A novel multiplex pyrosequencing assay for genotyping functionally relevant CTLA-4 polymorphisms: Potential applications in autoimmunity and cancer

Abstract CTLA-4 expression/function can be affected by single nucleotide polymorphisms ( SNPs ) of CTLA-4 gene, which have been widely associated with susceptibility or progression to autoimmune diseases and cancer development. In this study, we analyzed six CTLA-4 SNPs (−1661A > G, −1577G > A, −658C > T, −319C > T, +49A > G, CT60G > A) in 197 DNA samples from 43 B-lymphoblastoid cell lines (B-LCLs), 40 systemic sclerosis (SSc) patients, 14 pre-analyzed melanoma patients and 100 Italian healthy subjects. Genotyping of −1661A > G, −1577G > A, −658C > T and CT60G > A was performed by newly developed multiplex pyrosequencing (PSQ) assays, whereas −319C > T and +49A > G by T-ARMS PCR and direct sequencing. Genotype/allele frequency were analyzed using χ 2 or Fisher exact test. Our study provides the first multiplex PSQ method that allows simultaneous genotyping of two CTLA-4 SNP pairs (i.e. −1661A > G/−658C > T and −1577G > A/CT60G > A) by two multiplex PSQ reactions. Herein, we show the CTLA-4 genotype distribution in the B-LCLs providing the first and best characterized cell line panel typed for functionally relevant CTLA-4 SNPs . We also report the significant association of the −1661A/G genotype, −1661 & −319 AC-GT diplotype and −319 & CT60 TG haplotype with susceptibility to SSc without Hashimoto’s thyroiditis occurrence. Furthermore, we confirmed previous genotyping data referred to melanoma patients and provided new genotyping data for Italian healthy subjects.