When PGT was performed by expert teams, the negative impact was modest, but careful management is required in specific categories of subjects, whatever the genetic test result

A framework to identify contributing genes in patients with Phelan-McDermid syndrome

International audience[This corrects the article DOI: 10.1038/s41525-017-0035-2.]

A framework to identify contributing genes in patients with Phelan-McDermid syndrome

The underlying genetics of Phelan-McDermid syndrome Multiple chromosomal changes may impact the severity of symptoms in people with Phelan-McDermid syndrome (PMS). Thomas Bourgeron of the Institut Pasteur and colleagues in France conducted genomic analyses and explored the clinical features of 85 people with PMS, a condition caused by a deletion in the long arm of chromosome 22. It is often associated with severe symptoms such as intellectual disability, autism, and seizures. The chromosomal changes in 65% of those studied were not inherited. MRI brain scans showed visible abnormalities in 23 of 35 patients imaged. The size of the chromosomal deletion varied. Patients with small deletions were more likely to have autistic symptoms while those with large deletions were more likely to be unable to talk. The team also identified genes in chromosome 22 and in other regions of the genome that could modify the severity of PMS symptoms