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

Next generation sequencing increases precise diagnoses in non-syndromic cleft lip and/or palate

Cleft of the lip and/or palate (CLP) are among the most common birth defects. Affected children need multidisciplinary treatment and care from birth until adulthood. They most commonly occur as an isolated defect and have a complex and heterogeneous etiology with genetic predisposition as well as environmental factors acting in concert. Whole Exome Sequencing (WES) has now become a useful approach for proving clinical molecular diagnosis. We conducted WES in 79 non-syndromic (ns) CLP multiplex families to identify rare causing variants. Among families studied, seven were found to be mutated (~10%) in syndromic genes: TP63, TBX1, LRP6, GRHL3 and TBX22 providing further evidence of contribution of syndromic CLP genes in ns forms. Unraveling TBX22 mutations in 2 families allowed us to describe a new clinical oral sign: bony overgrowths on the posterior edge of the hard palate, on each side of the palatal midline, as well as to expand the TBX22-related phenotypic spectrum to choanal atresia and Pierre-Robin sequence. We also described mutations in a new gene, LRRC1 in 2 distinct families: one with Van der Woude syndrome and one with ns cleft palate. We then focused on a distinguishable CLP phenotype and performed WES on eight sporadic or familial patients with “discontinuous cleft” which combined a cleft of the primary and the secondary palate with preservation of part of the intermaxillary palate. Three causative mutations were found in genes previously implicated in human CLP phenotype and/or syndrome. Our work illustrates how well-defined phenotype combined with the power of high throughput technologies significantly contribute to the understanding of genetic factor underlying CLP, and how it could help more accurate clinical, epidemiological, and fundamental research, ultimately resulting in better diagnosis and care of CLP patients(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Likely Pathogenic Variants in One Third of Non-Syndromic Discontinuous Cleft Lip and Palate Patients

Oral clefts are composed of cleft of the lip, cleft of the lip and palate, or cleft of the palate, and they are associated with a wide range of expression and severity. When cleft of the palate is associated with cleft of the lip with preservation of the primary palate, it defines an atypical phenotype called discontinuous cleft. Although this phenotype may represent 5% of clefts of the lip and/or palate (CLP), it is rarely specifically referred to and its pathophysiology is unknown. We conducted whole exome sequencing (WES) and apply a candidate gene approach to non-syndromic discontinuous CLP individuals in order to identify genes and deleterious variants that could underlie this phenotype. We discovered loss-of-function variants in two out of the seven individuals, implicating FGFR1 and DLG1 genes, which represents almost one third of this cohort. Whole exome sequencing of clinically well-defined subgroups of CLP, such as discontinuous cleft, is a relevant approach to study CLP etiopathogenesis. It could facilitate more accurate clinical, epidemiological and fundamental research, ultimately resulting in better diagnosis and care of CLP patients. Non-syndromic discontinuous cleft lip and palate seems to have a strong genetic basis

Génétique et developpement oculaire : exemple de variabilité phénotypique au sein d'une famille avec mutation du gène PAX6

REIMS-BU Santé (514542104) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Unmasking Familial CPX by WES and Identification of Novel Clinical Signs

Mutations in the T-Box transcription factor gene TBX22 are found in X-linked Cleft Palate with or without Ankyloglossia syndrome (CPX syndrome). In addition to X-linked inheritance, ankyloglossia, present in the majority of CPX patients, is an important diagnostic marker, but it is frequently missed or unreported, as it is a "minor" feature. Other described anomalies include cleft lip, micro and/or hypodontia, and features of CHARGE syndrome. We conducted whole exome sequencing (WES) on 22 individuals from 17 "a priori" non-syndromic cleft lip and/or cleft palate (CL/P) families. We filtered the data for heterozygous pathogenic variants within a set of predefined candidate genes. Two canonical splice-site mutations were found in TBX22. Detailed re-phenotyping of the two probands and their families unravelled orofacial features previously not associated with the CPX phenotypic spectrum: choanal atresia, Pierre-Robin sequence, and overgrowths on the posterior edge of the hard palate, on each side of the palatal midline. This study emphasizes the importance of WES analysis in familial CLP cases, combined with deep (reverse) phenotyping in "a priori" non-syndromic cleft

Likely Pathogenic Variants in One Third of Non-Syndromic Discontinuous Cleft Lip and Palate Patients.

Oral clefts are composed of cleft of the lip, cleft of the lip and palate, or cleft of the palate, and they are associated with a wide range of expression and severity. When cleft of the palate is associated with cleft of the lip with preservation of the primary palate, it defines an atypical phenotype called discontinuous cleft. Although this phenotype may represent 5% of clefts of the lip and/or palate (CLP), it is rarely specifically referred to and its pathophysiology is unknown. We conducted whole exome sequencing (WES) and apply a candidate gene approach to non-syndromic discontinuous CLP individuals in order to identify genes and deleterious variants that could underlie this phenotype. We discovered loss-of-function variants in two out of the seven individuals, implicating and genes, which represents almost one third of this cohort. Whole exome sequencing of clinically well-defined subgroups of CLP, such as discontinuous cleft, is a relevant approach to study CLP etiopathogenesis. It could facilitate more accurate clinical, epidemiological and fundamental research, ultimately resulting in better diagnosis and care of CLP patients. Non-syndromic discontinuous cleft lip and palate seems to have a strong genetic basis

Whole exome sequencing identifies mutations in 10% of patients with familial non-syndromic cleft lip and/or palate in genes mutated in well-known syndromes.

Oral clefts, that is, clefts of the lip and/or cleft palate (CL/P), are the most common craniofacial birth defects with an approximate incidence of ~1/700. To date, physicians stratify patients with oral clefts into either syndromic CL/P (syCL/P) or non-syndromic CL/P (nsCL/P) depending on whether the CL/P is associated with another anomaly or not. In general, patients with syCL/P follow Mendelian inheritance, while those with nsCL/P have a complex aetiology and, as such, do not adhere to Mendelian inheritance. Genome-wide association studies have identified approximately 30 risk loci for nsCL/P, which could explain a small fraction of heritability. To identify variants causing nsCL/P, we conducted whole exome sequencing on 84 individuals with nsCL/P, drawn from multiplex families (n=46). We identified rare damaging variants in four genes known to be mutated in syCL/P: (one family), (one family), (one family) and (two families), and clinical reassessment confirmed the isolated nature of their CL/P. These data demonstrate that patients with CL/P without cardinal signs of a syndrome may still carry a mutation in a gene linked to syCL/P. Rare coding and non-coding variants in syCL/P genes could in part explain the controversial question of 'missing heritability' for nsCL/P. Therefore, gene panels designed for diagnostic testing of syCL/P should be used for patients with nsCL/P, especially when there is at least third-degree family history. This would allow a more precise management, follow-up and genetic counselling. Moreover, stratified cohorts would allow hunting for genetic modifiers

Pseudohypoparathyroïdie : Distorsion du ratio de transmission maternelle des mutations perte de fonction de GNAS

International audienceLa PseudoHypoParathyroïdie de type 1A (PHP1A) et la PseudoPseudoHypoparathyroïdie (PPHP) sont deux maladies rares à transmission autosomique dominante provoquées par des mutations perte de fonction du gène GNAS soumis à empreinte, codant la protéine Gsα. La PHP1A est causée par des mutations sur l’allèle maternel et entraîne une Ostéodystrophie Héréditaire d’Albright (AHO) et une résistance à la PTH, tandis que la PPHP avec AHO et sans résistance hormonale est liée à des mutations de l’allèle paternel. Cette étude visait à étudier la transmission des mutations de GNAS. Nous avons mené une étude rétrospective sur un grand nombre de familles mutées GNAS. Pour éviter un biais de constatation en faveur d’une proportion plus élevée d’enfants affectés du à la manière dont les patients ont été inclus, les cas index faisant partie des fratrie de descendants ont été exclus. Le ratio de distribution des allèles mutés a été calculé à partir des génotypes observés chez des descendants de familles nucléaires et a été comparé au ratio attendu de 50% selon l'héritage mendélien (z-test à un échantillon). La transmission en fonction de la sévérité et du phénotype du parent transmettant de la mutation a également été analysée, ainsi que le sex ratio. L'étude a été réalisée sur 114 familles nucléaires et a inclus 250 descendants. Nous avons montré un excès de transmission maternelle d’allèles mutés (59%, P=0,022), d’autant plus important que les mutations étaient sévères (61,7%, P=0,023) et que l’allèle provenait de la grand-mère (64,7%, P=0,036). Une distribution mendélienne a été observée lorsque les mutations étaient héritées du père. Un déséquilibre du sex ratio en faveur des femmes a été observé parmi les porteurs, avec un sex ratio équilibré chez les descendants sains. La distorsion du ratio de transmission sexe-spécifique observée ici suggère un rôle de Gsα dans la biologie des ovocytes ou dans l'embryogenèse, avec des implications pour le conseil génétique

Monoallelic CRMP1 gene variants cause neurodevelopmental disorder

Collapsin response mediator proteins (CRMPs) are key for brain development and function. Here, we link CRMP1 to a neurodevelopmental disorder. We report heterozygous de novo variants in the CRMP1 gene in three unrelated individuals with muscular hypotonia, intellectual disability, and/or autism spectrum disorder. Based on in silico analysis these variants are predicted to affect the CRMP1 structure. We further analyzed the effect of the variants on the protein structure/levels and cellular processes. We showed that the human CRMP1 variants impact the oligomerization of CRMP1 proteins. Moreover, overexpression of the CRMP1 variants affect neurite outgrowth of murine cortical neurons. While altered CRMP1 levels have been reported in psychiatric diseases, genetic variants in CRMP1 gene have never been linked to human disease. We report for the first-time variants in the CRMP1 gene and emphasize its key role in brain development and function by linking directly to a human neurodevelopmental disease