RESUMEN
Typhoid fever occurs in an endemic form in Brazil and is a serious public health problem in some regions. In this scenario, further research is urgently needed to identify the associations between socioeconomic factors and typhoid fever, contributing to guiding policy decisions in the country. We aimed to investigate the influence of socioeconomic disparities on the prevalence of typhoid fever and non-typhoidal Salmonella (NTS) in Brazil. A search for data from 2010 to 2019 was carried out with the national health and human development agencies. As milk and derivatives are the fourth food incriminated in food outbreaks in Brazil, analyses for detecting Salmonella spp. in commercial dairy products allowed us to assess whether the outbreaks associated with these foods are due to inadequacies in sanitary control in dairy establishments or whether they are mainly home-based outbreaks. Predictive models validated by the bootstrapping method demonstrate an association of NTS prevalence reduction with improvements in the Sanitation Service Index (Rv ≥ -8 0.686; p ≤ 0.01) and Municipal Human Development Index - MHDI - (Rv = -0.789; p ≤ 0.02). In the North, typhoid fever prevalence had seasonal variability with the rainfall, while sanitation services (Rv ≥-0.684; p ≤ 0.04) and MHDI (Rv ≥-0.949; p ≤ 0.003) directly influenced Northeast and South Brazil. Thus, the unequal distribution of investments in the sanitation sector contributed to disparities in typhoid fever prevalence among Brazilian regions. The absence of Salmonella spp. in commercial samples ratified the collected data that the outbreaks of Salmonella spp. in the Brazilian population occur mainly at residences. These findings show that implementing public health education and increasing investments in sanitation in regions with poor service can control outbreaks of Salmonella spp. in Brazilian endemic areas.
Asunto(s)
Fiebre Tifoidea , Humanos , Brasil/epidemiología , Fiebre Tifoidea/epidemiología , Brotes de Enfermedades , Salmonella , Factores SocioeconómicosRESUMEN
Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder characterized by an increased ristocetin-induced platelet aggregation (RIPA) and enhanced affinity of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF). To date, only seven variants have been described with this gain-of-function effect, most of them located in the C-terminal disulphide loop of the VWF-binding domain of GPIbα. We herein describe a patient with moderate bleeding symptoms, mild thrombocytopenia and increased RIPA. By direct sequencing of GP1BA, a novel leucine-rich repeat heterozygous variant was identified (c.580C>T; predictably p.Leu194Phe), strongly suggestive as being the underlying cause for the PT-VWD phenotype of our patient.
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Enfermedades de von Willebrand , Factor de von Willebrand , Humanos , Factor de von Willebrand/genética , Mutación con Ganancia de Función , Enfermedades de von Willebrand/diagnóstico , Plaquetas , Hemorragia/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/genéticaRESUMEN
We describe the phenotype of 22 male patients (20 probands) carrying a hemizygous missense variant in MED12. The phenotypic spectrum is very broad ranging from nonspecific intellectual disability (ID) to the three well-known syndromes: Opitz-Kaveggia syndrome, Lujan-Fryns syndrome, or Ohdo syndrome. The identified variants were randomly distributed throughout the gene (p = 0.993, χ2 test), but mostly outside the functional domains (p = 0.004; χ2 test). Statistical analyses did not show a correlation between the MED12-related phenotypes and the locations of the variants (p = 0.295; Pearson correlation), nor the protein domain involved (p = 0.422; Pearson correlation). In conclusion, establishing a genotype-phenotype correlation in MED12-related diseases remains challenging. Therefore, we think that patients with a causative MED12 variant are currently underdiagnosed due to the broad patients' clinical presentations.
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Blefarofimosis , Discapacidad Intelectual , Discapacidad Intelectual Ligada al Cromosoma X , Masculino , Humanos , Complejo Mediador/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Blefarofimosis/genética , Mutación Missense/genética , Fenotipo , SíndromeRESUMEN
Thrombocytopenia-absent radius (TAR) syndrome is a rare congenital disorder characterized by the bilateral absence of the radius and thrombocytopenia, and sometimes by other skeletal, gastrointestinal, cardiac, and renal abnormalities. The underlying genetic defect is usually the compound inheritance of a microdeletion in 1q21.1 (null allele) and a low-frequency, non-coding single nucleotide variant (SNV) in the RBM8A gene (hypomorphic allele). We report three new cases from two unrelated families. The two siblings presented the common genotype, namely the compound heterozygosity for a 1q21.1 microdeletion and the hypomorphic SNV c.-21G>A in RBM8A, whereas the third, unrelated patient presented a rare genotype comprised by two RBM8A variants: c.-21G>A (hypomorphic allele) and a novel pathogenic variant, c.343-2A>G (null allele). Of the eight documented RBM8A variants identified in TAR syndrome patients, four have hypomorphic expression and four behave as null alleles. The present report expands the RBM8A null allele spectrum and corroborates the particularities of RBM8A involvement in TAR syndrome pathogenesis.
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Trombocitopenia , Deformidades Congénitas de las Extremidades Superiores , Alelos , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Humanos , Proteínas de Unión al ARN/genética , Radio (Anatomía) , Trombocitopenia/patología , Deformidades Congénitas de las Extremidades Superiores/genética , Deformidades Congénitas de las Extremidades Superiores/patologíaRESUMEN
General cheese manufacturing involves high temperatures, fermentation and ripening steps that function as hurdles to microbial growth. On the other hand, the application of several different formulations and manufacturing techniques may create a bacterial protective environment. In cheese, the persistent behavior of Shiga toxin-producing Escherichia coli (STEC) relies on complex mechanisms that enable bacteria to respond to stressful conditions found in cheese matrix. In this review, we discuss how STEC manages to survive to high and low temperatures, hyperosmotic conditions, exposure to weak organic acids, and pH decreasing related to cheese manufacturing, the cheese matrix itself and storage. Moreover, we discuss how these stress responses interact with each other by enhancing adaptation and consequently, the persistence of STEC in cheese. Further, we show how virulence genes eae and tir are affected by stress response mechanisms, increasing either cell adherence or virulence factors production, which leads to a selection of more resistant and virulent pathogens in the cheese industry, leading to a public health issue.
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Queso , Infecciones por Escherichia coli , Escherichia coli Shiga-Toxigénica , Queso/análisis , Microbiología de Alimentos , Humanos , Amor , Toxina Shiga , VirulenciaRESUMEN
RASGRP2 encodes the calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) identified as a Rap1-activating molecule. Pathogenic variants previously identified in RASGRP2 allowed the characterization of CalDAG-GEFI deficiency as a non-syndromic, autosomal recessive platelet function disease. We report on the clinical manifestations and laboratory features of a Portuguese family with a likely pathogenic variant in RASGRP2 (c.999G>C leading to a p.Lys333Asn change in the CDC25 catalytic domain of CalDAG-GEFI) and discuss the contribution of this variant to the disease manifestations. Based on the study of this family with one homozygous patient and five heterozygous carriers and on a critical analysis of the literature, we challenge previous knowledge that CalDAG-GEFI deficiency only manifests in homozygous patients. Our data suggest that at least for the RASGRP2 variant reported herein, there is a phenotypic expression, albeit milder, in heterozygous carriers.
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Trastornos de las Plaquetas Sanguíneas/sangre , Trastornos de las Plaquetas Sanguíneas/genética , Familia , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Heterocigoto , Homocigoto , Adolescente , Adulto , Plaquetas/metabolismo , Dominio Catalítico/genética , Niño , Femenino , Tamización de Portadores Genéticos/métodos , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/química , Humanos , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Agregación Plaquetaria , Portugal , Adulto JovenRESUMEN
While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a ~8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics.
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Distrofina/genética , Genoma/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Adulto , Secuencia de Bases , Exones/genética , Genética , Humanos , Masculino , Secuenciación Completa del Genoma/métodos , Adulto JovenRESUMEN
Congenital muscular dystrophy type 1A (MDC1A) is one of the main subtypes of early-onset muscle disease, caused by disease-associated variants in the laminin-α2 (LAMA2) gene. MDC1A usually presents as a severe neonatal hypotonia and failure to thrive. Muscle weakness compromises normal motor development, leading to the inability to sit unsupported or to walk independently. The phenotype associated with LAMA2 defects has been expanded to include milder and atypical cases, being now collectively known as LAMA2-related muscular dystrophies (LAMA2-MD). Through an international multicenter collaborative effort, 61 new LAMA2 disease-associated variants were identified in 86 patients, representing the largest number of patients and new disease-causing variants in a single report. The collaborative variant collection was supported by the LOVD-powered LAMA2 gene variant database (https://www.LOVD.nl/LAMA2), updated as part of this work. As of December 2017, the database contains 486 unique LAMA2 variants (309 disease-associated), obtained from direct submissions and literature reports. Database content was systematically reviewed and further insights concerning LAMA2-MD are presented. We focus on the impact of missense changes, especially the c.2461A > C (p.Thr821Pro) variant and its association with late-onset LAMA2-MD. Finally, we report diagnostically challenging cases, highlighting the relevance of modern genetic analysis in the characterization of clinically heterogeneous muscle diseases.
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Estudios de Asociación Genética , Laminina/genética , Mutación , Fenotipo , Alelos , Biomarcadores , Encéfalo/anomalías , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Biología Computacional/métodos , Bases de Datos de Ácidos Nucleicos , Frecuencia de los Genes , Variación Genética , Genotipo , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética/métodos , Distrofias Musculares/diagnóstico , Distrofias Musculares/genéticaRESUMEN
BACKGROUND: We describe a female infant with Fragile-X syndrome, with a fully expanded FMR1 allele and preferential inactivation of the homologous X-chromosome carrying a de novo deletion. This unusual and rare case demonstrates the importance of a detailed genomic approach, the absence of which could be misguiding, and calls for reflection on the current clinical and diagnostic workup for developmental disabilities. CASE PRESENTATION: We present a female infant, referred for genetic testing due to psychomotor developmental delay without specific dysmorphic features or relevant family history. FMR1 mutation screening revealed a methylated full mutation and a normal but inactive FMR1 allele, which led to further investigation. Complete skewing of X-chromosome inactivation towards the paternally-inherited normal-sized FMR1 allele was found. No pathogenic variants were identified in the XIST promoter. Microarray analysis revealed a 439 kb deletion at Xq28, in a region known to be associated with extreme skewing of X-chromosome inactivation. CONCLUSIONS: Overall results enable us to conclude that the developmental delay is the cumulative result of a methylated FMR1 full mutation on the active X-chromosome and the inactivation of the other homologue carrying the de novo 439 kb deletion. Our findings should be taken into consideration in future guidelines for the diagnostic workup on the diagnosis of intellectual disabilities, particularly in female infant cases.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Genómica/métodos , Eliminación de Secuencia , Inactivación del Cromosoma X , Cromosomas Humanos X/genética , Metilación de ADN , Femenino , Pruebas Genéticas , Humanos , Lactante , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Herencia Paterna , LinajeRESUMEN
Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is due to the expansion over 200 CGGs and methylation of this polymorphic region, in the 5'-UTR (untranslated region) of FMR1 (Xq27.3). We have identified four FXS mosaic males: M1-(CGG)35/(CGG)>200; M2-(CGG)26/(CGG)>200; M3-(CGG)39/(CGG)>200; and M4-(CGG)18/(CGG)125/(CGG)>200. After genotyping their respective mothers, we suggested that normal alleles of these patients resulted from post-zygotic contractions of full expansions. The detection of these four rare independent cases led us to hypothesize the existence of a large-contraction predisposing haplotype in our population. Next, we questioned whether other normal pure CGGs would have arisen through similar contractions from fully expanded alleles. To address these questions, we identified stable single-nucleotide polymorphism (SNP) lineages and related short tandem repeat (STR) haplotypes (DXS998-DXS548-FRAXAC1-FRAXAC2) of the four mosaics, 123 unrelated FXS patients and 212 controls. An extended flanking haplotype (34-44-38-336) shared by mosaics from lineage A suggested a risk lineage-specific haplotype more prone to large contractions. Other normal pure FMR1 alleles from this SNP background also shared phylogenetically close STR haplotypes, although a single (CGG)exp>(CGG)24 contraction or the loss of AGG interruptions may explain their origin. In both scenarios, multistep FMR1 mutations involving the gain or loss of several CGGs seem to underlie the evolution of the repeat.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Repeticiones de Microsatélite/genética , Polimorfismo de Nucleótido Simple/genética , Repeticiones de Trinucleótidos/genética , Niño , Preescolar , Frecuencia de los Genes , Haplotipos/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Congenital myopathies (CMs) are a heterogeneous group of muscle diseases characterized by hypotonia, delayed motor skills and muscle weakness with onset during the first years of life. The diagnostic workup of CM is highly dependent on the interpretation of the muscle histology, where typical pathognomonic findings are suggestive of a CM but are not necessarily gene specific. Over 20 loci have been linked to these myopathies, including three exceptionally large genes (TTN, NEB and RYR1), which are a challenge for molecular diagnosis. We developed a new approach using massive parallel sequencing (MPS) technology to simultaneously analyze 20 genes linked to CMs. Assay design was based on the Ion AmpliSeq strategy and sequencing runs were performed on an Ion PGM system. A total of 12 patients were analyzed in this study. Among the 2534 variants detected, 14 pathogenic mutations were successfully identified in the DNM2, NEB, RYR1, SEPN1 and TTN genes. Most of these had not been documented and/or fully characterized, hereby contributing to expand the CM mutational spectrum. The utility of this approach was demonstrated by the identification of mutations in 70% of the patients included in this study, which is relevant for CMs especially considering its wide phenotypic and genetic heterogeneity.
Asunto(s)
Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedades Musculares/congénito , Enfermedades Musculares/diagnóstico , Adolescente , Adulto , Anciano , Alelos , Sustitución de Aminoácidos , Biopsia , Niño , Análisis Mutacional de ADN , Dinamina II/genética , Femenino , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Mutación , Linaje , Fenotipo , Canal Liberador de Calcio Receptor de Rianodina/genética , Adulto JovenRESUMEN
Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).
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Bases de Datos Genéticas , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Mutación , Humanos , Sistema de RegistrosRESUMEN
Muscular dystrophies (MDs) are a group of hereditary muscle disorders that include two particularly heterogeneous subgroups: limb-girdle MD and congenital MD, linked to 52 different genes (seven common to both subgroups). Massive parallel sequencing technology may avoid the usual stepwise gene-by-gene analysis. We report the whole-exome sequencing (WES) analysis of a patient with childhood-onset progressive MD, also presenting mental retardation and dilated cardiomyopathy. Conventional sequencing had excluded eight candidate genes. WES of the trio (patient and parents) was performed using the ion proton sequencing system. Data analysis resorted to filtering steps using the GEMINI software revealed a novel silent variant in the choline kinase beta (CHKB) gene. Inspection of sequence alignments ultimately identified the causal variant (CHKB:c.1031+3G>C). This splice site mutation was confirmed using Sanger sequencing and its effect was further evaluated with gene expression analysis. On reassessment of the muscle biopsy, typical abnormal mitochondrial oxidative changes were observed. Mutations in CHKB have been shown to cause phosphatidylcholine deficiency in myofibers, causing a rare form of CMD (only 21 patients reported). Notwithstanding interpretative difficulties that need to be overcome before the integration of WES in the diagnostic workflow, this work corroborates its utility in solving cases from highly heterogeneous groups of diseases, in which conventional diagnostic approaches fail to provide a definitive diagnosis.
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Colina Quinasa/genética , Distrofias Musculares/genética , Adulto , Secuencia de Bases , Colina Quinasa/metabolismo , Análisis Mutacional de ADN , Exoma , Femenino , Expresión Génica , Estudios de Asociación Genética , Humanos , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Sitios de Empalme de ARNRESUMEN
PURPOSE: Perform the genetic characterization of five patients with total sperm immotility using Sanger sequencing and Whole Exome Sequencing (WES), in order to increase the knowledge on the genetics of sperm immotility and, ultimately, allow the identification of potential genetic markers for infertility. METHODS: Prospective study at a University Medical school. We analysed five men with total sperm immotility, four with dysplasia of the fibrous sheath (DFS), associated with disruption of several axonemal structures, and one patient with situs inversus totalis, which showed absence of dynein arms (DA) and nexin bridges. We screened 7 genes by Sanger sequencing, involved in sperm motility and associated to ultrastructural defects found in these patients (CCDC39, CCDC40, DNAH5, DNAI1, RSPH1, AKAP3 and AKAP4). Additionally, we performed WES analysis in the patient with situs inversus. RESULTS: We identified nine new DNA sequence variants by WES. Two of these variants were considered particularly relevant: a homozygous missense change in CCDC103 gene (c.104G > C, p.R35P) probably related with absence of dynein arms; the other in the INSL6 gene (c.262_263delCC) is thought to be also involved in sperm immotility. CONCLUSIONS: Our work suggests that WES is an effective strategy, especially as compared with conventional sequencing, to study highly heterogenic genetic diseases, such as sperm immotility. For future work we expect to expand the analysis of WES to the other four patients and complement findings with expression analysis or functional studies to determine the impact of the novel variants.
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Infertilidad Masculina/genética , Mutación , Motilidad Espermática/genética , Análisis Mutacional de ADN , Exoma , Marcadores Genéticos , Humanos , Infertilidad Masculina/patología , MasculinoRESUMEN
Triggered by the sequencing of the human genome, personalized medicine has been one of the fastest growing research areas in the last decade. Multiple software and hardware technologies have been developed by several projects, culminating in the exponential growth of genetic data. Considering the technological developments in this field, it is now fairly easy and inexpensive to obtain genetic profiles for unique individuals, such as those performed by several genetic analysis companies. The availability of computational tools that simplify genetic data analysis and the disclosure of biomedical evidences are of utmost importance. We present Variobox, a desktop tool to annotate, analyze, and compare human genes. Variobox obtains variant annotation data from WAVe, protein metadata annotations from Protein Data Bank, and sequences are obtained from Locus Reference Genomic or RefSeq databases. To explore the data, Variobox provides an advanced sequence visualization that enables agile navigation through genetic regions. DNA sequencing data can be compared with reference sequences retrieved from LRG or RefSeq records, identifying and automatically annotating new potential variants. These features and data, ranging from patient sequences to HGVS-compliant variant descriptions, are combined in an intuitive interface to analyze genes and variants. Variobox is a Java application, available at http://bioinformatics.ua.pt/variobox.
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Biología Computacional/métodos , Bases de Datos Genéticas , Variación Genética , Genoma Humano , Anotación de Secuencia Molecular , Secuencia de Aminoácidos , Secuencia de Bases , Humanos , Medicina de Precisión , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Molecular characterization of patients with Duchenne or Becker muscular dystrophies is essential for establishing a differential diagnosis, allowing appropriate clinical follow-up, patient management and genetic counseling. In light of the recent mutation-based therapeutic approaches, DMD gene analysis has gained further relevance. Owing to the size and complexity of the DMD gene and the diversity of mutation types, molecular analysis is not always a straightforward task requiring the combination of several methodologies. Our national genetic diagnostic service genetically characterized 308 dystrophinopathy patients (284 unrelated families), leading to the identification of 175 distinct mutations, including 39 unpublished variants. These studies revealed several potential diagnostic pitfalls (because of technical limitations or related with DMD's genetic heterogeneity) that may be overlooked even considering the international disease-specific diagnostic guidelines. Comprehensive analysis involved expression studies at the mRNA level, the identification of splicing changes and ultimately providing evidence for apparent exceptions to the reading-frame rule. Besides increasing the mutation detection rate, this detailed molecular characterization is indispensable for the identification of suitable candidates for the new mutation-centered therapies. As patient registries are internationally recognized as essential for clinical trial recruitment, this led us to develop the Portuguese Duchenne and Becker Muscular Dystrophy registry in collaboration with the Translational Research in Europe-Assessment and Treatment of Neuromuscular Diseases network.
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Distrofina/genética , Distrofia Muscular de Duchenne/genética , Niño , Preescolar , Estudios de Cohortes , Biología Computacional , ADN Complementario/genética , Diagnóstico Diferencial , Europa (Continente) , Femenino , Genotipo , Humanos , Lactante , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/terapia , Mutación , Portugal , Pronóstico , Empalme del ARN , ARN Mensajero/genética , Sistemas de Lectura , Sistema de RegistrosRESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked genetic disease, caused by the absence of the dystrophin protein. Although many novel therapies are under development for DMD, there is currently no cure and affected individuals are often confined to a wheelchair by their teens and die in their twenties/thirties. DMD is a rare disease (prevalence <5/10,000). Even the largest countries do not have enough affected patients to rigorously assess novel therapies, unravel genetic complexities, and determine patient outcomes. TREAT-NMD is a worldwide network for neuromuscular diseases that provides an infrastructure to support the delivery of promising new therapies for patients. The harmonized implementation of national and ultimately global patient registries has been central to the success of TREAT-NMD. For the DMD registries within TREAT-NMD, individual countries have chosen to collect patient information in the form of standardized patient registries to increase the overall patient population on which clinical outcomes and new technologies can be assessed. The registries comprise more than 13,500 patients from 31 different countries. Here, we describe how the TREAT-NMD national patient registries for DMD were established. We look at their continued growth and assess how successful they have been at fostering collaboration between academia, patient organizations, and industry.
Asunto(s)
Bases de Datos Factuales , Distrofia Muscular de Duchenne , Sistema de Registros , Bases de Datos Factuales/economía , Geografía Médica , Salud Global , Humanos , Distrofia Muscular de Duchenne/economía , Distrofia Muscular de Duchenne/epidemiologíaRESUMEN
BACKGROUND: X-linked intellectual disability is a common cause of inherited cognitive deficit affecting mostly males. There are several genetic causes implicated in this condition, which has hampered the establishment of an accurate diagnosis. We developed a multiplex-PCR assay for the mutational hotspot regions of the FMR1, AFF2 and ARX genes. METHODS: The multiplex-PCR was validated in a cohort of 100 males selected to include known alleles for the FMR1 repetitive region: five full mutations (250-650 CGGs), ten premutations (70-165 CGGs) and eighty-five in the normal range (19-42 CGGs). Sequencing or Southern blotting was used to confirm the results, depending on the allele class. In this cohort, with the exception of one sample showing an AFF2 intermediate-sized allele, all other samples were normal (8-34 CCGs). No ARX variant was found besides the c.429_452dup. The validated assay was applied to 5000 samples (64.4% males and 35.6% females). RESULTS: The normal-allelic range of both FMR1 and AFF2 genes as well as the nature of ARX variants identified was similar in both genders. The rate of homozygosity observed in female samples, 27.5% for FMR1 and 17.8% for AFF2 alleles, is comparable to that published by others. Two FMR1 premutations were identified, in a male (58 CGGs) and a female case [(CGG)(47)/(CGG)(61)], as well as several FMR1 or AFF2 intermediate-sized alleles. One AFF2 premutation (68 CCGs) and two putative full expansions were picked up in male subjects, which seems relevant considering the rarity of reported AFF2 mutations found in the absence of a family history. CONCLUSIONS: We developed a robust multiplex-PCR that can be used to screen the mutational hotspot regions of FMR1, AFF2 and ARX genes. Moreover, this strategy led to the identification of variants in all three genes, representing not only an improvement in allele-sizing but also in achieving a differential diagnosis. Although the distinction between females who are truly homozygous and those with a second pre- or full mutation sized allele, as well as a definitive diagnosis, requires a specific downstream technique, the use of this multiplex-PCR for initial screening is a cost-effective approach which widens the scope of detection.
Asunto(s)
Genes Ligados a X , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Alelos , Southern Blotting , Estudios de Cohortes , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/diagnóstico , Síndrome del Cromosoma X Frágil/genética , Pruebas Genéticas , Proteínas de Homeodominio/genética , Homocigoto , Humanos , Masculino , Mutación , Proteínas Nucleares/genética , Fenotipo , Análisis de Secuencia de ADN , Factores de Transcripción/genéticaRESUMEN
The Gilbert syndrome is a benign form of unconjugated hyperbilirubinemia, mainly associated with alterations in UGT1A1 gene. This work investigated the effect of UGT1A1 variants on total bilirubin levels in Gilbert patients (n=45) and healthy controls (n=161). Total bilirubin levels were determined using a colorimetric method; molecular analysis of exons 1-5 and two UGT1A1 promoter regions were performed by direct sequencing and automatic analysis of fragments. Five in silico methods predicted the effect of new identified variants. A significant different allelic distribution, in Gilbert patients and in controls, was found for two promoter polymorphisms. Among patients, 82.2% were homozygous and 17.8% heterozygous for the c.-41_-40dupTA allele; in control group, 9.9% were homozygous and 43.5% heterozygous for this promoter variant, while 46.6% (n=75) presented the [A(TA)6TAA]. For the T>G transition at c.-3279 promoter region, in patients, 86.7% were homozygous and 13.3% heterozygous; in control group, 33.5% were homozygous for the wild type allele, 44.1% were heterozygous and 22.4% homozygous for the mutated allele. The two polymorphisms were in Hardy-Weinberg equilibrium in both groups. Sequencing of UGT1A1 coding region identified nine novel variants, five in patients and four in controls. In silico analysis of these amino acids replacements predicted four of them as benign and three as damaging. In conclusion, we demonstrated that total bilirubin levels are mainly determined by the TA duplication in the TATA-box promoter and by the c.-3279T>G variant. Alterations in the UGT1A1 coding region seem to be associated with increased bilirubin levels, and, therefore, with Gilbert syndrome.
Asunto(s)
Bilirrubina/sangre , Enfermedad de Gilbert/sangre , Enfermedad de Gilbert/genética , Glucuronosiltransferasa/genética , Polimorfismo Genético , Adulto , Secuencia de Bases , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Sistemas de Lectura Abierta , Regiones Promotoras GenéticasRESUMEN
X-chromosome inactivation (XCI) is a developmental process to compensate the imbalance in the dosage of X-chromosomal genes in females. A skewing of the XCI pattern may suggest a carrier status for an X-linked disease or explain the presence of a severe phenotype. In these cases, it is important to determine the XCI pattern, conventionally using the gold standard Human Androgen-Receptor Assay (HUMARA), based on the analysis of the methylation status at a polymorphic CAG region in the first exon of the human androgen receptor gene (AR). The aim of this study was to evaluate whether the methylation status of the fragile mental retardation protein translational regulator gene (FMR1) can provide an XCI pattern similar to that obtained by HUMARA. A set of 48 female carriers of FMR1 gene normal-sized alleles was examined using two assays: HUMARA and a FMR1 methylation PCR (mPCR). Ranges were defined to establish the XCI pattern using the methylation pattern of the FMR1 gene by mPCR. Overall, a 77% concordance of the XCI patterns was obtained between the two assays, which led us to propose a set of key points and a stepwise analysis towards obtaining an accurate result for the XCI pattern and to minimize the underlying pitfalls.