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

Barbara Banelli, Anna Morabito, Stefania Laurent, Patrizia Piccioli, Beatrice Dozin, Massimo Ghio, Paolo Antonio Ascierto, Stefano Monteghirfo, Antonella Marasco, Vincenzo Ottaviano, Paola Queirolo, Massimo Romani, Maria Pia Pistillo

Research output: Contribution to journalArticlepeer-review

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 χ2 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.

Original languageEnglish
Pages (from-to)730-739
Number of pages10
JournalHuman Immunology
Volume75
Issue number8
DOIs
Publication statusPublished - 2014

Keywords

  • B-LCLs
  • CTLA-4 single nucleotide polymorphisms
  • Melanoma patients
  • Pyrosequencing
  • Sclerodermic patients

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy
  • Medicine(all)

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