Association between the IVS4G > T mutation in the TCF7L2 gene and susceptibility to diabetes in cystic fibrosis patients

Background Clinical complications appear to be a decisive factor for the prognosis of patients. Diabetes is an important complication of cystic fibrosis(CF). In our study we evaluated the association between the IVS4G>T mutation in the TCF7L2 gene with the presence of diabetes in patients with CF. Findings We evaluated 145 patients with CF in relation to the genotype of the IVS4G>T mutation. For this, the PCR method associated with specific enzyme digestion was used. The genotypes G/G, G/T and T/T were observed to have frequencies of 54 (37.2%), 78 (53.8%) and 13 (9%), respectively. There was no association between genotype and the occurrence of diabetes among patients. Conclusions In our sample, no association was found between the IVS4G>T mutation in the TCF7L2 gene and diabetes.


Introduction
Cystic Fibrosis (CF) is a monogenic autosomal recessive disease more common in the Caucasian population. It has the overall prevalence of 1:2500 births [1].
CF occurs due to mutations in the gene CFTR ("Cystic Fibrosis Transmembrane Conductance Regulator") located in the 7q3.1 region, which encodes the CFTR protein. Over 1897 mutations were identified in the CFTR gene [2].
The disease is characterized by a higher viscosity of secretions leading to chronic obstructive pulmonary disease, recurrent respiratory tract infections, pancreatic insufficiency, increased concentration of chloride in sweat and male infertility [1].
The clinical features of CF are characterized by clinical heterogeneity; some individuals have early death, while others survive to adulthood [3]. Clinical complications appear to be a decisive factor for the prognosis of patients. Diabetes is an important complication of CF. Diabetes risk increases with age, affecting about 25% of adolescents and 40-50% of adults with CF [4][5][6]. As CF's patients are now living longer, diabetes has become the most common systemic complication of CF after lung disease. Diabetes is associated with a significantly worse CF prognosis [6,7], although treatment of diabetes improves nutritional status and pulmonary function [8]. Among CF's patients, the prevalence of diabetes is approximately tenfold greater (at about onethird the age) than is seen for type 2 diabetes in the general population [6,9].
Grant et al. [10] reported on the association of a common microsatellite (DG10S478) within intron 3 of the transcription factor 7-like 2 gene (TCF7L2) with type 2 diabetes in an Icelandic case-control sample and replicated this result in two additional case-control cohorts of white patients. The noncoding single-nucleotide polymorphisms rs12255372 and rs7903146 were in strong linkage disequilibrium with DG10S478 (r2 = 0.95 and r2 = 0.78, respectively) and showed similarly robust associations with type 2 diabetes. The authors recommended that these two single-nucleotide polymorphisms be genotyped in all attempts at replication [11].
In our study we evaluated the association of the rs12255372 polymorphism (IVS4G > T mutation) in TCF7L2 gene with the presence of diabetes in patients with CF.

Results
This study was approved by the Ethics Committee of the Faculty of Medical Sciences of University of Campinas -UNICAMP (#528/2008).
The study included 145 patients with CF who are monitored by the Pediatric Pulmonology Outpatient Clinic at UNICAMP.
Association between genotype of the IVS4G > T mutation and the development of diabetes and the presence of meconium ileus (Tables 1, 2) was not found. When the variable presence of the F508del mutation was introduced, an association was still not found in our sample (Tables 3,4). [11], examined whether the two most strongly associated variants (rs12255372 and rs7903146) predict the progression to diabetes in 3.548 persons with impaired glucose tolerance who were enrolled in the Diabetes Prevention Program, in which lifestyle intervention or treatment with metformin was compared with placebo. The data found showed that the risk alleles in rs7903146 and rs12255372 predict the risk of diabetes prospectively, beyond that conferred by the clinical risk factors.

Florez et al. (2006)
In 2009, Blackman et al., [6] had already tested whether a family history of type 2 diabetes affected diabetes risk in CF patients in 539 families in the CF Twin and Sibling family-based study. 998 patients were evaluated from the family-based study and 802 unrelated CF patients in an independent case-control study. Family history of type 2 diabetes was shown to increase the risk of diabetes in CF (OR 3.1; p = 0.0009). A variant in TCF7L2 associated with type 2 diabetes (the T allele at rs7903146) was associated with diabetes in CF in the family study (p = 0.004) and in the case-control study (p = 0.02; combined p = 0.0002). In the family-based   Given these results, it became interesting to evaluate the other polymorphism, rs12255372 that is also related to a risk of diabetes among CF's patients. Our analysis, however, found no such correlation.
The absence of a relationship may be due to the sample size, but since this correlation is of great importance, it should have appeared even in a small sample. In any case, with an analysis of a larger number of patients it is possible that a relationship between the studied polymorphism and CF risk can still be evidenced.

Patients and methods
Patients were included in the study from the Pediatric Clinic at the Faculty of Medical Sciences of UNICAMP. The sample size calculation was performed by G-POWER program version 3.1 (using 0.05 alpha, 0.8 beta and W size effect of 0.3). To have the statically power to all analyses using chi square test we need a population size of 143 patients. All patients were confirmed as having CF through two positive sodium and chloride sweat tests (value greater than 60 mmol/L) and by analysis of differential membrane epithelium of the intestine by the dosage of active CFTR through the Ussing chamber. The identification of mutations in the CFTR gene was performed in the laboratory of Table 4 Association IVS4G > T mutation in TCF7L2 gene after genotypic groupings, distributed by genotype for the mutation in the CFTR gene delF508 with clinical variables in cystic fibrosis patients followed at the Pediatric Clinic at UNICAMP with the distribution by mutations in the CFTR gene Molecular Genetics, FCM/UNICAMP, which has the routine analysis tests for major mutations found in the population of Brazil, which are: F508del, G542X, R1162X, N1303K, G551D and N1303K. Of the patients initially included in the survey, only those without clinical data for statistical analysis and/or those who did not sign the informed consent were excluded. The DNA was obtained by the extraction technique of phenol chloroform from 8 mL of venous blood. The concentration of DNA used for analysis was 50 ng/mL. Genotyping was performed using the PCR technique associated with specific enzyme digestion.
The product from the PCR had a size of 337 bp and after digestion by the restriction enzyme Tsp509I, fragments of 143 bp + 99 + 95 -G/G genotype, fragments of 126 + 17 + 99 + 95 bp -T/T genotype and fragments of 143 + 126 + 99 + 95 + 17 bp -genotype G/T were found.
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) v.17.0 and Open Epi v.5.0 program. Data was compared by different tests according to data distribution. Initially, data was compared the chi square and Fisher exact test. For all the analyses we adopted the value of p = 0.05.