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

Analysis and molecular characterisation of tumor hypoxia in the oropharyngeal squamous cell carcinoma

Les carcinomes épidermoïdes des voies aéro-digestives supérieures (VADS) se situent au sixième rang des cancers les plus fréquents dans le monde. Ces tumeurs sont liés à deux facteurs de risque : l’intoxication éthylo-tabagique (80% des cas) et l’infection de l’épithélium des VADS par les papillomavirus humain (HPV) à haut risque oncogène (20% des cas). Ces derniers définissent une sous-population de patients de meilleur pronostic. Une des hypothèses actuellement étudiées, afin d’expliquer la survie améliorée des patients HPV positifs, serait une hypoxie moindre dans ces tumeurs. En effet, les tumeurs des VADS sont fréquemment hypoxiques, et l’hypoxie intratumorale est un facteur de mauvais pronostic. Dans une première partie de cette thèse, nous avons entrepris une caractérisation moléculaire de l’hypoxie intratumorale dans les tumeurs humaines oropharyngées en fonction du statut HPV. Il apparaît que les tumeurs HPV positives présentent un statut hypoxique moindre comparées aux tumeurs HPV négatives. Ces tumeurs se caractérisent également par une abondante vascularisation intratumorale, qui pourrait être à l’origine de ce statut hypoxique moindre. Dans une deuxième partie, nous avons étudié l’adaptation à l’hypoxie de la lignée cellulaire HPV négative SQ20B et la lignée cellulaire HPV positive SCC90. De plus, des modèles de xénogreffes ont été établis à partir de ces mêmes lignées cellulaires et ont été analysés du point de vue de l’hypoxie intratumorale. De façon comparable aux tumeurs HPV positives, les xénogreffes obtenus à partir de la lignée SCC90 montre un statut hypoxique réduit comparés aux xénogreffes SQ20B. Les deux lignées cellulaires s’adaptent également différemment en hypoxie in vitro. La réponse à l’hypoxie dans la lignée SCC90 semble plus dynamique. En effet, la lignée SCC90 tente de s’adapter et de répondre à cet environnement hypoxique en induisant de fort niveau d’expression de gènes comparée à la lignée SQ20B.Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common malignancy worldwide. The major risk factors for HNSCC identified are tobacco use and alcohol consumption (80% of all HNSCC), which seem to have a synergistic effect. A subgroup of HNSCCs (20% of cases), particularly those of the oropharynx, is caused by infection with high-risk types of human papillomavirus (HPV). Human papillomavirus HPV-related oropharyngeal squamous cell carcinoma defines a distinct clinical subgroup of head and neck cancer patients with improved prognosis. Currently, one of the several hypothesis studied to account for their improved survival outcomes could be a distinct hypoxia status compared to their HPV-negative counterpart. Indeed, tumour hypoxia is common in solid tumours including head and neck tumours, and hypoxia is a well-known poor prognosis factor. In first part of this thesis, we have performed a molecular characterisation of tumor hypoxia on cohort of oropharyngeal tumours according to HPV status of the patients. The results support the hypothesis that HPV-related tumours display a lesser hypoxia status compared to HPV-negative oropharyngeal tumours. These HPV-related tumours also characterize by an abundant tumour vascularisation, which could be responsible for a lesser hypoxia status. In a second part, we have studied the ability of the adaptation to hypoxia of the HPV-positive SCC90 cell line and HPV-negative SQ20B cell line. Furthermore, HPV-positive and HPV-negative HNSCC xenograft models have been established and have been analysed about tumor hypoxia. Similar to HPV-related HNSCC, tumours-derived HPV positive cell lines display a reduced hypoxic status compared to tumours-derived HPV negative cell lines. The two cell lines adapt also differently to in vitro hypoxia. In the HPV-positive cell line, the hypoxia response pathways could be more dynamics. Indeed, SCC90 cell lines attempt to adapt and to reply to hypoxic environment inducing highly expression of all of the hypoxia related genes compared to SQ20B cell lines

Analyse et caractérisation moléculaire de l'hypoxie intratumorale de carcinomes épidermoïdes de l'oropharynx

Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common malignancy worldwide. The major risk factors for HNSCC identified are tobacco use and alcohol consumption (80% of all HNSCC), which seem to have a synergistic effect. A subgroup of HNSCCs (20% of cases), particularly those of the oropharynx, is caused by infection with high-risk types of human papillomavirus (HPV). Human papillomavirus HPV-related oropharyngeal squamous cell carcinoma defines a distinct clinical subgroup of head and neck cancer patients with improved prognosis. Currently, one of the several hypothesis studied to account for their improved survival outcomes could be a distinct hypoxia status compared to their HPV-negative counterpart. Indeed, tumour hypoxia is common in solid tumours including head and neck tumours, and hypoxia is a well-known poor prognosis factor. In first part of this thesis, we have performed a molecular characterisation of tumor hypoxia on cohort of oropharyngeal tumours according to HPV status of the patients. The results support the hypothesis that HPV-related tumours display a lesser hypoxia status compared to HPV-negative oropharyngeal tumours. These HPV-related tumours also characterize by an abundant tumour vascularisation, which could be responsible for a lesser hypoxia status. In a second part, we have studied the ability of the adaptation to hypoxia of the HPV-positive SCC90 cell line and HPV-negative SQ20B cell line. Furthermore, HPV-positive and HPV-negative HNSCC xenograft models have been established and have been analysed about tumor hypoxia. Similar to HPV-related HNSCC, tumours-derived HPV positive cell lines display a reduced hypoxic status compared to tumours-derived HPV negative cell lines. The two cell lines adapt also differently to in vitro hypoxia. In the HPV-positive cell line, the hypoxia response pathways could be more dynamics. Indeed, SCC90 cell lines attempt to adapt and to reply to hypoxic environment inducing highly expression of all of the hypoxia related genes compared to SQ20B cell lines.Les carcinomes épidermoïdes des voies aéro-digestives supérieures (VADS) se situent au sixième rang des cancers les plus fréquents dans le monde. Ces tumeurs sont liés à deux facteurs de risque : l’intoxication éthylo-tabagique (80% des cas) et l’infection de l’épithélium des VADS par les papillomavirus humain (HPV) à haut risque oncogène (20% des cas). Ces derniers définissent une sous-population de patients de meilleur pronostic. Une des hypothèses actuellement étudiées, afin d’expliquer la survie améliorée des patients HPV positifs, serait une hypoxie moindre dans ces tumeurs. En effet, les tumeurs des VADS sont fréquemment hypoxiques, et l’hypoxie intratumorale est un facteur de mauvais pronostic. Dans une première partie de cette thèse, nous avons entrepris une caractérisation moléculaire de l’hypoxie intratumorale dans les tumeurs humaines oropharyngées en fonction du statut HPV. Il apparaît que les tumeurs HPV positives présentent un statut hypoxique moindre comparées aux tumeurs HPV négatives. Ces tumeurs se caractérisent également par une abondante vascularisation intratumorale, qui pourrait être à l’origine de ce statut hypoxique moindre. Dans une deuxième partie, nous avons étudié l’adaptation à l’hypoxie de la lignée cellulaire HPV négative SQ20B et la lignée cellulaire HPV positive SCC90. De plus, des modèles de xénogreffes ont été établis à partir de ces mêmes lignées cellulaires et ont été analysés du point de vue de l’hypoxie intratumorale. De façon comparable aux tumeurs HPV positives, les xénogreffes obtenus à partir de la lignée SCC90 montre un statut hypoxique réduit comparés aux xénogreffes SQ20B. Les deux lignées cellulaires s’adaptent également différemment en hypoxie in vitro. La réponse à l’hypoxie dans la lignée SCC90 semble plus dynamique. En effet, la lignée SCC90 tente de s’adapter et de répondre à cet environnement hypoxique en induisant de fort niveau d’expression de gènes comparée à la lignée SQ20B

Analysis and molecular characterisation of tumor hypoxia in the oropharyngeal squamous cell carcinoma

Les carcinomes épidermoïdes des voies aéro-digestives supérieures (VADS) se situent au sixième rang des cancers les plus fréquents dans le monde. Ces tumeurs sont liés à deux facteurs de risque : l’intoxication éthylo-tabagique (80% des cas) et l’infection de l’épithélium des VADS par les papillomavirus humain (HPV) à haut risque oncogène (20% des cas). Ces derniers définissent une sous-population de patients de meilleur pronostic. Une des hypothèses actuellement étudiées, afin d’expliquer la survie améliorée des patients HPV positifs, serait une hypoxie moindre dans ces tumeurs. En effet, les tumeurs des VADS sont fréquemment hypoxiques, et l’hypoxie intratumorale est un facteur de mauvais pronostic. Dans une première partie de cette thèse, nous avons entrepris une caractérisation moléculaire de l’hypoxie intratumorale dans les tumeurs humaines oropharyngées en fonction du statut HPV. Il apparaît que les tumeurs HPV positives présentent un statut hypoxique moindre comparées aux tumeurs HPV négatives. Ces tumeurs se caractérisent également par une abondante vascularisation intratumorale, qui pourrait être à l’origine de ce statut hypoxique moindre. Dans une deuxième partie, nous avons étudié l’adaptation à l’hypoxie de la lignée cellulaire HPV négative SQ20B et la lignée cellulaire HPV positive SCC90. De plus, des modèles de xénogreffes ont été établis à partir de ces mêmes lignées cellulaires et ont été analysés du point de vue de l’hypoxie intratumorale. De façon comparable aux tumeurs HPV positives, les xénogreffes obtenus à partir de la lignée SCC90 montre un statut hypoxique réduit comparés aux xénogreffes SQ20B. Les deux lignées cellulaires s’adaptent également différemment en hypoxie in vitro. La réponse à l’hypoxie dans la lignée SCC90 semble plus dynamique. En effet, la lignée SCC90 tente de s’adapter et de répondre à cet environnement hypoxique en induisant de fort niveau d’expression de gènes comparée à la lignée SQ20B.Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common malignancy worldwide. The major risk factors for HNSCC identified are tobacco use and alcohol consumption (80% of all HNSCC), which seem to have a synergistic effect. A subgroup of HNSCCs (20% of cases), particularly those of the oropharynx, is caused by infection with high-risk types of human papillomavirus (HPV). Human papillomavirus HPV-related oropharyngeal squamous cell carcinoma defines a distinct clinical subgroup of head and neck cancer patients with improved prognosis. Currently, one of the several hypothesis studied to account for their improved survival outcomes could be a distinct hypoxia status compared to their HPV-negative counterpart. Indeed, tumour hypoxia is common in solid tumours including head and neck tumours, and hypoxia is a well-known poor prognosis factor. In first part of this thesis, we have performed a molecular characterisation of tumor hypoxia on cohort of oropharyngeal tumours according to HPV status of the patients. The results support the hypothesis that HPV-related tumours display a lesser hypoxia status compared to HPV-negative oropharyngeal tumours. These HPV-related tumours also characterize by an abundant tumour vascularisation, which could be responsible for a lesser hypoxia status. In a second part, we have studied the ability of the adaptation to hypoxia of the HPV-positive SCC90 cell line and HPV-negative SQ20B cell line. Furthermore, HPV-positive and HPV-negative HNSCC xenograft models have been established and have been analysed about tumor hypoxia. Similar to HPV-related HNSCC, tumours-derived HPV positive cell lines display a reduced hypoxic status compared to tumours-derived HPV negative cell lines. The two cell lines adapt also differently to in vitro hypoxia. In the HPV-positive cell line, the hypoxia response pathways could be more dynamics. Indeed, SCC90 cell lines attempt to adapt and to reply to hypoxic environment inducing highly expression of all of the hypoxia related genes compared to SQ20B cell lines

Human Papillomavirus-related tumours of the oropharynx display a lower tumour hypoxia signature

OBJECTIVES: Human Papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OSCC) patients have improved prognosis compared to other head and neck (HNSCC) cancers. Since poor prognosis is associated with tumour hypoxia, we studied whether the hypoxic response is different in HPV-related cells and tumours. MATERIAL AND METHODS: HPV-positive and -negative cells were incubated in hypoxia and analyzed by qRTPCR, western blotting and cell proliferation assays. Tumours formed by xenografting these cells in nude mice were studied by IHC. HNSCC patient samples were analyzed by unsupervised clustering of hypoxia-related gene expression, quantitative real-time PCR (qRTPCR) and immunohistochemical (IHC) detection of neo-blood vessels. RESULTS AND CONCLUSION: HPV-positive and -negative cells responded differently to hypoxia, in terms of gene expression (HIF-1α, PHD-3, GLUT-1 and VEGF-A) and cell survival. Tumour xenografts formed by HPV-positive cells had fewer hypoxic areas than those formed by HPV-negative cells. HPV related tumours were less hypoxic, expressed lower levels of hypoxia-responsive genes, and had a higher density of neo-blood vessels. HPV-related OSCC display lower tumour hypoxia, which could be linked to the distinct intrinsic abilities of HPV-positive tumour cells to adapt to hypoxia and to their better prognosis

The FBN2 gene: new mutations, locus-specific database (Universal Mutation Database FBN2), and genotype-phenotype correlations.

International audienceCongenital contractural arachnodactyly (CCA) is an extremely rare disease, due to mutations in the FBN2 gene encoding fibrillin-2. Another member of the fibrillin family, the FBN1 gene, is involved in a broad phenotypic continuum of connective-tissue disorders including Marfan syndrome. Identifying not only what is in common but also what differentiates these two proteins should enable us to better comprehend their respective functions and better understand the multitude of diseases in which these two genes are involved. In 1995 we created a locus-specific database (LSDB) for FBN1 mutations with the Universal Mutation Database (UMD) tool. To facilitate comparison of identified mutations in these two genes and search for specific functional areas, we created an LSDB for the FBN2 gene: the UMD-FBN2 database. This database lists 26 published and six newly identified mutations that mainly comprise missense and splice-site mutations. Although the number of described FBN2 mutations was low, the frequency of joint dislocation was significantly higher with missense mutations when compared to splice site mutations

Agrin mutations lead to a congenital myasthenic syndrome with distal muscle weakness and atrophy

International audienceCongenital myasthenic syndromes are a clinically and genetically heterogeneous group of rare diseases resulting from impaired neuromuscular transmission. Their clinical hallmark is fatigable muscle weakness associated with a decremental muscle response to repetitive nerve stimulation and frequently related to postsynaptic defects. Distal myopathies form another clinically and genetically heterogeneous group of primary muscle disorders where weakness and atrophy are restricted to distal muscles, at least initially. In both congenital myasthenic syndromes and distal myopathies, a significant number of patients remain genetically undiagnosed. Here, we report five patients from three unrelated families with a strikingly homogenous clinical entity combining congenital myasthenia with distal muscle weakness and atrophy reminiscent of a distal myopathy. MRI and neurophysiological studies were compatible with mild myopathy restricted to distal limb muscles, but decrement (up to 72%) in response to 3 Hz repetitive nerve stimulation pointed towards a neuromuscular transmission defect. Post-exercise increment (up to 285%) was observed in the distal limb muscles in all cases suggesting presynaptic congenital myasthenic syndrome. Immunofluorescence and ultrastructural analyses of muscle end-plate regions showed synaptic remodelling with denervation-reinnervation events. We performed whole-exome sequencing in two kinships and Sanger sequencing in one isolated case and identified five new recessive mutations in the gene encoding agrin. This synaptic proteoglycan with critical function at the neuromuscular junction was previously found mutated in more typical forms of congenital myasthenic syndrome. In our patients, we found two missense mutations residing in the N-terminal agrin domain, which reduced acetylcholine receptors clustering activity of agrin in vitro. Our findings expand the spectrum of congenital myasthenic syndromes due to agrin mutations and show an unexpected correlation between the mutated gene and the associated phenotype. This provides a good rationale for examining patients with apparent distal myopathy for a neuromuscular transmission disorder and agrin mutations

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata511 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata511 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype

Factors associated with COVID-19-related death in people with rheumatic diseases: results from the COVID-19 Global Rheumatology Alliance physician-reported registry

Objectives: To determine factors associated with COVID-19-related death in people with rheumatic diseases. Methods: Physician-reported registry of adults with rheumatic disease and confirmed or presumptive COVID-19 (from 24 March to 1 July 2020). The primary outcome was COVID-19-related death. Age, sex, smoking status, comorbidities, rheumatic disease diagnosis, disease activity and medications were included as covariates in multivariable logistic regression models. Analyses were further stratified according to rheumatic disease category. Results: Of 3729 patients (mean age 57 years, 68% female), 390 (10.5%) died. Independent factors associated with COVID-19-related death were age (66-75 years: OR 3.00, 95% CI 2.13 to 4.22; >75 years: 6.18, 4.47 to 8.53; both vs ≤65 years), male sex (1.46, 1.11 to 1.91), hypertension combined with cardiovascular disease (1.89, 1.31 to 2.73), chronic lung disease (1.68, 1.26 to 2.25) and prednisolone-equivalent dosage >10 mg/day (1.69, 1.18 to 2.41; vs no glucocorticoid intake). Moderate/high disease activity (vs remission/low disease activity) was associated with higher odds of death (1.87, 1.27 to 2.77). Rituximab (4.04, 2.32 to 7.03), sulfasalazine (3.60, 1.66 to 7.78), immunosuppressants (azathioprine, cyclophosphamide, ciclosporin, mycophenolate or tacrolimus: 2.22, 1.43 to 3.46) and not receiving any disease-modifying anti-rheumatic drug (DMARD) (2.11, 1.48 to 3.01) were associated with higher odds of death, compared with methotrexate monotherapy. Other synthetic/biological DMARDs were not associated with COVID-19-related death. Conclusion: Among people with rheumatic disease, COVID-19-related death was associated with known general factors (older age, male sex and specific comorbidities) and disease-specific factors (disease activity and specific medications). The association with moderate/high disease activity highlights the importance of adequate disease control with DMARDs, preferably without increasing glucocorticoid dosages. Caution may be required with rituximab, sulfasalazine and some immunosuppressants