An evaluation of pipelines for DNA variant detection can guide a reanalysis protocol to increase the diagnostic ratio of genetic diseases

Clinical exome (CE) sequencing has become a first-tier diagnostic test for hereditary diseases; however, its diagnostic rate is around 30–50%. In this study, we aimed to increase the diagnostic yield of CE using a custom reanalysis algorithm. Sequencing data were available for three cohorts using two commercial protocols applied as part of the diagnostic process. Using these cohorts, we compared the performance of general and clinically relevant variant calling and the efficacy of an in-house bioinformatic protocol (FJD-pipeline) in detecting causal variants as compared to commercial protocols. On the whole, the FJD-pipeline detected 99.74% of the causal variants identified by the commercial protocol in previously solved cases. In the unsolved cases, FJD-pipeline detects more INDELs and non-exonic variants, and is able to increase the diagnostic yield in 2.5% and 3.2% in the re-analysis of 78 cancer and 62 cardiovascular cases. These results were considered to design a reanalysis, filtering and prioritization algorithm that was tested by reassessing 68 inconclusive cases of monoallelic autosomal recessive retinal dystrophies increasing the diagnosis by 4.4%. In conclusion, a guided NGS reanalysis of unsolved cases increases the diagnostic yield in genetic disorders, making it a useful diagnostic tool in medical geneticsWe want to thank the participants for consenting to the use of their data for the study. We would like to thank all technical staff in the genetics service of the Fundación Jiménez Díaz University Hospital for conducting the sequencing and segregation analysis. We also thank Oliver Shaw (IIS-FJD) for editorial assistance. This work was supported by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (FIS; PI16/00425, PI19/00321, PI18/00579 and PI20/00851), Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER, 06/07/0036), IIS-FJD BioBank (PT13/0010/0012), Comunidad de Madrid (CAM, RAREGenomics Project, B2017/BMD-3721), Ramón Areces Foundation (4019/012), Conchita Rábago Foundation, and the University Chair UAM-IIS-FJD of Genomic Medicine. R.R. is supported by a postdoctoral fellowship of the Comunidad de Madrid (2019-T2/BMD-13714), L.d.l.F. is supported by the platform technician contract of ISCIII (CA18/00017), IPR is supported by a PhD studentship from the predoctoral program from ISCIII (FI17/ 00192), I.F.I. is supported by a grant from the Comunidad de Madrid (CAM, PEJ-2017- AI/BMD7256), G.N.M. is supported by a grant from the Comunidad de Madrid (PEJ2020-AI/BMD-18610), A.D. is supported by a PhD studentship from the predoctoral program from ISCIII (FI18/00123), B.A. is supported by a Juan Rodes program from ISCIII (JR17/00020), C.R. is supported by a PhD studentship from the Conchita Rabago Foundation and PM and MC are supported by a Miguel Servet program contract from ISCIII (CP16/00116 and CPII17/00006, respectively). The funders played no role in study design, data collection, data analysis, manuscript preparation, and/or publication decision

Desarrollo de métodos bioinformáticos para la priorización de variantes y genes en casos no resueltos de distrofias hereditarias de retina

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 18-07-2022Esta Tesis tiene embargado el acceso al texto completo hasta el 18-01-2024Las enfermedades raras (EERR) se caracterizan por tener una baja prevalencia en la población general, aunque en su conjunto afectan a más de 400 millones de personas en todo el mundo. El 80% de estas enfermedades son genéticas. Desde la aparición de las tecnologías de secuenciación masiva (next generation sequencing, NGS) se ha producido un aumento considerable en el conocimiento de su etiología y en sus tasas de diagnóstico, disminuyendo el tiempo de espera de los pacientes. Las NGS también ha traído varios retos: por un lado, en cada experimento se detectan miles de variantes genómicas que hay que filtrar y priorizar para encontrar aquellas causales del fenotipo; por otro, el conocimiento clínico de las variantes no ha aumentado a la misma velocidad y se necesitan métodos concluyentes y predictivos para disminuir la incertidumbre en la valoración clínica de estas variantes. En esta tesis doctoral se ha tomado a las distrofias hereditarias de retina (DHR) como ejemplo de EERR y se han desarrollado métodos bioinformáticos para priorizar variantes y genes en casos no resueltos de la cohorte de pacientes del Hospital Universitario Fundación Jiménez Díaz. Así, se ha desarrollado una metodología para priorizar variantes de significado incierto (variants of uncertain significance, VUS) monitorizadas en casos de DHR, estudiando características específicas de variante, enfermedad y laboratorio y comparándolo con las variantes patogénicas de la misma enfermedad. Esto ha permitido priorizar un total de 49 VUS, de las cuáles se han conseguido reclasificar a probablemente patogénicas un total de 5 variantes (10%). Además, se ha desarrollado una base de datos con información genómica agregada de una cohorte heterogénea de >5.500 pacientes con varias enfermedades raras y unos 5.000 genes secuenciados. A partir de este recurso y utilizando comparaciones de frecuencias entre casos de DHR y pacientes con enfermedades no relacionadas (pseudocontroles) se han desarrollado métodos para priorizar variantes y genes en casos no resueltos de DHR. De esta forma, se han resuelto parcial o totalmente 5 casos y reclasificado 10 VUS a probablemente patogénicas (~91%). También se han priorizado 18 genes en casos no resueltos que han contribuido a caracterizar parcial o totalmente otros 4 casos previamente no resueltos. Por último, se ha utilizado la información genómica agregada disponible para realizar el cálculo de la frecuencia alélica en portadores, para los genes con un patrón de herencia autosómico recesivo de DHR. Así, los genes con una mayor frecuencia en portadores resultaron ser ABCA4 y USH2A con una frecuencia del 7% y 3% respectivamente. Esta tesis demuestra por un lado la utilidad de desarrollar soluciones específicas adaptadas al contexto clínico y técnico de un laboratorio de diagnóstico genético y un grupo de enfermedades concreto, como las DHR, permitiendo detectar sesgos en la tasa de reporte de VUS, que permiten su priorización y posterior reclasificación. Por otro lado, también se ha demostrado la utilidad de la información genómica agregada como fuente de hipótesis, que ha permitido el desarrollo de metodologías para priorizar variantes y genes en casos no resueltos de DHR, que en última instancia permiten incrementar la tasa diagnóstica de esta enfermeda

Biocompatible and Antimicrobial Cellulose Acetate-Collagen Films Containing MWCNTs Decorated with TiO2 Nanoparticles for Potential Biomedical Applications

This research focuses on the synthesis of multi-walled carbon nanotubes (MWCNTs) decorated with TiO2 nanoparticles (NPs) and incorporated in cellulose acetate-collagen film in order to obtain a new biomaterial with potential biomedical applications and improved antimicrobial activity. The successful decoration of the MWCNTs with TiO2 NPs was confirmed by several structural and morphological analysis, such as Fourier transformed infrared spectroscopy, Raman spectroscopy, X-ray diffraction and transmission electron microscopy. The obtained nanocomposites were further incorporated into cellulose acetate-collagen films, at different concentrations and absorption kinetics, antimicrobial activity and in vitro biocompatibility of the obtained films was investigated. The antimicrobial tests sustained that the presence of the nanocomposites into the polymeric matrix is an important aspect in increasing and maintaining the antimicrobial activity of the polymeric wound dressings over time. The biocompatibility and cytotoxicity of the obtained films was evaluated using cellular viability/proliferation assay and fluorescent microscopy which revealed the ability of the obtained materials as potential wound dressing biomaterial

Diagnostics of HNSCC Patients: An Analysis of Cell Lines and Patient-Derived Xenograft Models for Personalized Therapeutical Medicine

Head and neck squamous cell carcinomas (HNSCC) are very frequent worldwide, and smoking and chronic alcohol use are recognized as the main risk factors. For oropharyngeal cancers, HPV 16 infection is known to be a risk factor as well. By employing next-generation sequencing, both HPV-positive and negative HNSCC patients were detected as positive for PI3K mutation, which was considered an optimal molecular target. We analyzed scientific literature published in the last 5 years regarding the newly available diagnostic platform for targeted therapy of HNSCC HPV+/−, using HNSCC-derived cell lines cultures and HNSCC pdx (patient-derived xenografts). The research results are promising and require optimal implementation in the management of HNSCC patients

Genotype-Phenotype Correlations in a Spanish Cohort of 506 Families With Biallelic ABCA4 Pathogenic Variants

To define genotype-phenotype correlations in the largest cohort study worldwide of patients with biallelic ABCA4 variants, including 434 patients with Stargardt disease (STGD1) and 72 with cone-rod dystrophy (CRD).; Cohort study.; We characterized 506 patients with ABCA4 variants using conventional genetic tools and next-generation sequencing technologies. Medical history and ophthalmologic data were obtained from 372 patients. Genotype-phenotype correlation studies were carried out for the following variables: variant type, age at symptom onset (AO), and clinical phenotype.; A total of 228 different pathogenic variants were identified in 506 ABCA4 patients, 50 of which were novel. Genotype-phenotype correlations showed that most of the patients with biallelic truncating variants presented with CRD and that these cases had a significantly earlier AO than patients with STGD1. Three missense variants are associated with CRD for the first time (c.1804C>T; p.[Arg602Trp], c.3056C>T; p.[Thr1019Met], and c.6320G>C; p.[Arg2107Pro]). Analysis of the most prevalent ABCA4 variant in Spain, c.3386G>T; p.(Arg1129Leu), revealed that is correlated to STGD1, later AO, and foveal sparing.; Our study, conducted in the largest ABCA4-associated disease cohort reported to date, updates the genotype-phenotype model established for ABCA4 variants and broadens the mutational spectrum of the gene. According to our observations, patients with ABCA4 presenting with 2 truncating variants may first present features of STGD1 but eventually develop rod dysfunction, and specific missense variants may be associated with a different phenotype, underscoring the importance of an accurate genetic diagnosis. Also, it is a prerequisite for enrollment in clinical trials, and to date, no other treatment has been approved for STGD1

Aggregated Genomic Data as Cohort-Specific Allelic Frequencies can Boost Variants and Genes Prioritization in Non-Solved Cases of Inherited Retinal Dystrophies

The introduction of NGS in genetic diagnosis has increased the repertoire of variants and genes involved and the amount of genomic information produced. We built an allelic-frequency (AF) database for a heterogeneous cohort of genetic diseases to explore the aggregated genomic information and boost diagnosis in inherited retinal dystrophies (IRD). We retrospectively selected 5683 index-cases with clinical exome sequencing tests available, 1766 with IRD and the rest with diverse genetic diseases. We calculated a subcohort’s IRD-specific AF and compared it with suitable pseudocontrols. For non-solved IRD cases, we prioritized variants with a significant increment of frequencies, with eight variants that may help to explain the phenotype, and 10/11 of uncertain significance that were reclassified as probably pathogenic according to ACMG. Moreover, we developed a method to highlight genes with more frequent pathogenic variants in IRD cases than in pseudocontrols weighted by the increment of benign variants in the same comparison. We identified 18 genes for further studies that provided new insights in five cases. This resource can also help one to calculate the carrier frequency in IRD genes. A cohort-specific AF database assists with variants and genes prioritization and operates as an engine that provides a new hypothesis in non-solved cases, augmenting the diagnosis rate