Analysis of single nucleotide polymorphisms in the promoter region of interleukin-10 by denaturing high-performance liquid chromatography.
Interleukin-10 (IL10), an anti-inflammatory cytokine, has been implicated in a variety of immune- and inflammatory-related diseases. We investigated the following SNPs: -1082, -819, -592 in the promoter region of IL10 in a normal (control) population and selected diseases: breast cancer (BrCa), systemic lupus erythematosus (SLE), and B-cell chronic lymphocytic leukemia (B-CLL) by denaturing high-performance liquid chromatography (DHPLC) and found distinct genotype and haplotype patterns. DHPLC was performed using the Transgenomic WAVE instrument, a mutational discovery tool that allows for high throughout analysis of SNPs. The principle of DHPLC is based on separation of homo- and heteroduplex formation of individual polymerase chain reaction products at specific melting temperatures and set gradients. The melting temperature selected for each SNP was based on size and sequence of the polymerase chain reaction product (for -1082, 57 degrees C; for -819, 58 degrees C; and for -592, 59.2 degrees C). Before fragment mutational analysis, all samples were denatured at 95 degrees C and slowly reannealed to allow for reassociation of different strands. Heteroduplex samples were easily distinguished from homoduplex samples. In order to identify wild type from homozygous mutant, two homoduplex polymerase chain reaction samples had to be mixed together, denatured at 95 degrees C and reannealed. The homozygous mutant, when combined with wild type, displayed a double peak on chromatogram. Once distinct chromatograms were established for each of the SNPs and the nucleotide changes confirmed by sequencing, genotype and haplotype frequencies were tabulated for the groups studied.
Guzowski
Dorothy
D
North Shore-LIJ Research Institute, 350 Community Drive, Manhasset, New York 11030, USA. dguzowsk@nshs.edu
Chandrasekaran
Alamelu
A
Gawel
Craig
C
Palma
Jacqueline
J
Koenig
Jonathan
J
Wang
Xue Ping
XP
Dosik
Michael
M
Kaplan
Mark
M
Chu
Charles C
CC
Chavan
Sangeeta
S
Furie
Richard
R
Albesiano
Emilia
E
Chiorazzi
Nicholas
N
Goodwin
Leslie
L
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics; D008180:Lupus Erythematosus, Systemic / Q000235:genetics; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic
D016753:Interleukin-10