Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 49: 1050-1057, 2003; 10.1373/49.7.1050
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Killeen, A. A.
Right arrow Articles by Hixson, C. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Killeen, A. A.
Right arrow Articles by Hixson, C. S.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2003;49:1050-1057.)
© 2003 American Association for Clinical Chemistry, Inc.

Linked Linear Amplification for Simultaneous Analysis of the Two Most Common Hemochromatosis Mutations

Anthony A. Killeen1,a, John W. Breneman, III2, Arlene R. Carillo2, Jason Liu2 and Craig S. Hixson2,4

1 Department of Pathology, University of Michigan, Ann Arbor, MI 48109.

2 Clinical Diagnostics Group, Nucleic Acid Technology, Bio-Rad Laboratories, Hercules, CA 94547.

aAddress correspondence to this author at: Department of Laboratory Medicine & Pathology, University of Minnesota, Mayo Mail Code 609, 420 Delaware St. SE, Minneapolis, MN 55455. E-mail kille001{at}umn.edu.

Background: Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations.

Methods: Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin–horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared.

Results: The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63.

Conclusions: LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.




eLetters:

Read all eLetters

Authorship correction
John W. Breneman
Clinical Chemistry Online, 13 Aug 2003 [Full text]

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2003 by the American Association for Clinical Chemistry.