The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Wnt/ß-catenin pathway and cell adhesion deregulation in CSDE1-related intellectual disability and autism spectrum disorders.

Among the genetic factors playing a key role in the etiology of intellectual disabilities (IDs) and autism spectrum disorders (ASDs), several encode RNA-binding proteins (RBPs). In this study, we deciphered the molecular and cellular bases of ID-ASD in a patient followed from birth to the age of 21, in whom we identified a de novo CSDE1 (Cold Shock Domain-containing E1) nonsense variation. CSDE1 encodes an RBP that regulates multiple cellular pathways by monitoring the translation and abundance of target transcripts. Analyses performed on the patient's primary fibroblasts showed that the identified CSDE1 variation leads to haploinsufficiency. We identified through RNA-seq assays the Wnt/ß-catenin signaling and cellular adhesion as two major deregulated pathways. These results were further confirmed by functional studies involving Wnt-specific luciferase and substrate adhesion assays. Additional data support a disease model involving APC Down-Regulated-1 (APCDD1) and cadherin-2 (CDH2), two components of the Wnt/ß-catenin pathway, CDH2 being also pivotal for cellular adhesion. Our study, which relies on both the deep phenotyping and long-term follow-up of a patient with CSDE1 haploinsufficiency and on ex vivo studies, sheds new light on the CSDE1-dependent deregulated pathways in ID-ASD.

[Health professionals facing the coronavirus disease 2019 (COVID-19) pandemic: What are the mental health risks?] / Les professionnels de santé face à la pandémie de la maladie à coronavirus (COVID-19) : quels risques pour leur santé mentale ?

OBJECTIVES: The coronavirus disease 2019 (COVID-19) pandemic has caused major sanitary crisis worldwide. Half of the world has been placed in quarantine. In France, this large-scale health crisis urgently triggered the restructuring and reorganization of health service delivery to support emergency services, medical intensive care units and continuing care units. Health professionals mobilized all their resources to provide emergency aid in a general climate of uncertainty. Concerns about the mental health, psychological adjustment, and recovery of health care workers treating and caring for patients with COVID-19 are now arising. The goal of the present article is to provide up-to-date information on potential mental health risks associated with exposure of health professionals to the COVID-19 pandemic. METHODS: Authors performed a narrative review identifying relevant results in the scientific and medical literature considering previous epidemics of 2003 (SARS-CoV-1) and 2009 (H1N1) with the more recent data about the COVID-19 pandemic. We highlighted most relevant data concerning the disease characteristics, the organizational factors and personal factors that may contribute to developing psychological distress and other mental health symptoms. RESULTS: The disease characteristics of the current COVID-19 pandemic provoked a generalized climate of wariness and uncertainty, particularly among health professionals, due to a range of causes such as the rapid spread of COVID-19, the severity of symptoms it can cause in a segment of infected individuals, the lack of knowledge of the disease, and deaths among health professionals. Stress may also be caused by organizational factors, such as depletion of personal protection equipment, concerns about not being able to provide competent care if deployed to new area, concerns about rapidly changing information, lack of access to up-to-date information and communication, lack of specific drugs, the shortage of ventilators and intensive care unit beds necessary to care for the surge of critically ill patients, and significant change in their daily social and family life. Further risk factors have been identified, including feelings of being inadequately supported, concerns about health of self, fear of taking home infection to family members or others, and not having rapid access to testing through occupational health if needed, being isolated, feelings of uncertainty and social stigmatization, overwhelming workload, or insecure attachment. Additionally, we discussed positive social and organizational factors that contribute to enhance resilience in the face of the pandemic. There is a consensus in all the relevant literature that health care professionals are at an increased risk of high levels of stress, anxiety, depression, burnout, addiction and post-traumatic stress disorder, which could have long-term psychological implications. CONCLUSIONS: In the long run, this tragic health crisis should significantly enhance our understanding of the mental health risk factors among the health care professionals facing the COVID-19 pandemic. Reporting information such as this is essential to plan future prevention strategies. Protecting health care professionals is indeed an important component of public health measures to address large-scale health crisis. Thus, interventions to promote mental well-being in health care professionals exposed to COVID-19 need to be immediately implemented, and to strengthen prevention and response strategies by training health care professionals on mental help and crisis management.

Penetrance is a critical parameter for assessing the disease liability of CFTR variants.

BACKGROUND: Major issues of newborn screening (NBS) for CF are the assessment of disease liability of variants and of the penetrance of clinical CF, notably in inconclusive diagnosis. The penetrance of CF is defined as the risk of a particular genotype to lead to a CF phenotype. METHODS: We aimed to get insight into the penetrance of CF for fifteen CFTR variants: 5 frequent CF-causing and 10 classified as of varying clinical consequence (VCC) or associated with a CFTR-related disorder (CFTR-RD) in CFTR2 or CFTR-France databases. The penetrance was approached by: (1) comparison of variant allelic frequencies in CF patients (CFTR2) and in the general population; (2) estimation of the likelihood of a positive NBS test for the 14 compound heterozygous with F508del and the F508del homozygous genotypes, defined as the ratio of detected/expected number of neonates with a given genotype in the 2002-2017 period. RESULTS: A full penetrance was observed for severe CF-causing variants. Five variants were more frequently found in the general population than in CF patients: TG11T5, TG12T5, TG13T5, L997F and R117H;T7. The likelihood of a positive NBS test was 0.03% for TG11T5, 0.3% for TG12T5, 1.9% for TG13T5, 0.6% for L997F, 11.7% for D1152H, and 17.8% for R117H;T7. Penetrance varied greatly for variants with discrepant classification between CFTR2 and CFTR-France: 5.1% for R117C, 12.3% for T338I, 43.5% for D110H and 52.6% for L206W. CONCLUSION: These results illustrate the contribution of genetics population data to assess the disease liability of variants for diagnosis and genetic counselling purposes.

Current and future diagnosis of cystic fibrosis: Performance and limitations.

Cystic fibrosis (CF) is the most frequent genetic disorder in the Caucasian population benefiting from systematic newborn screening tests. It is also the most frequent indication of prenatal and preimplantation genetic diagnosis for a single gene disorder. During the past thirty years, thanks in part to the evolution of diagnostic techniques, our knowledge on CFTR genetics and pathophysiological mechanisms involved in CF have significantly improved. With the implementation of newborn screening in France and in several countries, the diagnosis now often occurs in clinically asymptomatic infants and this has modified the criteria for CF diagnosis. Recently, guidelines for CF diagnosis have been reformulated in Europe and the US, in regard to sweat chloride usual values and disease terminology. This review describes the methods and molecular approaches that are used in routine practice or are being developed to detect CFTR protein dysfunction and to identify disease-causing CFTR variants. Ultimately, an optimal use of all these functional and genetic resources may improve patient care and therapeutic decision-making. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

Functional characterization and phenotypic spectrum of three recurrent disease-causing deep intronic variants of the CFTR gene.

BACKGROUND: The CFTR genotype remains incomplete in 1% of Cystic Fibrosis (CF) cases, because only one or no disease-causing variants is detected after extended analysis. This fraction is probably higher in CFTR-Related Disorders (CFTR-RD). Deep-intronic CFTR variants are putative candidates to fill this gap. However, the recurrence, phenotypic spectrum and full molecular characterization of newly reported variants are unknown. METHODS: Minigenes and analysis of CFTR transcripts in nasal epithelial cells were used to determine the impact on CFTR splicing of intronic variants that we previously identified by next generation sequencing of the whole CFTR locus. Phenotypic data were collected in 19 patients with CF and CFTR-RD, in whom one of the deep intronic variants has been detected. RESULTS: Three deep-intronic variants promoted the inclusion of pseudo-exons (PE) in the CFTR transcript, hindering the synthesis of a functional protein. The c.2989-313A > T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118 bp PE. The c.3469-1304C > G variant promoted the inclusion of a 214 bp-PE and was identified in five patients with CF from four families. Haplotype analysis confirmed that this variant was associated with one CF chromosome of African origin. The most represented variant in our cohort was the c.3874-4522A > G, detected in 10 patients with various phenotypes, from male infertility to CF with pancreatic insufficiency. CONCLUSION: These three deep intronic CFTR variants are associated with a large phenotypic spectrum, including typical CF. They should be included in CF diagnostic testing and carrier screening strategies.

Wiedemann-Steiner syndrome as a major cause of syndromic intellectual disability: A study of 33 French cases.

Wiedemann-Steiner syndrome (WSS) is a rare syndromic condition in which intellectual disability (ID) is associated with hypertrichosis cubiti, short stature, and characteristic facies. Following the identification of the causative gene (KMT2A) in 2012, only 31 cases of WSS have been described precisely in the literature. We report on 33 French individuals with a KMT2A mutation confirmed by targeted gene sequencing, high-throughput sequencing or exome sequencing. Patients' molecular and clinical features were recorded and compared with the literature data. On the molecular level, we found 29 novel mutations. We observed autosomal dominant transmission of WSS in 3 families and mosaicism in one family. Clinically, we observed a broad phenotypic spectrum with regard to ID (mild to severe), the facies (typical or not of WSS) and associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Hypertrichosis cubiti that was supposed to be pathognomonic in the literature was found only in 61% of our cases. This is the largest series of WSS cases yet described to date. A majority of patients exhibited suggestive features, but others were less characteristic, only identified by molecular diagnosis. The prevalence of WSS was higher than expected in patients with ID, suggesting than KMT2A is a major gene in ID.

The association of severe encephalopathy and question mark ear is highly suggestive of loss of MEF2C function.

Auriculocondylar syndrome and isolated question mark ear result from dysregulation of the endothelin 1-endothelin receptor type A signaling pathway. Animal models have highlighted the role of the transcription factor MEF2C as an effector of this pathway. We report heterozygous MEF2C loss-of-function as a possible cause of question mark ear associated with intellectual deficiency.

Autism spectrum disorder recurrence, resulting of germline mosaicism for a CHD2 gene missense variant.

Germline mosaicism for a novel missense variant p.Thr645Met located in the SNF2-related ATP dependent helicase domain of CHD2 in 2 affected siblings with autism spectrum disorder.

Xq28 duplication including MECP2 in six unreported affected females: what can we learn for diagnosis and genetic counselling?

Duplication of the Xq28 region, involving MECP2 (dupMECP2), has been primarily described in males with severe developmental delay, spasticity, epilepsy, stereotyped movements and recurrent infections. Carrier mothers are usually asymptomatic with an extremely skewed X chromosome inactivation (XCI) pattern. We report a series of six novel symptomatic females carrying a de novo interstitial dupMECP2, and review the 14 symptomatic females reported to date, with the aim to further delineate their phenotype and give clues for genetic counselling. One patient was adopted and among the other 19 patients, seven (37%) had inherited their duplication from their mother, including three mildly (XCI: 70/30, 63/37, 100/0 in blood and random in saliva), one moderately (XCI: random) and three severely (XCI: uninformative and 88/12) affected patients. After combining our data with data from the literature, we could not show a correlation between XCI in the blood or duplication size and the severity of the phenotype, or explain the presence of a phenotype in these females. These findings confirm that an abnormal phenotype, even severe, can be a rare event in females born to asymptomatic carrier mothers, making genetic counselling difficult in couples at risk in terms of prognosis, in particular in prenatal cases.

Identification of intellectual disability genes showing circadian clock-dependent expression in the mouse hippocampus.

Sleep is strongly implicated in learning, especially in the reprocessing of recently acquired memory. Children with intellectual disability (ID) tend to have sleep-wake disturbances, which may contribute to the pathophysiology of the disease. Given that sleep is partly controlled by the circadian clock, we decided to study the rhythmic expression of genes in the hippocampus, a brain structure which plays a key role in memory in humans and rodents. By investigating the hippocampal transcriptome of adult mice, we identified 663 circadian clock controlled (CCC) genes, which we divided into four categories based on their temporal pattern of expression. In addition to the standard core clock genes, enrichment analysis identified several transcription factors among these hippocampal CCC genes, and our findings suggest that genes from one cluster regulate the expression of those in another. Interestingly, these hippocampal CCC genes were highly enriched in sleep/wakefulness-related genes. We show here that several genes in the glucocorticoid signaling pathway, which is involved in memory, show a CCC pattern of expression. However, ID genes were not enriched among these CCC genes, suggesting that sleep or learning and memory disturbances observed in patients with ID are probably not related to the circadian clock in the hippocampus.

Medial prefrontal cortex neuronal circuits in fear behavior.

The medial prefrontal cortex (mPFC) has emerged as a key structure involved in the modulation of fear behavior over the past few decades. Anatomical, functional and electrophysiological studies have begun to shed light on the precise mechanisms by which different prefrontal regions regulate the expression and inhibition of fear behavior. These studies have established a canonical view of mPFC functions during fear behavior with dorsal regions selectively involved in the expression of fear behavior and ventral regions linked to the inhibition of fear behavior. Although numerous reports support this view, recent data have refined this model and suggested that dorsal prefrontal regions might also play an important role in the encoding of fear behavior itself. The recent development of sophisticated approaches such as large scale neuronal recordings, simultaneous multisite recordings of spiking activity and local field potentials (LFPs) along with optogenetic approaches will facilitate the testing of these new hypotheses in the near future. Here we provide an extensive review of the literature on the role of mPFC in fear behavior and propose further directions to dissect the contribution of specific prefrontal neuronal elements and circuits in the regulation of fear behavior.

FOXG1-Related Disorders: From Clinical Description to Molecular Genetics.

Rett syndrome (RTT) is a severe neurodevelopmental disease that affects approximately 1 in 10,000 live female births and is often caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MECP2). Mutations in loci other than MECP2 have also been found in individuals that have been labeled as atypical RTT. Among them, a mutation in the gene forkhead box G1 (FOXG1) has been involved in the molecular aetiology of the congenital variant of RTT. The FOXG1 gene encodes a winged-helix transcriptional repressor essential for the development of the ventral telencephalon in embryonic forebrain. Later, FOXG1 continues to be expressed in neurogenetic zones of the postnatal brain. Although RTT affects quasi-exclusively girls, FOXG1 mutations have also been identified in male patients. As far as we know, about 12 point mutations and 13 cases with FOXG1 molecular abnormalities (including translocation, duplication and large deletion on the chromosome 14q12) have been described in the literature. Affected individuals with FOXG1 mutations have shown dysmorphic features and Rett-like clinical course, including normal perinatal period, postnatal microcephaly, seizures and severe mental retardation. Interestingly, the existing animal models of FOXG1 deficiency showed similar phenotype, suggesting that animal models may be a fascinating model to understand this human disease. Here, we describe the impacts of FOXG1 mutations and their associated phenotypes in human and mouse models.

CDKL5-Related Disorders: From Clinical Description to Molecular Genetics.

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been described in girls with Rett-like features and early-onset epileptic encephalopathy including infantile spasms. To date, with more than 80 reported cases, the phenotype of CDKL5-related encephalopathy is better defined. The main features consist of early-onset seizures starting before 5 months of age, severe mental retardation with absent speech and Rett-like features such as hand stereotypies and deceleration of head growth. On the other hand, neuro-vegetative signs and developmental regression are rare in CDKL5 mutation patients. The CDKL5 gene encodes a serine threonine kinase protein which is characterized by a catalytic domain and a long C-terminal extension involved in the regulation of the catalytic activity of CDKL5 and in the sub-nuclear localization of the protein. To our knowledge, more than 70 different point mutations have been described including missense mutations within the catalytic domain, nonsense mutations causing the premature termination of the protein distributed in the entire open reading frame, splice variants, and frameshift mutations. Additionally, CDKL5 mutations have recently been described in 7 males with a more severe epileptic encephalopathy and a worse outcome compared to female patients. Finally, about 23 male and female patients have been identified with gross rearrangements encompassing all or part of the CDKL5 gene, with a phenotype reminiscent of CDKL5-related encephalopathy combined with dysmorphic features. Even if recent data clearly indicate that CDKL5 plays an important role in brain function, the protein remains largely uncharacterized. Phenotype-genotype correlation is additionally hampered by the relatively small number of patients described.

De novo SCN1A mutations in migrating partial seizures of infancy.

OBJECTIVE: To determine the genetic etiology of the severe early infantile onset syndrome of malignant migrating partial seizures of infancy (MPSI). METHODS: Fifteen unrelated children with MPSI were screened for mutations in genes associated with infantile epileptic encephalopathies: SCN1A, CDKL5, STXBP1, PCDH19, and POLG. Microarray studies were performed to identify copy number variations. RESULTS: One patient had a de novo SCN1A missense mutation p.R862G that affects the voltage sensor segment of SCN1A. A second patient had a de novo 11.06 Mb deletion of chromosome 2q24.2q31.1 encompassing more than 40 genes that included SCN1A. Screening of CDKL5 (13/15 patients), STXBP1 (13/15), PCDH19 (9/11 females), and the 3 common European mutations of POLG (11/15) was negative. Pathogenic copy number variations were not detected in 11/12 cases. CONCLUSION: Epilepsies associated with SCN1A mutations range in severity from febrile seizures to severe epileptic encephalopathies including Dravet syndrome and severe infantile multifocal epilepsy. MPSI is now the most severe SCN1A phenotype described to date. While not a common cause of MPSI, SCN1A screening should now be considered in patients with this devastating epileptic encephalopathy.

Cell cloning-based transcriptome analysis in Rett patients: relevance to the pathogenesis of Rett syndrome of new human MeCP2 target genes.

More than 90% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene that encodes the methyl-CpG-binding protein 2, a transcriptional modulator. Because MECP2 is subjected to X chromosome inactivation (XCI), girls with RTT either express the wild-type or mutant allele in each individual cell. To test the consequences of MECP2 mutations resulting from a genome-wide transcriptional dysregulation and to identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we carried out gene expression profiling of clonal populations derived from fibroblast primary cultures expressing exclusively either the wild-type or the mutant MECP2 allele. Clonal cultures were obtained from skin biopsy of three RTT patients carrying either a non-sense or a frameshift MECP2 mutation. For each patient, gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis. Firstly, clustering analysis classified the RTT patients according to their genetic background and MECP2 mutation. Secondly, expression profiling by microarray analysis and quantitative RT-PCR indicated four up-regulated genes and five down-regulated genes significantly dysregulated in all our statistical analysis, including excellent potential candidate genes for the understanding of the pathophysiology of this neurodevelopmental disease. Thirdly, chromatin immunoprecipitation analysis confirmed MeCP2 binding to respective CpG islands in three out of four up-regulated candidate genes and sequencing of bisulphite-converted DNA indicated that MeCP2 preferentially binds to methylated-DNA sequences. Most importantly, the finding that at least two of these genes (BMCC1 and RNF182) were shown to be involved in cell survival and/or apoptosis may suggest that impaired MeCP2 function could alter the survival of neurons thus compromising brain function without inducing cell death.