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Next generation sequencing in research and diagnostics of ocular birth defects.

ABSTRACT: Sequence capture enrichment (SCE) strategies and massively parallel next generation sequencing (NGS) are expected to increase the rate of gene discovery for genetically heterogeneous hereditary diseases, but at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. Our study assessed whether array based target enrichment followed by re-sequencing on the Roche Genome Sequencer FLX (GS FLX) system could be used for novel mutation identification in more than 1000 exons representing 100 candidate genes for ocular birth defects, and as a control, whether these methods could detect two known mutations in the PAX2 gene. We assayed two samples with heterozygous sequence changes in PAX2 that were previously identified by conventional Sanger sequencing. These changes were a c.527G>C (S176T) substitution and a single basepair deletion c.77delG. The nucleotide substitution c.527G>C was easily identified by NGS. A deletion of one base in a long polyG stretch (c.77delG) was not registered initially by the GS Reference Mapper, but was detected in repeated analysis using two different software packages. Different approaches were evaluated for distinguishing false positives (sequencing errors) and benign polymorphisms from potentially pathogenic sequence changes that require further follow-up. Although improvements will be necessary in accuracy, speed, ease of data analysis and cost, our study confirms that NGS can be used in research and diagnostic settings to screen for mutations in hundreds of loci in genetically heterogeneous human diseases.

SUBMITTER: Raca G

PROVIDER: S-EPMC2871986 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Next generation sequencing in research and diagnostics of ocular birth defects.

Raca Gordana G Jackson Craig C Warman Berta B Bair Tom T Schimmenti Lisa A LA

Molecular genetics and metabolism 20100315 2

Sequence capture enrichment (SCE) strategies and massively parallel next generation sequencing (NGS) are expected to increase the rate of gene discovery for genetically heterogeneous hereditary diseases, but at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. Our study assessed whether array based target enrichment followed by re-sequencing on the Roche Genome Sequencer FLX (GS FLX) system could be used f ...[more]

PMID: 20359920

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Project description:BackgroundRecently, a growing number of novel genetic defects underlying primary immunodeficiencies (PIDs) have been identified, increasing the number of PID up to more than 250 well-defined forms. Next-generation sequencing (NGS) technologies and proper filtering strategies greatly contributed to this rapid evolution, providing the possibility to rapidly and simultaneously analyze large numbers of genes or the whole exome.ObjectiveTo evaluate the role of targeted NGS and whole exome sequencing (WES) in the diagnosis of a case series, characterized by complex or atypical clinical features suggesting a PID, difficult to diagnose using the current diagnostic procedures.MethodsWe retrospectively analyzed genetic variants identified through targeted NGS or WES in 45 patients with complex PID of unknown etiology.ResultsForty-seven variants were identified using targeted NGS, while 5 were identified using WES. Newly identified genetic variants were classified into four groups: (I) variations associated with a well-defined PID, (II) variations associated with atypical features of a well-defined PID, (III) functionally relevant variations potentially involved in the immunological features, and (IV) non-diagnostic genotype, in whom the link with phenotype is missing. We reached a conclusive genetic diagnosis in 7/45 patients (~16%). Among them, four patients presented with a typical well-defined PID. In the remaining three cases, mutations were associated with unexpected clinical features, expanding the phenotypic spectrum of typical PIDs. In addition, we identified 31 variants in 10 patients with complex phenotype, individually not causative per se of the disorder.ConclusionNGS technologies represent a cost-effective and rapid first-line genetic approach for the evaluation of complex PIDs. WES, despite a moderate higher cost compared to targeted, is emerging as a valuable tool to reach in a timely manner, a PID diagnosis with a considerable potential to draw genotype-phenotype correlation. Nevertheless, a large fraction of patients still remains without a conclusive diagnosis. In these patients, the sum of non-diagnostic variants might be proven informative in future studies with larger cohorts of patients.

Project description:IntroductionSepsis and septic shock are the most severe forms of infection affecting predominantly elderly people, preterm and term neonates, and young infants. Even in high-income countries sepsis causes about 8% of admissions to pediatric intensive care units (PICUs). Early diagnosis, rapid anti-infective treatment, and prompt hemodynamic stabilization are crucial for patient survival. In this context, it is essential to identify the causative pathogen as soon as possible to optimize antimicrobial treatment. To date, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care. However, they have 2 major problems: on the one hand, in the case of very small sample volumes (and thus usually in children), they are not sufficiently sensitive. On the other hand, with a time-to-result of 2 to 5 days, blood cultures need a relatively long time for the anti-infective therapy to be calculated. To overcome these problems, culture-independent molecular diagnostic procedures such as unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) have been tested successfully in adult septic patients. However, these results still need to be transferred to the pediatric setting.MethodsThe Next GeneSiPS-Trial is a prospective, observational, non-interventional, multicenter study used to assess the diagnostic performance of an NGS-based approach for the identification of causative pathogens in (preterm and term) neonates (d1-d28, n = 50), infants (d29 to <1 yr, n = 50), and toddlers (1 yr to <5 yr, n = 50) with suspected or proven severe sepsis or septic shock (according to the pediatric sepsis definition) by the use of the quantitative sepsis indicating quantifier (SIQ) score in comparison to standard of care (culture-based) microbiological diagnostics. Potential changes in anti-infective treatment regimens based on these NGS results will be estimated retrospectively by a panel of 3 independent clinical specialists.DiscussionNeonates, infants, and young children are significantly affected by sepsis. Fast and more sensitive diagnostic approaches are urgently needed. This prospective, observational, non-interventional, multicenter study seeks to evaluate an NGS-based approach in critically ill children suffering from sepsis.Trial registrationDRKS-ID: DRKS00015705 (registered October 24, 2018). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00015705.

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Next generation sequencing in research and diagnostics of ocular birth defects.

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Next generation sequencing in research and diagnostics of ocular birth defects.

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