Rapid genetic analysis of x-linked chronic granulomatous disease by high-resolution melting.
High-resolution melting analysis was applied to X-linked chronic granulomatous disease, a rare disorder resulting from mutations in CYBB. Melting curves of the 13 PCR products bracketing CYBB exons were predicted by Poland's algorithm and compared with observed curves from 96 normal individuals. Primer plates were prepared robotically in batches and dried, greatly simplifying the 3- to 6-hour workflow that included DNA isolation, PCR, melting, and cycle sequencing of any positive products. Small point mutations or insertions/deletions were detected by mixing the hemizygous male DNA with normal male DNA to produce artificial heterozygotes, whereas detection of gross deletions was performed on unmixed samples. Eighteen validation samples and 22 clinical kindreds were analyzed for CYBB mutations. All blinded validation samples were correctly identified. The clinical probands were identified after screening for neutrophil oxidase activity. Nineteen different mutations were found, including seven near intron-exon boundaries predicting splicing defects, five substitutions within exons, three small deletions predicting premature termination, and four gross deletions of multiple exons. Ten novel mutations were found, including (c.) two missense (730T>A, 134T>G), one nonsense (90C>A), four splice site defects (45 + 1G>T, 674 + 4A>G, 1461 + 2delT, and 1462-2A>C), two small deletions (636delT, 1661_1662delCT), and one gross deletion of exons 6 to 8. High-resolution melting can provide timely diagnosis at low cost for effective clinical management of rare, genetic primary immunodeficiency disorders.
Department of Pathology, Pediatrics and Medicine, 5B114, University of Utah School of Medicine, 50 N. Medical Dr., Salt Lake City, UT 84132, USA. harry.hill@path.utah.edu
Harry R
HR
Hill
Nancy H
NH
Augustine
Robert J
RJ
Pryor
Gudrun H
GH
Reed
Joshua D
JD
Bagnato
Anne E
AE
Tebo
Jeffrey M
JM
Bender
Brian M
BM
Pasi
Javier
J
Chinen
I Celine
IC
Hanson
Martin
M
de Boer
Dirk
D
Roos
Carl T
CT
Wittwer
D005260:Female; D050172:Genes, X-Linked / Q000235:genetics*; D006105:Granulomatous Disease, Chronic / Q000235:genetics*; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D008562:Membrane Glycoproteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases