RESUMEN
BACKGROUND: Dystrophic epidermolysis bullosa pruriginosa (DEB-Pr) is a rare subtype of hereditary epidermolysis bullosa, with a poorly understood pathogenesis and no satisfactory treatment. OBJECTIVES: To assess the clinical and biological features, genetic basis and therapeutic management, to better characterize this rare genodermatosis. METHODS: We have conducted a retrospective study, reviewing the clinical presentation, genetic diagnosis, immunohistopathological findings and biological characteristics and management of patients with dystrophic epidermolysis bullosa pruriginosa. This study was conducted in the Department of Dermatology at Saint-Louis Hospital and the Department of Genetics at Necker Hospital (Paris, France). All patients with a diagnosis of DEB-Pr seen between 2010 and 2020 were included. RESULTS: Seven patients were included, the average age of 50.1 years [range 36-76]. Pruriginous-lichenified papules, plaques or nodules appeared at 27.6 years on average [range 7-66] on pretibial areas and forearms, associated with milia and toenails dystrophy. All patients received multiple treatments, but none could sustainably reduce pruritus. Immunohistopathological analysis of lesion skin revealed subepidermal blister with fibrosis, milia and mast cell infiltration. Serum TNFα, IL1ß and IL6 levels were elevated in 2/6 patients. Total serum IgE levels were increased in 7/7 patients, with no history of atopy. Immunophenotyping of circulating T-cells revealed an increased Th2 subset in 4/4 patients, with reduced Th1 and Th17 subpopulations. Genetic analysis of COL7A1 identified 7 distinct causative mutations, six of which were new. Intra-familial clinical variability was documented in 5/7 patients and was associated with the co-inheritance of a recessive COL7A1 mutation or an FLG2 mutation in 2 families. CONCLUSION: Our study confirms the stereotyped presentation of DEB-Pr with large intra-familial variability in disease expression. Mast cell infiltration, elevated IgE and increased Th2 subset without atopy strongly support a role of Th2-mediated immunity in DEB-Pr, and further argue for new targeted therapeutic options such as dupilumab.
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Colágeno Tipo VII , Epidermólisis Ampollosa Distrófica , Proteínas Filagrina/genética , Adulto , Anciano , Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/genética , Humanos , Persona de Mediana Edad , Mutación , Fenotipo , Estudios RetrospectivosRESUMEN
This study provides an overview of 10 years of experience of preimplantation genetic diagnosis (PGD) for cystic fibrosis (CF) in our center. Owing to the high allelic heterogeneity of CF transmembrane conductance regulator (CFTR) mutations in south of France, we have set up a powerful universal test based on haplotyping eight short tandem repeats (STR) markers together with the major mutation p.Phe508del. Of 142 couples requesting PGD for CF, 76 have been so far enrolled in the genetic work-up, and 53 had 114 PGD cycles performed. Twenty-nine cycles were canceled upon in vitro fertilization (IVF) treatment because of hyper- or hypostimulation. Of the remaining 85 cycles, a total of 493 embryos were biopsied and a genetic diagnosis was obtained in 463 (93.9%), of which 262 (without or with a single CF-causing mutation) were transferable. Twenty-eight clinical pregnancies were established, yielding a pregnancy rate per transfer of 30.8% in the group of seven couples with one member affected with CF, and 38.3% in the group of couples whose both members are carriers of a CF-causing mutation [including six couples with congenital bilateral absence of the vas deferens (CBAVD)]. So far, 25 children were born free of CF and no misdiagnosis was recorded. Our test is applicable to 98% of couples at risk of transmitting CF.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Diagnóstico Preimplantación , Niño , Fibrosis Quística/genética , Fibrosis Quística/patología , Femenino , Francia , Asesoramiento Genético , Genotipo , Haplotipos , Heterocigoto , Humanos , Masculino , EmbarazoRESUMEN
The objective of this work was to study the natural history of dystrophinopathies and the genotype-phenotype correlations made possible by the development of the clinical part of the French DMD database. The collection of 70,000 clinical data for 600 patients with an average longitudinal follow-up of 12years enabled clarification of the natural history of Duchenne and Becker muscular dystrophies and clinical presentations in symptomatic females. We were able to specify the phenotypic heterogeneity of motor, orthopedic and respiratory involvements (severe, standard and intermediary form), of the cardiac disorder (severe, standard or absent cardiomyopathy, absence of correlation between motor and cardiac involvements), and of brain function (mental deficiency in the patients with Becker muscular dystrophy, psychopathological disorders in dystrophinopathies). Phenotypic variability did not correlate with a specific mutational spectrum. We propose a model of phenotypic analysis based on the presence or not of muscular and cardiac involvements (described by age at onset and rate of progression) and brain involvement (described by the type and the severity of the cognitive impairment and of the psychological disorders). The methodology developed for the DMD gene can be generalized and used for other databases dedicated to genetic diseases. Application of this model of phenotypic analysis for each patient and further development of the database should contribute substantially to clinical research providing useful tools for future clinical trials.
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Distrofina/genética , Estudios de Asociación Genética , Heterogeneidad Genética , Distrofia Muscular de Duchenne/genética , Adolescente , Edad de Inicio , Niño , Preescolar , Estudios de Cohortes , Bases de Datos Factuales , Femenino , Francia/epidemiología , Técnicas Genéticas , Humanos , Masculino , Actividad Motora , Distrofia Muscular de Duchenne/epidemiología , FenotipoRESUMEN
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.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Exones , Empalme del ARN/genética , Mutación Missense , MutaciónRESUMEN
Bilateral sensorineural hearing loss (HL), classically described as mild to severe with a typically down-sloping audiometric configuration, is the earliest symptom occurring in Usher syndrome type II (USH2). Audiological findings were analyzed in a total of 100 USH2 patients (92 families) divided into three groups according to the gene involved: 88 USH2A, 10 GPR98 and 2 DFNB31 patients. A fine analysis of audiograms was performed (pure tone average, degree of severity, configuration). The median age of HL diagnosis was 5 years (range 8 months-31 years) although the median age at USH2 diagnosis was 34.5 (range 8-76). Moderate HL was predominant (76%) and a gently down-sloping configuration characterized most audiograms (66%). No statistically significant difference was found between USH2A and GPR98 patients but a tendency was clearly noted for more GPR98 patients to present with severe hearing loss. It is not possible to predict the mutated gene from audiograms.
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Audiología/métodos , Proteínas de la Matriz Extracelular/genética , Pérdida Auditiva Sensorineural/diagnóstico , Adolescente , Adulto , Audiometría/métodos , Niño , Preescolar , Estudios Transversales , Femenino , Genotipo , Pérdida Auditiva Sensorineural/genética , Humanos , Lactante , Masculino , Proteínas de la Membrana/genética , Mutación , Receptores Acoplados a Proteínas G/genética , Adulto JovenRESUMEN
BACKGROUND: Cystic fibrosis (CF) is caused by compound heterozygosity or homozygosity of CF transmembrane conductance regulator gene (CFTR) mutations. Phenotypic variability associated with certain mutations makes genetic counselling difficult, notably for R117H, whose disease phenotype varies from asymptomatic to classical CF. The high frequency of R117H observed in CF newborn screening has also introduced diagnostic dilemmas. The aim of this study was to evaluate the disease penetrance for R117H in order to improve clinical practice. METHODS: The phenotypes in all individuals identified in France as compound heterozygous for R117H and F508del, the most frequent CF mutation, were described. The allelic prevalences of R117H (p(R117H)), on either intron 8 T5 or T7 background, and F508del (p(F508del)) were determined in the French population, to permit an evaluation of the penetrance of CF for the [R117H]+[F508del] genotype. RESULTS: Clinical details were documented for 184 [R117H]+[F508del] individuals, including 72 newborns. The disease phenotype was predominantly mild; one child had classical CF, and three adults' severe pulmonary symptoms. In 5245 healthy adults, p(F508del) was 1.06%, p(R117H;T7) 0.27% and p(R117H;T5)<0.01%. The theoretical number of [R117H;T7]+[F508del] individuals in the French population was estimated at 3650, whereas only 112 were known with CF related symptoms (3.1%). The penetrance of classical CF for [R117H;T7]+[F508del] was estimated at 0.03% and that of severe CF in adulthood at 0.06%. CONCLUSIONS: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes. The real impact of so-called disease mutations should be assessed before including them in newborn or preconceptional carrier screening programmes.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Asesoramiento Genético , Heterocigoto , Tamizaje Neonatal , Penetrancia , Estudios Transversales , Fibrosis Quística/diagnóstico , Fibrosis Quística/epidemiología , Humanos , Recién Nacido , Estimación de Kaplan-Meier , Mutación , FenotipoRESUMEN
New technologies, which constantly become available for mutation detection and gene analysis, have contributed to an exponential rate of discovery of disease genes and variation in the human genome. The task of collecting and documenting this enormous amount of data in genetic databases represents a major challenge for the future of biological and medical science. The Locus Specific Databases (LSDBs) are so far the most efficient mutation databases. This review presents the main types of databases available for the analysis of mutations responsible for genetic disorders, as well as open perspectives for new therapeutic research or challenges for future medicine. Accurate and exhaustive collection of variations in human genomes will be crucial for research and personalized delivery of healthcare.
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Bases de Datos Genéticas , Enfermedades Genéticas Congénitas/genética , Mutación , Enfermedades Raras/genética , Codón de Terminación , Etnicidad/genética , Predicción , Enfermedades Genéticas Congénitas/clasificación , Enfermedades Genéticas Congénitas/terapia , Terapia Genética , Genética Médica/ética , Genotipo , Humanos , Internet , Fenotipo , ARN sin Sentido/uso terapéutico , Enfermedades Raras/clasificación , Enfermedades Raras/terapia , Terminología como Asunto , Transcripción Genética/efectos de los fármacosRESUMEN
Dystonias are clinically and genetically heterogeneous neurological disorders that affect movement, and are the focus of much investigative work. The recent identification of mutations in the gene THAP1 in DYT6 dystonia reopens the very interesting question of the in fine involvement of dopamine in the different types of dystonia. In this review, we will go through the recent literature in order to evaluate the many contributions to this theory as well as to highlight the difficulties in identifying a global regulatory pathway for the different forms of this disease that we are just starting to decipher.
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Dopamina/fisiología , Distonía Muscular Deformante/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al ADN/genética , Distonía Muscular Deformante/fisiopatología , Heterogeneidad Genética , Humanos , Chaperonas Moleculares/genética , Mutación/fisiología , Proteínas Nucleares/genéticaRESUMEN
Mutations in the FBN1 gene cause Marfan syndrome (MFS) and have been associated with a wide range of milder overlapping phenotypes. A proportion of patients carrying a FBN1 mutation does not meet diagnostic criteria for MFS, and are diagnosed with "other type I fibrillinopathy." In order to better describe this entity, we analyzed a subgroup of 146 out of 689 adult propositi with incomplete "clinical" international criteria (Ghent nosology) from a large collaborative international study including 1,009 propositi with a pathogenic FBN1 mutation. We focused on patients with only one major clinical criterion, [including isolated ectopia lentis (EL; 12 patients), isolated ascending aortic dilatation (17 patients), and isolated major skeletal manifestations (1 patient)] or with no major criterion but only minor criteria in 1 or more organ systems (16 patients). At least one component of the Ghent nosology, insufficient alone to make a minor criterion, was found in the majority of patients with isolated ascending aortic dilatation and isolated EL. In patients with isolated EL, missense mutations involving a cysteine were predominant, mutations in exons 24-32 were underrepresented, and no mutations leading to a premature truncation were found. Studies of recurrent mutations and affected family members of propositi with only one major clinical criterion argue for a clinical continuum between such phenotypes and classical MFS. Using strict definitions, we conclude that patients with FBN1 mutation and only one major clinical criterion or with only minor clinical criteria of one or more organ system do exist but represent only 5% of the adult cohort.
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Síndrome de Marfan/diagnóstico , Síndrome de Marfan/genética , Proteínas de Microfilamentos/genética , Adulto , Estudios de Cohortes , Desplazamiento del Cristalino/diagnóstico , Desplazamiento del Cristalino/genética , Desplazamiento del Cristalino/patología , Fibrilina-1 , Fibrilinas , Humanos , Masculino , Síndrome de Marfan/clasificación , Síndrome de Marfan/patología , Mutación , FenotipoRESUMEN
Duchenne muscular dystrophy (DMD) is a common childhood lethal X-linked recessive disorder, resulting from deletions, duplications and point mutations in the dystrophin gene. Single-cell protocols for preimplantation genetic diagnosis (PGD) still remain challenging due to the enormous size of the gene and the high risk of intragenic recombination, limitations that often lead to sex determination and selection of female embryos. This study describes direct and rapid decaplex and dodecaplex polymerase chain reaction protocols enabling the analysis of five or seven exons and four microsatellite markers scattered along the dystrophin gene, chosen to be located in the two deletion hotspots, and the analysis of amelogenin sequences for gender determination. The dodecaplex protocol may be applied to most of the couples requesting PGD for DMD in whom the female partner is a carrier of a deletion. This generic approach will allow prompt response to the PGD referrals by reducing the pre-clinical PGD work-up. It was successfully applied in three DMD families, resulting in the birth of a girl as well as in a healthy ongoing pregnancy.
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Distrofina/genética , Distrofia Muscular de Duchenne/genética , Reacción en Cadena de la Polimerasa/métodos , Diagnóstico Preimplantación/métodos , Amelogenina/genética , Femenino , Eliminación de Gen , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Linaje , EmbarazoRESUMEN
BACKGROUND: The diagnosis of Marfan syndrome (MFS) is usually initially based on clinical criteria according to the number of major and minor systems affected following international nosology. The number of FBN1 mutation carriers, at risk of aortic complications who would not be properly diagnosed based only on clinical grounds, is of growing importance owing to the increased availability of molecular screening. The aim of the study was to identify patients who should be considered for FBN1 mutation screening. METHODS: Our international series included 1009 probands with a known FBN1 mutation. Patients were classified as either fulfilling or not fulfilling "clinical" criteria. In patients with unfulfilled "clinical" criteria, we evaluated the percentage of additional patients who became positive for international criteria when the FBN1 mutation was considered. The aortic risk was evaluated and compared in patients fulfilling or not fulfilling the "clinical" international criteria. RESULTS: Diagnosis of MFS was possible on clinical grounds in 79% of the adults, whereas 90% fulfilled the international criteria when including the FBN1 mutation. Corresponding figures for children were 56% and 85%, respectively. Aortic dilatation occurred later in adults with unfulfilled "clinical criteria" when compared to the Marfan syndrome group (44% vs 73% at 40 years, p<0.001), but the lifelong risk for ascending aortic dissection or surgery was not significantly different in both groups. CONCLUSIONS: Because of its implications for aortic follow-up, FBN1 molecular analysis is recommended in newly suspected MFS when two systems are involved with at least one major system affected. This is of utmost importance in patients without aortic dilatation and in children.
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Cooperación Internacional , Síndrome de Marfan/diagnóstico , Síndrome de Marfan/genética , Proteínas de Microfilamentos/genética , Adolescente , Adulto , Anciano , Aorta/patología , Niño , Femenino , Fibrilina-1 , Fibrilinas , Humanos , Masculino , Mutación/genéticaRESUMEN
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.2989-313Aâ¯>â¯T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118â¯bp PE. The c.3469-1304Câ¯>â¯G variant promoted the inclusion of a 214â¯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.3874-4522Aâ¯>â¯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.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/complicaciones , Fibrosis Quística/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Variación Genética , Humanos , Lactante , Intrones , Masculino , Fenotipo , RecurrenciaRESUMEN
Early onset torsion dystonia are rare movement disorders. Molecular defect is known for only a subgroup, consisting of a unique and recurrent mutation in the TOR1A gene. We undertook a nationwide census of French TOR1A-mutation carriers and the assessment of clinical associated signs. Overall, 53 index cases and 104 relatives were studied and haplotypes linked to the mutation constructed. The previously reported Ashkenazi-Jewish haplotype was found in 11 families with the remainder carrying distinct haplotypes suggesting independent mutation events. This study demonstrates the scarcity of this disease in France with estimated disease frequency of 0.13:100,000 and mutation frequency of 0.17:100,000.
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Distonía Muscular Deformante/genética , Chaperonas Moleculares/genética , Eliminación de Secuencia , Adolescente , Edad de Inicio , Estudios de Casos y Controles , Niño , Femenino , Francia , Frecuencia de los Genes , Ligamiento Genético , Haplotipos , Heterocigoto , Humanos , Judíos/genética , Masculino , FenotipoRESUMEN
It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Análisis Mutacional de ADN , Humanos , Estado Nutricional/genética , Polimorfismo Genético , Pronóstico , Empalme de Proteína , Control de Calidad , Pruebas de Función Respiratoria , Terminología como AsuntoAsunto(s)
Ceguera/congénito , Ceguera/genética , Proteínas del Ojo/genética , Mutación , Atrofias Ópticas Hereditarias/genética , Proteínas , Adolescente , Adulto , Secuencia de Bases , Proteínas Portadoras , ADN/genética , Análisis Mutacional de ADN , Cartilla de ADN/genética , Femenino , Humanos , Masculino , Linaje , Reacción en Cadena de la Polimerasa , cis-trans-IsomerasasAsunto(s)
Cromosomas Humanos Par 19 , Fibrosis Quística/genética , Obstrucción Intestinal/genética , Meconio , Niño , Mapeo Cromosómico , Fibrosis Quística/fisiopatología , Volumen Espiratorio Forzado , Marcadores Genéticos , Humanos , Repeticiones de Microsatélite , Núcleo Familiar , Polimorfismo GenéticoRESUMEN
Researchers and clinicians ideally need instant access to all the variation in their gene/locus of interest to efficiently conduct their research and genetic healthcare to the highest standards. Currently much key data resides in the laboratory books or patient records around the world, as there are many impediments to submitting this data. It would be ideal therefore if a semiautomated pathway was available, with a minimum of effort, to make the deidentified data publicly available for others to use. The Human Variome Project (HVP) meeting listed 96 recommendations to work toward this situation. This article is planned to initiate a strategy to enhance the collection of phenotype and genotype data from the clinician/diagnostic laboratory nexus. Thus, the aim is to develop universally applicable forms that people can use when investigating patients for each inherited disease, to assist in satisfying many of the recommendations of the HVP Meeting [Cotton et al., 2007]. We call for comment and collaboration in this article.
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Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/genética , Técnicas Genéticas , Genómica/normas , Mutación , Bases de Datos Genéticas , Genoma Humano , Genotipo , Humanos , Fenotipo , PublicacionesRESUMEN
BACKGROUND: Usher syndrome, a devastating recessive disorder which combines hearing loss with retinitis pigmentosa, is clinically and genetically heterogeneous. Usher syndrome type 1 (USH1) is the most severe form, characterised by profound congenital hearing loss and vestibular dysfunction. OBJECTIVE: To describe an efficient protocol which has identified the mutated gene in more than 90% of a cohort of patients currently living in France. RESULTS: The five genes currently known to cause USH1 (MYO7A, USH1C, CDH23, PCDH15, and USH1G) were tested for. Disease causing mutations were identified in 31 of the 34 families referred: 17 in MYO7A, 6 in CDH23, 6 in PCDH15, and 2 in USH1C. As mutations in genes other than myosin VIIA form nearly 50% of the total, this shows that a comprehensive approach to sequencing is required. Twenty nine of the 46 identified mutations were novel. In view of the complexity of the genes involved, and to minimise sequencing, a protocol for efficient testing of samples was developed. This includes a preliminary linkage and haplotype analysis to indicate which genes to target. It proved very useful and demonstrated consanguinity in several unsuspected cases. In contrast to CDH23 and PCDH15, where most of the changes are truncating mutations, myosin VIIA has both nonsense and missense mutations. Methods for deciding whether a missense mutation is pathogenic are discussed. CONCLUSIONS: Diagnostic testing for USH1 is feasible with a high rate of detection and can be made more efficient by selecting a candidate gene by preliminary linkage and haplotype analysis.
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Cadherinas/genética , Mutación/genética , Síndromes de Usher/diagnóstico , Síndromes de Usher/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Adulto , Proteínas Relacionadas con las Cadherinas , Proteínas de Ciclo Celular , Niño , Preescolar , Estudios de Cohortes , Proteínas del Citoesqueleto , Análisis Mutacional de ADN , Dineínas/genética , Exones/genética , Haplotipos , Humanos , Intrones/genética , Miosina VIIa , Miosinas/genética , Proteínas del Tejido Nervioso/genéticaRESUMEN
Hearing loss is the most common sensory disorder and because of its high genetic heterogeneity, implementation of Massively Parallel Sequencing (MPS) in diagnostic laboratories is greatly improving the possibilities of offering optimal care to patients. We present the results of a two-year period of molecular diagnosis that included 207 French families referred for non-syndromic hearing loss. Our multi-step strategy involved (i) DFNB1 locus analysis, (ii) MPS of 74 genes, and (iii) additional approaches including Copy Number Variations, in silico analyses, minigene studies coupled when appropriate with complete gene sequencing, and a specific assay for STRC. This comprehensive screening yielded an overall diagnostic rate of 48%, equally distributed between DFNB1 (24%) and the other genes (24%). Pathogenic genotypes were identified in 19 different genes, with a high prevalence of GJB2, STRC, MYO15A, OTOF, TMC1, MYO7A and USH2A. Involvement of an Usher gene was reported in 16% of the genotyped cohort. Four de novo variants were identified. This study highlights the need to develop several molecular approaches for efficient molecular diagnosis of hearing loss, as this is crucial for genetic counselling, audiological rehabilitation and the detection of syndromic forms.
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Conexinas/genética , Variaciones en el Número de Copia de ADN , Pérdida Auditiva/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Población Blanca/genética , Estudios de Cohortes , Simulación por Computador , Conexina 26 , Diagnóstico Precoz , Francia , Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Pérdida Auditiva/genética , Humanos , Masculino , Mutación , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodosRESUMEN
BACKGROUND: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. METHODS: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. RESULTS: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. CONCLUSIONS: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.