Diagnostic approaches to apparent homozygosity

Landsverk, Megan; Douglas, Ganka; Tang, Sha; Zhang, Victor W.; Wang, Guoli; Wang, Jing; Wong, Lee‐Jun C.

doi:10.1038/gim.2012.58

Cited by 35 publications

(30 citation statements)

References 14 publications

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“…Primer based target enrichment techniques used in Sanger sequencing and NGS often produce false negatives due to polymorphisms located under primer binding sequences, which interrupts primer hybridization resulting in amplicon dropout [18]–[19]. If the polymorphism occurs on the same allele as the causative mutation, the mutation will go undetected.…”

Section: Resultsmentioning

confidence: 99%

The Validation and Clinical Implementation of BRCAplus: A Comprehensive High-Risk Breast Cancer Diagnostic Assay

Chong

Wang

et al. 2014

PLoS ONE

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Breast cancer is the most commonly diagnosed cancer in women, with 10% of disease attributed to hereditary factors. Although BRCA1 and BRCA2 account for a high percentage of hereditary cases, there are more than 25 susceptibility genes that differentially impact the risk for breast cancer. Traditionally, germline testing for breast cancer was performed by Sanger dideoxy terminator sequencing in a reflexive manner, beginning with BRCA1 and BRCA2. The introduction of next-generation sequencing (NGS) has enabled the simultaneous testing of all genes implicated in breast cancer resulting in diagnostic labs offering large, comprehensive gene panels. However, some physicians prefer to only test for those genes in which established surveillance and treatment protocol exists. The NGS based BRCAplus test utilizes a custom tiled PCR based target enrichment design and bioinformatics pipeline coupled with array comparative genomic hybridization (aCGH) to identify mutations in the six high-risk genes: BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11. Validation of the assay with 250 previously characterized samples resulted in 100% detection of 3,025 known variants and analytical specificity of 99.99%. Analysis of the clinical performance of the first 3,000 BRCAplus samples referred for testing revealed an average coverage greater than 9,000X per target base pair resulting in excellent specificity and the sensitivity to detect low level mosaicism and allele-drop out. The unique design of the assay enabled the detection of pathogenic mutations missed by previous testing. With the abundance of NGS diagnostic tests being released, it is essential that clinicians understand the advantages and limitations of different test designs.

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Section: Resultsmentioning

confidence: 99%

The Validation and Clinical Implementation of BRCAplus: A Comprehensive High-Risk Breast Cancer Diagnostic Assay

Chong

Wang

et al. 2014

PLoS ONE

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“…In patients with MPS VI, multiple examples of patients with two deleterious mutations in cis have been reported (Karageorgos et al, 2007). At one molecular testing center, parental genotyping of patients with 40 different autosomal recessive disorders revealed that of 75 apparently homozygous patients, four were incorrectly assessed as homozygotes due to allele dropout and two were genuine homozygotes but resulted from UPD (Landsverk et al, 2012). Additionally, parental genotyping can aid in differentiating alleles that are genuinely associated with disease from benign polymorphisms, as recently shown in MPS VI (Zanetti et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

“…In standard DNA sequencing approaches, separate PCR reactions amplify the GALNS exons and short stretches of the adjacent sequence, followed by sequencing of the individual PCR products. However, standard sequencing approaches can yield misleading results when one patient allele contains deletions or point mutations that eliminate PCR primer binding sites, resulting in apparent homozygosity due to allele dropout (Landsverk et al, 2012). Parental testing can confirm biallelic inheritance and indicate cases where deletion/duplication testing is necessary or uniparental isodisomy (UPD) may be a possibility (Catarzi et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Molecular testing of 163 patients with Morquio A (Mucopolysaccharidosis IVA) identifies 39 novel GALNS mutations

Morrone

Tylee²,

AlSayed

et al. 2014

Molecular Genetics and Metabolism

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Morquio A (Mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by partial or total deficiency of the enzyme galactosamine-6-sulfate sulfatase (GALNS; also known as N-acetylgalactosamine-6-sulfate sulfatase) encoded by the GALNS gene. Patients who inherit two mutated GALNS gene alleles produce protein with decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. GALNS mutations occur throughout the gene and many mutations are identified only in single patients or families, causing difficulties both in mutation detection and interpretation. In this study, molecular analysis of 163 patients with Morquio A identified 99 unique mutations in the GALNS gene believed to negatively impact GALNS protein function, of which 39 are previously unpublished, together with 26 single-nucleotide polymorphisms. Recommendations for the molecular testing of patients, clear reporting of sequence findings, and interpretation of sequencing data are provided.

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“…Despite extensive optimization and near ubiquitous usage, PCR is still prone to failure under certain circumstances. For diploid organisms, the failure of one allele to amplify can result in allelic dropout (ADO), causing apparent homozygosity (Askree et al 2011; Boán et al 2004; Lam and Mak 2013; Landsverk et al 2012; Piyamongkol et al 2003; Saunders et al 2010; Wenzel et al 2009). ADO is an insidious problem that is difficult to recognize because the PCR appears successful, but half of the genetic information is missing.…”

mentioning

confidence: 99%

Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

Stevens

Taylor

Pearce

et al. 2017

G3 Genes|Genomes|Genetics

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Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.

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Diagnostic approaches to apparent homozygosity

Cited by 35 publications

References 14 publications

The Validation and Clinical Implementation of BRCAplus: A Comprehensive High-Risk Breast Cancer Diagnostic Assay

The Validation and Clinical Implementation of BRCAplus: A Comprehensive High-Risk Breast Cancer Diagnostic Assay

Molecular testing of 163 patients with Morquio A (Mucopolysaccharidosis IVA) identifies 39 novel GALNS mutations

Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

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