Analysis of Candidate Genes in Celiac Disease: A Tool to Identify Life-Threatening Associated Genes? 

DOMENICO NUZZO, FRANCESCO CATALDO, LETIZIA SCOLA, GIUSI IRMA FORTE, ANTONIO CRIVELLO, ANTONIO GIACALONE, SALVATORE ACCOMANDO, RAINER BARBIERI, GIUSEPPINA CANDORE, CALOGERO CARUSO 

The authors have recently reported that celiac patients show a proinflammatory cytokine genetic profile characterized by the contemporaneous presence of both the tumor necrosis factor-308A and the interferon-874T allele-positive genotypes. The same alleles are considered risk factors for aging-associated disease, whereas an anti-inflammatory cytokine genotype profile might be associated with an extended life expectancy. This paper reports data on the 1249-1250 InsACAA/Non-Ins transforming growth factor (TGF)-2, a multifunctional anti-inflammatory cytokine polymorphism distribution in 88 celiac disease (CD) patients, 99 age- and sex-matched controls, and 28 95-year-old healthy subjects living in western Sicily. These data demonstrate that genotype frequencies of CD patients are not different from those of age-matched and 95-year-old healthy control subjects. These data might suggest that TGF-2 polymorphism is not involved in the complex genotypes associated with successful or unsuccessful aging. In addition, one can speculate that the genotype profile associated with CD susceptibility might be detrimental to longevity, and studies of this CD genetic asset might point to a candidate gene for anti-aging strategies.

Because CD is a multisystem disorder, these patients are at risk of chronic ill health, permanent stunted growth, infertility, skeletal disorders, and malignancy. Moreover, the mortality rate at every age is twofold greater in untreated CD patients. As is well known, CD has a strong genetic component, with a sibling relative risk of about 10% to 15%. The major susceptibility locus is in the major histocompatibility complex (MHC), encompassing the human leukocyte antigen (HLA), and the HLA C (CD) is an inflammatory condition of the small intestine precipitated by the ingestion of wheat in individuals with a certain genetic makeup. CD can begin at any time in life. In susceptible individuals, the wheat protein gluten triggers an inflammatory reaction in the small bowel, which results in a decrease in the amount of surface area available for nutrient, fluid, and electrolyte absorption.

Immunosenescence Unit, Department of Pathobiology and Biomedical Methodology, University of Palermo, Clinic, Maternity and Child Department, University of Palermo, Palermo, Italy. and Development Biology Department, University of Palermo, Palermo, Italy.

This study investigated the role of TGF-2 gene polymorphism in CD susceptibility. Different SNP or insertion/deletion have been found in TGF-2 sequence. In particular, a four-nucleotide polymorphism between 1249 and 1250 nucleotides (1249-1250 InsACAA) from the transcription start site (GenBank: M87843), is able to influence cytokine production levels and functions. Herein are reported on the evaluation of the frequencies of the genotypes and alleles in CD patients and healthy subjects.

MATERIALS AND METHODS 

Subjects 

The 1249-1250 InsACAA/Non-Ins genotype distribution was typed on DNA samples from blood of three groups of subjects living in western Sicily: 88 CD patients (37 men and 51 women; mean age 25.8); 99 age- and sex-matched controls (39 men and 60 women; mean age 26.9) and 28 95-year-old healthy subjects (9 men and 19 women). Informed consent was obtained for all the subjects according to Italian law.

TGF-2 typing 

The sequences of PCR primer were: Non-Ins: TCG CAT CAA AAA CAA CAA CAA C; Ins: CAAA AGT TTC GCA TCA AAA AAC AAA CAA; Gen: TCA GGA TAG TCT TCT GGG GG. Polymerase chain reaction (PCR) was carried out in a total volume of 25 L, 5 pmol each primer, 200 M each dNTP, 20 mM Tris-MgCl and IU Taq polymerase. The mixes were incubated for 5 min at 95C, followed by 35 cycles of 40 sec at 95C, 40 sec at 64C, 40 sec at 42C, and an additional 5 min extension at 72C at the end of the 35 cycles. A 10-L aliquot of PCR products was mixed with 2 L of loading bufferGenotype frequencies were compared between control and 95-year-old healthy subject groups using 3x2 contingency tables and a chi-squared test. No significant differences were observed. The authors previously reported that celiac patients have a proinflammatory cytokine genetic profile characterized by the simultaneous presence of both the tumor necrosis factor-308A and the interferon-(cid:4) =_+6 874T allele-positive genotypes, which are associated with increased production of proinflammatory cytokines. A high proinflammatory profile is detrimental to life expectancy and can lead to age-associated diseases such as neurologic diseases, osteoporosis, and malignancy, which are major late celiac disease complications. An anti-inflammatory cytokine genotype profile may be associated with extended life expectancy. The lack of difference in genetic frequencies of ACAA Ins/Non-Ins alleles between CD patients and 95-year-old healthy control groups suggests that the TGF-2 polymorphism is not involved in the complex genotypes associated with successful or unsuccessful aging. Additionally, studies of CD genetic assets might suggest candidate genes for anti-aging strategies. Allele frequencies were evaluated by gene count, and the data were tested for the goodness of fit between the observed and expected genotype values (Hardy-Weinberg equilibrium). Chi-squared tests were performed to calculate significant different genotype distribution among the three groups of subjects using Graphpad Instat package by Graphpad Software Inc., San Diego, CA. Bonferroni's correction was applied because multiple comparisons were made by multiplying obtained p-values for the number of genotypes detected.