Molecular Diagnosis of Neonatal Diabetes Mellitus Using Next-Generation Sequencing of the Whole Exome

CNRS-UMR-8199, Univ Lille Nord de France, UDSL, Lille, France.
PLoS ONE (Impact Factor: 3.23). 10/2010; 5(10):e13630. DOI: 10.1371/journal.pone.0013630
Source: PubMed

ABSTRACT Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis.
We carried out WES for a patient presenting with permanent NDM, for whom mutations in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patient's mother, father and young brother, all of whom are normoglycemic.
WES identified a novel de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.

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Available from: Martine Vaxillaire, Sep 26, 2015
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    • "First, a majority of identified variants are present in dbSNP [8,9] and therefore represent more common variation. For example, exome and genome sequencing reports have shown that 88–99% of observed SNVs reside in dbSNP [1-3,5,10]. Second, the number of SNVs is dependent on individual genetic variability, ethnicity, and the reference sequence to which results are aligned and compared. "
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    ABSTRACT: Background The recent introduction of high throughput sequencing technologies into clinical genetics has made it practical to simultaneously sequence many genes. In contrast, previous technologies limited sequencing based tests to only a handful of genes. While the ability to more accurately diagnose inherited diseases is a great benefit it introduces specific challenges. Interpretation of missense mutations continues to be challenging and the number of variants of uncertain significance continues to grow. Results We leveraged the data available at ARUP Laboratories, a major reference laboratory, for the CFTR gene to explore specific challenges related to variant interpretation, including a focus on understanding ethnic-specific variants and an evaluation of existing databases for clinical interpretation of variants. In this study we analyzed 555 patients representing eight different ethnic groups. We observed 184 different variants, most of which were ethnic group specific. Eighty-five percent of these variants were present in the Cystic Fibrosis Mutation Database, whereas the Human Mutation Database and dbSNP/1000 Genomes had far fewer of the observed variants. Finally, 21 of the variants were novel and we report these variants and their clinical classifications. Conclusions Based on our analyses of data from six years of CFTR testing at ARUP Laboratories a more comprehensive, clinical grade database is needed for the accurate interpretation of observed variants. Furthermore, there is a particular need for more and better information regarding variants from individuals of non-Caucasian ethnicity.
    Journal of Clinical Bioinformatics 01/2013; 3(1):3. DOI:10.1186/2043-9113-3-3
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    • "Compared to whole-genome sequencing, the major advantage of exome sequencing is that the cost has been reduced significantly. Whole-exome sequencing has been used to identify genes associated with cancer[19], diabetes[20], immunologic disorders[21], and other conditions. Transcriptome sequencing involves sequencing cDNA fragments generated by reverse transcription of RNA. "
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    Chinese journal of cancer 09/2012; 31(10):463-70. DOI:10.5732/cjc.012.10216 · 2.16 Impact Factor
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    • "The chromatogram corresponding to the remaining variant had a high level of noise throughout. Other laboratories have noted similarly missed variants [31]. While currently considered the “gold standard” for molecular genetic diagnosis of SNV-type mutations, PCR-based dideoxy sequencing relies heavily on manual sequence trace inspection and is thus subject to human error. "
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    ABSTRACT: The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.
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