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Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing.

ABSTRACT: Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899?bp in intron 3. Our study is a proof-of-principle that high-throughput-targeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth.

SUBMITTER: Trujillano D

PROVIDER: S-EPMC3953903 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing.

Trujillano Daniel D Perez Belén B González Justo J Tornador Cristian C Navarrete Rosa R Escaramis Georgia G Ossowski Stephan S Armengol Lluís L Cornejo Verónica V Desviat Lourdes R LR Ugarte Magdalena M Estivill Xavier X

European journal of human genetics : EJHG 20130814 4

Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 9 ...[more]

PMID: 23942198

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Project description:BackgroundIntellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation.MethodsWe report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases.ResultsWe identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients' clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders.ConclusionsWith a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism.

Project description:BackgroundBovine tuberculosis (bTB) is an enduring contagious disease of cattle that has caused substantial losses to the global livestock industry. Despite large-scale eradication efforts, bTB continues to persist. Current bTB tests rely on the measurement of immune responses in vivo (skin tests), and in vitro (bovine interferon-γ release assay). Recent developments are characterized by interrogating the expression of an increasing number of genes that participate in the immune response. Currently used assays have the disadvantages of limited sensitivity and specificity, which may lead to incomplete eradication of bTB. Moreover, bTB that reemerges from wild disease reservoirs requires early and reliable diagnostics to prevent further spread. In this work, we use high-throughput sequencing of the peripheral blood mononuclear cells (PBMCs) transcriptome to identify an extensive panel of genes that participate in the immune response. We also investigate the possibility of developing a reliable bTB classification framework based on RNA-Seq reads.Methodology/principal findingsPooled PBMC mRNA samples from unaffected calves as well as from those with disease progression of 1 and 2 months were sequenced using the Illumina Genome Analyzer II. More than 90 million reads were splice-aligned against the reference genome, and deposited to the database for further expression analysis and visualization. Using this database, we identified 2,312 genes that were differentially expressed in response to bTB infection (p<10(-8)). We achieved a bTB infected status classification accuracy of more than 99% with split-sample validation on newly designed and learned mixtures of expression profiles.Conclusions/significanceWe demonstrated that bTB can be accurately diagnosed at the early stages of disease progression based on RNA-Seq high-throughput sequencing. The inclusion of multiple genes in the diagnostic panel, combined with the superior sensitivity and broader dynamic range of RNA-Seq, has the potential to improve the accuracy of bTB diagnostics. The computational pipeline used for the project is available from http://code.google.com/p/bovine-tb-prediction.

Dataset Information

Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing.

Publications

Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing.

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