Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing.

A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.

Diagnosis of becker muscular dystrophy: Results of Re-analysis of DNA samples.

INTRODUCTION: The phenotype of Becker muscular dystrophy (BMD) is highly variable, and the disease may be underdiagnosed. We searched for new mutations in the DMD gene in a cohort of previously undiagnosed patients who had been referred in the period 1985-1995. METHODS: All requests for DNA analysis of the DMD gene in probands with suspected BMD were re-evaluated. If the phenotype was compatible with BMD, and no deletions or duplications were detected, DNA samples were screened for small mutations. RESULTS: In 79 of 185 referrals, no mutation was found. Analysis could be performed on 31 DNA samples. Seven different mutations, including 3 novel ones, were found. Long-term clinical follow-up is described. CONCLUSIONS: Refining DNA analysis in previously undiagnosed cases can identify mutations in the DMD gene and provide genetic diagnosis of BMD. A delayed diagnosis can still be valuable for the proband or the relatives of BMD patients.

Discordancia genotipo-fenotipo en un paciente con distrofia muscular de Duchenne por una nueva mutación: hipótesis de la función de amortiguación de la distrofina / Genotype-phenotype discordance in a Duchenne muscular dystrophy patient due to a novel mutation: insights into the shock absorber function of dystrophin

Introducción. La distrofia muscular de Duchenne (DMD) es un trastorno caracterizado por atrofia muscular progresiva y debilidad, debido a la ausencia o alteración de la función de la distrofina, una proteína que protege a las células del músculo de la tensión mecánica inducida durante la contracción. Las mutaciones en el gen DMD pueden dar lugar a diferentes fenotipos clínicos, conocidos colectivamente como distrofinopatías, de los cuales la DMD es el más grave. Caso clínico. Se presenta una deleción nueva de los exones 24-41, que no interrumpe el marco de lectura y se espera que origine un fenotipo leve. Por el contrario, el paciente tiene un fenotipo DMD grave. Conclusiones. Nuestra comunicación apoya la hipótesis de que la interrupción del sitio de unión a gamma-actina situado en el dominio cilíndrico central desempeña un papel crucial en la función de amortiguador de distrofina en las células musculares. La descripción de las variantes patogénicas en el gen DMD y los fenotipos resultantes tienen importantes implicaciones en el diseño de estrategias terapéuticas moleculares para la DMD (AU)

Introduction. Duchenne muscular dystrophy (DMD) is a genomic disorder characterized by progressive muscle wasting and weakness due to the absence or abnormal function of dystrophin; a protein that protects muscle cells from mechanical induced stress during contraction. Mutations in the DMD gene, may lead to different clinical phenotypes, collectively known as dystrophinopathies, of which DMD has the earliest onset and most severe progression. Case report. We report a novel deletion of exons 24-41, predicted to maintain the reading frame and expected to result in a mild phenotype. Conversely, the patient has a severe DMD phenotype. Conclusions. Our report supports the hypothesis that disruption of the gamma-actin-binding site located in the central rod domain plays a crucial role in the shock absorber function of dystrophin in muscle cells. Description of pathogenic variants in the DMD gene and the resulting phenotypes has important implications on the designing of molecular therapeutic approaches for DMD (AU)

[Genotype-phenotype discordance in a Duchenne muscular dystrophy patient due to a novel mutation: insights into the shock absorber function of dystrophin]. / Discordancia genotipo-fenotipo en un paciente con distrofia muscular de Duchenne por una nueva mutación: hipótesis de la función de amortiguación de la distrofina.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is a genomic disorder characterized by progressive muscle wasting and weakness due to the absence or abnormal function of dystrophin; a protein that protects muscle cells from mechanical induced stress during contraction. Mutations in the DMD gene, may lead to different clinical phenotypes, collectively known as dystrophinopathies, of which DMD has the earliest onset and most severe progression. CASE REPORT: We report a novel deletion of exons 24-41, predicted to maintain the reading frame and expected to result in a mild phenotype. Conversely, the patient has a severe DMD phenotype. CONCLUSIONS: Our report supports the hypothesis that disruption of the gamma-actin-binding site located in the central rod domain plays a crucial role in the shock absorber function of dystrophin in muscle cells. Description of pathogenic variants in the DMD gene and the resulting phenotypes has important implications on the designing of molecular therapeutic approaches for DMD.

[Improvements in the diagnosis of dystrophinopathies: what have we learnt in these last 20 years?]. / Mejoras en el diagnostico de distrofinopatias: que hemos aprendido despues de 20 anos?

INTRODUCTION. Dystrophinopathies are X-linked genetic disorders caused by mutations in the DMD gene. Genetic tests are of utmost importance for management and genetic counseling of these diseases. However, the complexity of the DMD gene is a challenge for diagnosis. AIM. To describe recent advances in the diagnosis of dystrophinopathies, after 20 years since the firsts molecular assays for genetic screening for these diseases. DEVELOPMENT. Currently, a variety of strategies such as automated mutation detection, cell-based methods and high throughput haplotyping have been developed to facilitate diagnosis of dystrophinopathies, carrier detection, prenatal and preimplantation diagnosis. CONCLUSION. New technologies have improved early detection and optimal management of dystrophinopathies and have established the basis for future molecular medicine. The most significant advances in dystrophinopathy diagnosis are reviewed herein.