Allele dropout in PCR-based diagnosis of Wilson disease: mechanisms and solutions

Clinical Chemistry
Ching-Wan Lam, Chloe M Mak

Abstract

We investigated the mechanisms leading to allele dropout-the nonamplification of 1 of the alleles-in PCR-based diagnosis of Wilson disease (WD). We extracted genomic DNA from blood samples from 6 WD patients (P1-P6) with allele dropouts detected in a previous study of WD in a Hong Kong Chinese population. We amplified the ATP7B gene by PCR and performed direct DNA sequencing of all exons of the ATP7B gene. To support the proposed mechanism of allele dropout, we used proofreading DNA polymerase, primer design avoiding single-nucleotide polymorphism sites, and duplex PCR. Patients P1-P4 were all apparently homozygous for a known disease-causing mutation, c.2975C > T (p.P992L) in exon 13. Patient P5 was apparently homozygous for a novel mutation, c.2524G > A, and patient P6 was apparently homozygous for another known mutation, c.522_523insA (p.K175K-fs). In all cases, we determined that the patients were actually heterozygous for these mutations. Our results confirm that allele dropout is the mechanism causing apparent homozygosity of heterozygous mutations in these WD patients.

Citations

May 19, 2012·Genetics in Medicine : Official Journal of the American College of Medical Genetics·Megan L LandsverkLee-Jun C Wong
Jun 24, 2008·Critical Reviews in Clinical Laboratory Sciences·Chloe M Mak, Ching-Wan Lam
May 15, 2010·Clinica Chimica Acta; International Journal of Clinical Chemistry·Chin-Wen LinJan-Gowth Chang
Jun 9, 2006·Biomolecular Engineering·Fakhraddin Naghibalhossaini, Afsaneh Moaddeb
Jul 7, 2015·The Journal of Molecular Diagnostics : JMD·Jonatan BlaisFrançois Rousseau
Mar 26, 2013·Clinica Chimica Acta; International Journal of Clinical Chemistry·Ching-wan Lam, Chloe Miu Mak
Apr 11, 2014·The Canadian Journal of Neurological Sciences. Le Journal Canadien Des Sciences Neurologiques·Nan ChengYongzhu Han
May 12, 2016·Hereditary Cancer in Clinical Practice·Steffen PistoriusHans K Schackert
Apr 7, 2007·Current Opinion in Gastroenterology·Scott Fink, Michael L Schilsky

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