Parallel sequencing used in detection of mosaic mutations: Comparison with four diagnostic DNA screening techniques

Rohlin, Anna; Wernersson, Josephine; Engwall, Yvonne; Wiklund, Leif; Björk, Jan; Nordling, Margareta

doi:10.1002/humu.20980

Cited by 151 publications

(132 citation statements)

References 38 publications

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“…However, although the assessed sensitivity for point mutation detection in mosaics of cells may vary according to different mutation types, the techniques used in this study can detect very low levels of mosaic mutations, such as 5% of mutated cells for DHPLC and 15% for direct sequencing (Rohlin et al, 2009). …”

Section: Discussionmentioning

confidence: 99%

Genetic models of osteochondroma onset and neoplastic progression: evidence for mechanisms alternative to EXT genes inactivation

et al. 2010

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Osteochondroma, the most common benign bone tumor, may occur as a sporadic lesion or as multiple neoplasms in the context of multiple osteochondromas syndrome. The most severe complication is malignant transformation into peripheral secondary chondrosarcoma. Although both benign conditions have been linked to defects in EXT1 or EXT2 genes, contradictory reports are present in the literature regarding the requirement of their biallelic inactivation for osteochondroma development. A major limitation of these studies is represented by the small number of samples available for the screening. Taking advantage of a large series of tissues, our aim was to contribute to the definition of a genetic model for osteochondromas onset and transformation. EXT genes point mutations and big deletions were analyzed in 64 tissue samples. A double hit was found in 5 out of 35 hereditary cases, 6 out of 16 chondrosarcomas and 2 recurrences; none of the 11 sporadic osteochondromas showed two somatic mutations. Our results clearly indicate that, in most cases, biallelic inactivation of EXT genes does not account for osteochondromas formation; this mechanism should be regarded as a common feature for hereditary osteochondromas transformation and as an event that occurs later in tumor progression of solitary cases. These findings suggest that mechanisms alternative to EXT genetic alteration likely have a role in osteochondromas pathogenesis.

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Section: Discussionmentioning

confidence: 99%

Genetic models of osteochondroma onset and neoplastic progression: evidence for mechanisms alternative to EXT genes inactivation

et al. 2010

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“…If the screening results are negative, sequencing of the whole mitochondrial genome is performed to find causative rare variants or novel mutations. Although the gold standard, Sanger sequencing, reliably detects most nucleotide changes, it does not provide quantitative information and is not adequately sensitive for detecting mutant heteroplasmy below 15% (11 ). Large mtDNA deletions are analyzed separately by Southern blotting (12 ) or by custom-designed array comparative genome hybridization (aCGH) (13,14 ).…”

confidence: 99%

Comprehensive One-Step Molecular Analyses of Mitochondrial Genome by Massively Parallel Sequencing

2012

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BACKGROUND:Mitochondrial diseases are clinically and genetically heterogeneous, with variable penetrance, expressivity, and differing age of onset. Diseasecausing point mutations and large deletions in the mitochondrial genome often exist in a heteroplasmic state. Current molecular analyses require multiple different and complementary methods for the detection and quantification of mitochondrial DNA (mtDNA) mutations. We developed a novel approach to analyze the mtDNA in 1 step.

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“…21 Although specific primers or probes can be designed for the quantification of target positions, 7,22 it is laborious to validate the method for every novel mtDNA point mutation. In addition, primers may contain modifications and probes that are specific for either the wild-type or mutant allele, [23][24][25] thus, a difference in PCR amplification efficiency for these two alleles is expected, leading to inherent inaccuracies with the measurement of the degree of heteroplasmy.…”

Section: Low-level Mutation Heteroplasmy and Its Clinical And Geneticmentioning

confidence: 99%

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

Cui

Chen

et al. 2013

Genetics in Medicine

111

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1-3Pathogenic mtDNA mutations, ranging from point mutations to large deletions, are often present in mixed proportions with wildtype mtDNA molecules within the same cell, a biological phenomenon termed heteroplasmy. The degree of mtDNA mutant heteroplasmy can vary significantly across different tissues of the same individual, and the percentage of a mutation is an important contributor to the clinical phenotype.4,5 Determination of the heteroplasmic status of any mtDNA mutation is clinically important to providing a family with informative genetic counseling regarding recurrence risk. Therefore, accurate measurement of heteroplasmy is an essential component of the molecular diagnostic scheme for mtDNA-related disorders.The diagnosis of an individual with suspected mtDNA-related disorders begins with a thorough clinical evaluation and assessment of family history.6 If a disorder with matrilineal inheritance can be established, common mtDNA point mutations and large deletions are typically analyzed using PCR-based targeted assays and Southern blotting methodology. If the screening for common point mutations and large deletions is negative, then the entire mitochondrial genome is usually analyzed by a conventional PCR-based Sanger sequencing approach with multiple pairs of primers. When a mutation is identified, analysis of the degree of heteroplasmy is carried out using various methods, including the commonly used allele refractory mutation system-based quantitative PCR (ARMS qPCR) for quantitative measurement.7 Large deletions are usually confirmed by PCR using primers encompassing the deletion break points followed by sequence analysis to map the break points, or, alternatively, by oligonucleotide array comparative genome hybridization.8 These sequential approaches are time consuming and costly. In an effort to improve efficiency and sensitivity of mtDNA mutation detection, we recently developed a comprehensive one-step long range/massively parallel sequencing (LR-PCR/MPS)-based approach that uses a single pair of PCR primers to amplify the entire mitochondrial genome. This method allows the simultaneous detection of point mutations with quantified heteroplasmy as well as large mtDNA deletions with accurately mapped break points. Purpose: The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA-related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome. Methods:The entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR-based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR-based Sanger sequencing using multiple pairs of primers. Results:Our results demo...

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Parallel sequencing used in detection of mosaic mutations: Comparison with four diagnostic DNA screening techniques

Cited by 151 publications

References 38 publications

Genetic models of osteochondroma onset and neoplastic progression: evidence for mechanisms alternative to EXT genes inactivation

Genetic models of osteochondroma onset and neoplastic progression: evidence for mechanisms alternative to EXT genes inactivation

Comprehensive One-Step Molecular Analyses of Mitochondrial Genome by Massively Parallel Sequencing

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

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