Biology subjects

Navarro-Gomez, D.

Publications and source records attributed to Navarro-Gomez, D..

5 recordsLinked to original sources

bioRxiv2020

Evaluating Methods for Differential Gene Expression And Alternative Splicing Using Internal Synthetic Controls.

High-throughput transcriptome sequencing has become a powerful tool in the study of human diseases. Identification of causal mechanisms may entail analysis of differential gene expression (DGE), differential transcript/isoform expression (DTE) and identification, classification and quantification of alternative splicing (AS) and/or detection of novel AS events. For such a global transcriptome profiling execution of multi-level data analysis methodologies is required. Each level presents its own unique challenges and the questions about their performance remains. In this work we present results from systematic and consistent assessing and comparing a number of widely used methods for detecting DGE, DTE and AS using internal control "spike-in" sequences (Sequins) in RNA-seq data. We demonstrated that inclusion of internal controls in RNA-seq experiments allows accurate determination of lower bounds detection levels, and better assessment of DGE, DTE and AS accuracy and sensitivity. Tools for RNA-seq read alignment and detection of DGE performed reasonably. More efforts are needed to improve specificity and sensitivity of DTE and AS detection. Low expression of isoforms accompanied with sequencing depth does impact sensitivity and specificity of DTE and AS tools.

bioinformatics↗

bioRxiv2019

A combined RNA-seq and whole genome sequencing approach for identification of non-coding pathogenic variants in single families.

Inherited retinal degenerations (IRDs) are at the focus of current genetic therapeutic advancements. For a genetic treatment such as gene therapy to be successful an accurate genetic diagnostic is required. Genetic diagnostics relies on the assessment of the probability that a given DNA variant is pathogenic. Non-coding variants present a unique challenge for such assessments as compared to coding variants. For one, non-coding variants are present at much higher number in the genome than coding variants. In addition, our understanding of the rules that govern the non-coding regions of the genome is less complete than our understanding of the coding regions. Methods that allow for both the identification of candidate non-coding pathogenic variants and their functional validation may help overcome these caveats allowing for a greater number of patients to benefit from advancements in genetic therapeutics. We present here an unbiased approach combining whole genome sequencing (WGS) with patient induced pluripotent stem cell (iPSC) derived retinal organoids (ROs) transcriptome analysis. With this approach we identified and functionally validated a novel pathogenic non-coding variant in a small family with a previously unresolved genetic diagnosis.

genetics↗

bioRxiv2019

Copy-number variation contributes 9% of pathogenicity in the inherited retinal degenerations

PurposeCurrent sequencing strategies can genetically solve 55-60% of inherited retinal degeneration (IRD) cases, despite recent progress in sequencing. This can partially be attributed to elusive pathogenic variants (PVs) in known IRD genes, including copy number variations (CNVs), which we believe are a major contributor to unsolved IRD cases.\n\nMethodsFive hundred IRD patients were analyzed with targeted next generation sequencing (NGS). The NGS data was used to detect CNVs with ExomeDepth and gCNV and the results were compared to CNV detection with a SNP-Array. Likely causal CNV predictions were validated by quantitative (q)PCR.\n\nResultsLikely disease-causing single nucleotide variants (SNVs) and small indels were found in 55.8% of subjects. PVs in USH2A (11.6%), RPGR (4%) and EYS (4%) were the most common. Likely causal CNVs were found in an additional 8.8% of patients. Of the three CNV detection methods, gCNV showed the highest accuracy. Approximately 30% of unsolved subjects had a single likely PV in a recessive IRD gene.\n\nConclusionsCNV detection using NGS-based algorithms is a reliable method that greatly increases the genetic diagnostic rate of IRDs. Experimentally validating CNVs helps estimate the rate at which IRDs might be solved by a CNV plus a more elusive variant.

genomics↗

bioRxiv2017

Contribution of structural and intronic mutations to RPGRIP1-mediated inherited retinal dystrophies.

PurposeWith the advent of gene therapies for inherited retinal degenerations (IRDs), genetic diagnostics will have an increasing role in clinical decision-making. Yet the genetic cause of disease cannot be identified using exon-based sequencing for a significant portion of patients. We hypothesized that non-coding mutations contribute significantly to the genetic causality of IRDs and evaluated patients with single coding mutations in RPGRIP1 to test this hypothesis.\n\nMethodsIRD families underwent targeted panel sequencing. Unsolved cases were explored by whole exome and genome sequencing looking for additional mutations. Candidate mutations were then validated by Sanger sequencing, quantitative PCR, and in vitro splicing assays in two cell lines analyzed through amplicon sequencing.\n\nResultsAmong 1722 families, three had biallelic loss of function mutations in RPGRIP1 while seven had a single disruptive coding mutation. Whole exome and genome sequencing revealed potential non-coding mutations in these seven families. In six, the non-coding mutations were shown to lead to loss of function in vitro.\n\nConclusionNon-coding mutations were identified in 6 of 7 families with single coding mutations in RPGRIP1. The results suggest that non-coding mutations contribute significantly to the genetic causality of IRDs and RPGRIP1-mediated IRDs are more common than previously thought.

genomics↗

bioRxiv2017

Allele-specific editing of rhodopsin P23H knock-in mice broadens therapeutic potential of CRISPR/Cas for dominant genetic diseases

Treatment strategies for dominantly inherited disorders typically involve silencing or ablating the pathogenic allele. CRISPR/Cas nucleases have shown promise in allele-specific knockout approaches when the dominant allele creates unique protospacer adjacent motifs (PAMs) that can lead to allele restricted targeting. Here, we present a spacer-mediated allele-specific knockout approach that utilizes both SpCas9 variants and truncated single guide RNAs (trusgRNAs) to achieve efficient discrimination of a single-nucleotide mutation in rhodopsin (Rho)-P23H mice, a model of dominant retinitis pigmentosa (RP). We found that approximately 45% of the mutant P23H allele was edited at DNA level, and that the relative RNA expression of wild-type Rho was about 2.8 times more than that of mutant Rho in treated retinas. Furthermore, the progression of photoreceptor cell degeneration in outer nuclear layer was significantly delayed in treated regions of the Rho-P23H retinas at five weeks of age. Our proof-of-concept study therefore outlines a general strategy that could potentially be expanded to examine the therapeutic benefit of allele-specific gene editing approach to treat human P23H patient. Our study also extends allele-specific editing strategies beyond discrimination within the PAM sites, with potentially broad applicability to other dominant diseases.

genetics↗