Single-nucleotide polymorphisms (SNPs) in human beta-defensin 1: high-throughput SNP assays and association with Candida carriage in type I diabetics and nondiabetic controls

Abstract

beta-Defensins are cationic antimicrobial peptides expressed in epithelia. They exhibit antibacterial, antifungal, and antiviral properties. Defensins are a component of the innate immune response, and it has been proposed that they have a protective role in the oral cavity. Previous studies have shown that human beta-defensin 1 (hBD-1) is constitutively expressed in oral epithelial cells but that expression varies between individuals. We tested the hypothesis that genetic variations in defensin peptide expression may be associated with opportunistic infections. This may be critical in the immunocompromised patient population, in which innate immune responses may have a relatively more important role. Oral Candida carriage status and the presence of six single-nucleotide polymorphisms (SNPs) in the DEFB1 gene encoding hBD-1 were evaluated in type I diabetic patients (n = 43) and nondiabetic controls (n = 50). Genomic DNA was obtained from buccal swabs. Portions of the DEFB1 gene were amplified, and each SNP was analyzed by a TaqMan assay, standardized with control DNA of known genotype. Candida carriage status was determined from unstimulated saliva on CHROMagar plating medium. A low level of Candida carriage was defined as < or = 350 CFU/ml. A high level of Candida carriage was seen in 44% of the diabetic subjects but only in 28% of the nondiabetic controls (P < 0.05). C. albicans predominated; however, diabetic subjects, especially those with high levels of carriage, showed an increased proportion of Candida glabrata and C. tropicalis. There was a strong association between an SNP in the 5' untranslated region (C-->G at position -44) and Candida carriage in both groups. Among individuals in the diabetic population who had the SNP allele 2 (G), 58% had low CFU, while 6% had high CFU. The C-->G SNP at position -44 is associated with low levels of Candida carriage. The resultant odd ratios are statistically significant for a protective effect (odd ratios, 25 for diabetic subjects and 8.5 for nondiabetic subjects). These results indicate that genetic variations in the DEFB1 gene encoding hBD-1 may have a major role in mediating and/or contributing to susceptibility to oral infection.

Figures

FIG. 1.

Candida species distribution in type I diabetic subjects and nondiabetic subjects with high levels (>350 CFU/ml) (A) and low levels (<350 CFU/ml) (B) of Candida carriage. (A) The mean numbers of CFU per milliliter are indicated. Note that the diabetic group had significantly larger numbers of CFU of C. tropicalis and C. glabrata per milliliter than the nondiabetic group (P < 0.01). The mean numbers of CFU of C. albicans per milliliter between the diabetic subjects and the nondiabetic subjects were not significantly different. (B) The mean numbers of CFU per milliliter are indicated on an expanded scale. The C. albicans distribution in individuals with low levels of carriage differed between diabetic subjects and nondiabetic subjects (P < 0.05).

FIG. 2.

Frequency of SNP at site −44 (C224G) in type I diabetic subjects and nondiabetic controls with high and low levels of Candida carriage. The groups with the low levels of Candida carriage had a high frequency of SNP 668 (−44), suggesting an association of this SNP with protection from Candida carriage (P < 0.01).