Genotyping the GGGCGG tandem repeat promoter polymorphism in the 5-lipoxygenase enzyme gene (ALOX5) by pyrosequencing assay.
OBJECTIVE
Efficient genotyping methods for many biologically significant repeat genetic polymorphisms, particularly in GC-rich regions of the genome, are limited. In particular, a short tandem repeat polymorphism [GGCGGG] in the promoter region of ALOX5 has been implicated as an important marker for inflammatory diseases. We developed a pyrosequencing assay to genotype the ALOX5 short tandem repeat polymorphism using pyrosequencing technology that will make assessing this important genetic marker in large, diverse populations more accessible than using current methods.


METHODS
We used a nested polymerase chain reaction approach to amplify DNA for pyrosequencing. Population allele frequencies were assessed in two cohorts of previously collected human DNA samples with 188 and 1032 samples, respectively. Sixteen genetic samples with known genotypes were used to confirm the accuracy of the method.


CONCLUSIONS
Genotypes were 100% concordant with samples of known genotype. Genotype frequencies in European American, Hispanic, and African American agreed with previously published results (wild-type homozygotes 66%, 64%, and 19%, respectively). The method presented here will facilitate both genetic association and pharmacogenomic research on this polymorphism in large samples that are ethnically and/or racially admixed.
Schentrup
Anzeela M
AM
Center for Pharmacogenomics, College of Pharmacy, University of Florida, Gainesville, Florida 32610, USA.
Allayee
Hooman
H
Lima
John J
JJ
Johnson
Julie A
JA
Langaee
Taimour Y
TY
D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics*; D005838:Genotype; D006801:Humans; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences
D001094:Arachidonate 5-Lipoxygenase