title: 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.
authors.affiliation: UOS Epigenetica dei Tumori, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Banelli
authors.affiliation: UOS Epigenetica dei Tumori, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Morabito
authors.affiliation: UOS Epigenetica dei Tumori, IRCCS A.O.U. San Martino-IST, Genova, Italy; Dipartimento di Medicina Interna e Specialità Mediche (DIMI), Università degli Studi di Genova, Genova, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Laurent
authors.affiliation: UOC Biologia cellulare, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Piccioli
authors.affiliation: UOC Epidemiologia Clinica, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Dozin
authors.affiliation: Dipartimento di Medicina Interna e Specialità Mediche (DIMI), Università degli Studi di Genova, Genova, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Ghio
authors.affiliation: Unità di Oncologia Medica e Terapie Innovative, Istituto Nazionale Tumori Fondazione G. Pascale, Napoli, Italy.
authors.forename: Paolo Antonio
authors.initials: PA
authors.lastname: Ascierto
authors.affiliation: UOC Oncologia Medica 2, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Monteghirfo
authors.affiliation: UOC Oncologia Medica 2, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Marasco
authors.affiliation: UOC Oncologia Medica 2, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Ottaviano
authors.affiliation: UOC Oncologia Medica 2, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Queirolo
authors.affiliation: UOS Epigenetica dei Tumori, IRCCS A.O.U. San Martino-IST, Genova, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Romani
authors.affiliation: UOS Epigenetica dei Tumori, IRCCS A.O.U. San Martino-IST, Genova, Italy. Electronic address: mariapia.pistillo@hsanmartino.it.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Pistillo
mesh_terms: D000483:Alleles; D015551:Autoimmunity / Q000235:genetics*; D060908:CTLA-4 Antigen / Q000235:genetics* / Q000276:immunology; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D004198:Disease Susceptibility; D015870:Gene Expression; D005787:Gene Frequency; D060005:Genotyping Techniques; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008545:Melanoma / Q000175:diagnosis / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D020641:Polymorphism, Single Nucleotide; D012595:Scleroderma, Systemic / Q000175:diagnosis / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D012878:Skin Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000276:immunology / Q000473:pathology
chemical_list: D060908:CTLA-4 Antigen
keywords: B-LCLs; CTLA-4 single nucleotide polymorphisms; Melanoma patients; Pyrosequencing; Sclerodermic patients