Association of -318 C/T and +49 A/G cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms with a clinical subset of Italian patients with systemic sclerosis.

Systemic sclerosis (SSc) is a complex and heterogeneous autoimmune disorder with a multi-factorial pathogenesis. Like other autoimmune disorders, the possible role of specific cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms in predisposing to SSc has been hypothesized, but it remains controversial. CTLA-4 promoter (-318C/T) and exon 1 (+49 A/G) polymorphisms have been analysed in 43 Italian females with SSc and in 93 unrelated matched healthy controls by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. No significant association has been found with either polymorphisms.Nevertheless, SSc patients without concomitant Hashimoto's thyroiditis (HT) were carrying both the -318T allele (P = 0.031) and the +49 G allele (P = 0.076) more frequently than SSc patients with HT [defined by positivity for anti-thyroperoxidase (TPO) and anti-thyroglobulin (TGA) autoantibodies] than controls. Haplotype analysis confirms this association (P = 0.028), and suggests the predominant role of the -318T, whereas that of the +49 G, if any, seems weak. Thus, in Italian SSc patients the CTLA-4 -318C/T promoter polymorphism appears to be associated with the susceptibility to develop SSc without thyroid involvement. Larger studies are needed to confirm these findings and to clarify whether the -318C/T polymorphism is the functional responsible or whether it reflects the presence of another linked genetic element in the same chromosomal region.

Novel germline APC variants in patients with multiple adenomas.

Chain-terminating germline APC mutations are responsible for adenomatous polyposis coli (APC). In the present work, we tested the hypothesis that germline APC mutations may be present in some patients with a milder phenotype, i.e., multiple synchronous colorectal adenomas. Eighteen patients with 3 or more colorectal adenomas at endoscopy (within a 6-month period) were ascertained from a series of subjects undergoing endoscopic examination. Their blood DNAs were analysed for the presence of germline mutations in the APC coding region by single-strand polymorphism analysis. Ten unrelated polyp-free subjects and 101 unrelated APC patients were used as controls in the molecular analyses. Five of the eighteen patients carried novel germline APC variants or rare polymorphisms. These were various in site (from the splice acceptor site of intron 7 to the end of exon 15) and type (splice-site, missense, and chain-terminating mutations). Only one of ten polyp-free individuals carried a silent APC variant and none of these variants was found in the 101 APC controls. A first- or second-degree family history of colorectal cancer was reported by 4 of the 5 patients carrying a germline APC variant. In conclusion, novel APC germline variants were detected in patients with multiple synchronous adenomas. This suggests that the development of sporadic adenomas, in some instances, is associated with the presence of minor germline variants of the APC gene and that the spectrum of germline APC functional mutations may be larger than previously thought.

Chain-terminating mutations in the APC gene lead to alterations in APC RNA and protein concentration.

APC mutations introduce premature stop codons into the open reading frame of the gene, leading to the formation of truncated tumor suppressor proteins. Both RNA and protein levels are likely to be profoundly altered by such nonsense mutations. To test this hypothesis, Western blotting and RT-PCR strategies were used to characterize mutant and normal APC protein and APC RNA concentrations in lymphoblastoid cell lines from 22 unrelated polyposis patients carrying different APC mutations. Variable levels of truncated APC peptides were observed in 14 of 14 cell lines with APC mutations within exon 15. No truncated APC protein was detected in six of eight cell lines with APC mutations located 5' of exon 15. Mutations located in exon 15 showed mutant RNA underrepresentation in four of eight cell lines, whereas mutations located 5' of exon 15 showed RNA reduction in five of six cell lines. These findings indicate that a two- to threefold decrease in RNA concentration is common when APC alleles carry chain-terminating mutations. They also suggest that the severe decrease of truncated APC protein observed in some cell lines is due to mechanisms acting at the protein level.