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Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis.

ABSTRACT: Massively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient's phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100-300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations.To overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene.We identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45-250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing.We showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.

SUBMITTER: Barseghyan H

PROVIDER: S-EPMC5655859 | biostudies-other | 2017 Oct

REPOSITORIES: biostudies-other

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Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis.

Barseghyan Hayk H Tang Wilson W Wang Richard T RT Almalvez Miguel M Segura Eva E Bramble Matthew S MS Lipson Allen A Douine Emilie D ED Lee Hane H Délot Emmanuèle C EC Nelson Stanley F SF Vilain Eric E

Genome medicine 20171025 1

<h4>Background</h4>Massively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient's phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100-300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as in ...[more]

PMID: 29070057

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Project description:Retinoblastoma (RB, OMIM:180200) is the most common malignant childhood tumor of the eye with an estimated incidence between 1 in 16,000 and 1 in 18,000 live births [1,2]. RB is the first disease for which a genetic etiology of cancer has been described [3] being caused by mutations in the first tumor suppressor gene identified (RB1, Genbank accession # L11910). Mutations in both alleles of the RB1 gene are required for the development of this neoplasm [4], and, depending on the germ-line or somatic origin of the defect, a heritable or sporadic form can be distinguished. RB is unilateral in 60% of cases and only 15% of these are heritable [5]; in contrast, 40% of retinoblastomas are bilateral with risk of transmission to the offspring. Heritable retinoblastoma constitutes a cancer predisposition syndrome [6]. RB1 is located on chromosome 13 at band q14 and can be affected by a heterogeneous spectrum of genetic abnormalities, including chromosome translocation/deletion, genomic rearrangements, ranging from whole gene microdeletion to intragenic exons loss or duplication, and more than 900 different point mutations [7]. Mutational analysis is performed to search for the predisposing RB1 gene mutation in peripheral blood of patients with RB, but the molecular diagnosis requires several technical approaches to cover the entire field of oncogenic RB1 defects, frequently resulting in numerous, expensive and time consuming procedures. In particular, cytogenetic tools, such as classical chromosome investigations and Fluorescent In Situ Hybridization (FISH), in addition to Multiplex Ligation-dependent Probe Amplification (MLPA) technique, may account for detection of about 16% of RB1 abnormalities [8], while the remaining large amount of point mutations need to be investigated using sequencing analysis. Since the 1970s, Sanger sequencing has been recognized as the gold standard for mutation analysis in molecular diagnostics; however, its low-throughput, long turnaround time and overall cost [9] have called for new paradigms. Next Generation Sequencing (NGS) can massively sequence millions of DNA segments, promising low costs, increased workflow speed and enhanced sensitivity in mutation detection [9,10,11] On the other hand, conventional and molecular cytogenetic analysis, have been replaced by modern high-throughput investigations, such as array Comparative Genomic Hybridization (aCGH), that can reveal and measure cryptic genomic imbalances. In addition, aCGH can be focused on specific DNA segments or genes maximizing the resolution via a customized process. Based on these observations, we have recruited a cohort of retinoblastoma patients we previously investigated with conventional cytogenetics and MLPA. Patients diagnosed with RB but negative to the above standard screening have been tested with NGS to assess its ability in identifying RB causative mutations. On the other hand, patients positive to standard screening have been further investigated with RB1-custom array CGH analysis to characterize the genomic rearrangements with a better resolution compared to the conventional techniques. The complementary aCGH data set related to this study has also been deposited at ArrayExpress, under accession number E-MTAB-3492: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3492/

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Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis.

Publications

Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis.

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