Vulvovaginal candidiasis (VVC) is a frequent disease affecting approximately more than 75% of all childbearing women at least once in their lifetime by overgrowth of opportunistic Candida species. This condition is characterized by itching, soreness, bright red rash, and heavy white vaginal discharge [1-4]. It is usually caused by Candida albicans, but other species, i.e., Candida glabrata, Candida tropicalis, and Candida krusei, may also be involved [5, 6]. However, recurrent vulvovaginal candidiasis (RVVC) characterized by at least four times infections within a year has had a modest increase in the frequency of attacks due to non-albicans species reaching approximately 10% to 15% [2-4]. It is more common in otherwise healthy individuals. Several risk factors, i.e., prolonged use of antibiotics, pregnancy, intensive immunosuppressive therapy, diabetes mellitus ketoacidosis, corticosteroid abusers, and stress were reported to contribute to RVVC susceptibility.
However, the majority of patients with RVVC were healthy individuals, suggesting that a genetic component most likely plays an important role in their susceptibility [2, 7]. Several studies proposed that patients with genetic polymorphism and familial history have increased susceptibility to Candida colonization, which leads to RVVC . Mannose, β-glucans, and chitin are the main carbohydrates at the surface of cell walls of Candida species, which bind to the pattern recognition receptors (PRRs) that are expressed by the innate immune cells as myeloid and epithelial cells.
The first step in the initiation of an immune response is the recognition of conserved structures of the pathogen (PAMPs) by PRRs [2, 3, 7, 8]. PRRs have several families, e.g., Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RIG-І helicases . TLRs and C-type lectin receptors are the main recognition receptors for C. albicans .
Among the PRRs, C-type lectin receptor Dectin-1 recognizes β-glucans on the cell wall. Ferwerda et al. reported an important link between Dectin-1 Y238X polymorphism and increased predisposition to fungal infections . A polymorphism in the gene for human Dectin-1, Y238X, which results in an early stop codon and leads to abrogated Dectin-1 expression, has been identified in patients with RVVC [7, 10]. In 2009, Plantinga et al. showed that a polym-orphism in Dectin-1 (Y238X, rs 16910526) was responsible for recurrent mucocutaneous fungal infections in a Dutch family. This polymorphism resulted in an early stop codon, which led to the loss of the last 10 amino acids of the extracellular domain and a diminished capacity to bind to β- glucans .
Studies on Dectin-1 deficient mice demonstrated Dectin-1’s protective role during infection with C. albicans, Aspergillus fumigatus, and Pneumocystis jirovecii . Therefore, the aim of the current study was to investigate the impact of single nucleotide polymorphisms (SNPs) in the genes coding Dectin-1 (Y238X) on susceptibility to RVVC in the Iranian population.
Materials and Methods
Patients and control samplings
One-hundred vaginal swab samples were obtained from patients with history of vaginal discharge. VVC and RVVC were diagnosed based on their clinical manifestation and mycological investigations as previously described . Twenty-five patients who had frequent VVC relapses and were diagnosed as RVVC were involved in this experiment. The control group consisted of healthy individuals (n=25) without any history of RVVC, vaginal discharge, or itching on the day of examination. Blood samples were obtained from the participants and transferred into sterile tubes with sodium-EDTA (0.5 mmol/l) to investigate Dectin-1 Y238X gene polymorphism. We collected the subjects’ personal information such as age, pregnancy, delivery type, contraception methods (coitus interruptus, condom, intra-uterine device [IUD], and combined oral contraceptives), broad antibiotic use, chronic disorders, history of familial diabetes mellitus, and RVVC. This study was approved by the Ethics Committee (nr. 566/92) of Mazandaran University of Medical Sciences, Sari, Iran, and written informed consent was obtained from the patient’s next of kin for publication of this report.
Genotyping of the SNPs
Genomic DNA was extracted from peripheral venous blood samples using the YTzol pure genomic DNA kit (YTA, Y9204, IR) according to the manufacturer’s instructions. DNA extracts were stored at -80°C prior to use. Genotyping was adjusted using bidirectional polymerase chain reaction (PCR) amplification of specific alleles (Bi-PASA), as previously described by Carvalho et al. [13, 14]. The PCR primers used were as follows: P1 (5’GTAGTCCCAGCTACTTGAGG3’), P2 (5’ACCACTTGAGATTCACAACA3’), P3 (5’ggcggcggggAGTGTGCCCTCATAT3’), and P4 (5’ gggccgggggTTCTTCTCACAAATACTC3’).
PCR reactions were performed on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) in 50 ml volumes containing 25 ng of template DNA, 5 ml of reaction buffer (0.1 M Trise HCl, pH 8.0, 0.5 M KCl, 15 mM MgCl2, 0.1% gelatin, 1% Triton X-100), 0.2 mM of each dNTP, and 2.0 U Taq DNA polymerase (ITK Diagnostics, Leiden, the Netherlands). Amplification was performed with cycles of 5 min at 94°C for primary denaturation, followed by 35 cycles at 94°C (30 s), 58°C (30 s), and 72°C (45 s), with a final 5 min extension step at 72°C. PCR products were run on 3% agarose gel to confirm the amplification of the genetic locus. Gel was run at 110 V for 15 min and visualized on a UV transilluminator, and the results were compared between the two groups (patient and control) .
Dectin-1 Y238X polymorphism region was amplified using forward (5-CCAAGAAAACCCATCTCCAA-3) and reverse (5-CTCCTTCTCCACCCTTCCTC-3) primers and subsequent sequencing was conducted as previously described . Briefly, amplification was performed with cycles of 1 min at 96°C for primary denaturation, followed by 25 cycles at 96°C (15 s), 50°C (15 s), and 60°C (60 s), with a final 5 min extension step at 72°C. Amplification products were first run on 2.5% agarose gel and visualized with UV after ethidiumbromide staining and then subsequently were purified using GFX PCR DNA (GE Healthcare, Buckinghamshire, UK).
Sequencing was performed as follows: 95°C for 1 min, followed by 35 cycles consisting of 95°C for 10 s, 50°C for 5 s, and 60°C on an ABI 3730xl automatic sequencer (Applied Biosystems, Foster City, CA). The obtained sequence data were adjusted using Lasergene SeqMan software (DNAStar, Madison, WI, USA) and the results were evaluated. Dectin-1 genotypes were grouped as wild-type (adenine, adenine), homozygous (cytosine, cytosine), and heterozygous (adenine, cytosine) as previously described .
Statistical analysis was performed using SPSS, version 17. Categorical parameters were evaluated using Pearson’s correlation coefficient, Chi-square, and Fisher’s exact test. P-value less than 0.05 was considered statistically significant.
By use of two outer primers (P & Q) and two inner allele-specific primers (M &W), two or three overlapping fragments were amplified based on genotyping. PQ is always produced as a positive control. PW and MQ are present in a heterozygote individual, but PW is only produced in wild-type homozygote and MQ only in homozygous mutant samples (Table 1).
|PQ (675 bp)||ــــــــ||ــــــــ||ــــــــ|
|PW (423 bp)||ــــــــ||ــــــــ|
|MQ (283 pb)||ــــــــ||ــــــــ|
The age range of the experimental and control groups were 20-60 years and 19-58 years, respectively. There was no significant difference in the mean age between these groups (P=1). In both groups, the number of cesarean sections was higher than vaginal deliveries. Remarkably, no underling abnormalities were reported in neither groups. Most of the patients used contraceptives, e.g., coitus interrupts, condom, IUD, and combined oral contraceptives. Pruritus vulvae, pain, itching, and vaginal discharge were observed in the experimental group. Table 2 Summarizes the results of bidirectional PCR amplification of specific alleles (Bi-PASA) among the tested individuals.
Results revealed that 100% of the cases were wild-type homozygous for Y238X polymorphism, and none of the individuals revealed a heterozygous Dectin-1 Y238X polymorphism (Figure 1). Randomly, five experimental groups were sequenced and the results were in concordance. These analyses showed that polymorphism in Dectin-1 Tyr238X, rs16910526 was observed in RVVC patients. When Dectin-1 genotypes were compared, there was no significant difference between the groups (P=1; Table 2).
VVC is one of the most common forms of Candida infection that affects up to 75% of women at least once in their lifetime [4, 15]. It is mostly caused by C. albicans, but other species includingC. glabrata, C. tropicalis, and C. krusei may also be involved. Despite the known risk factors (i.e., host status, diabetes, pregnancy, and immunosuppressive therapy), the majority of the patients are immune-competent individuals.
In the present investigation, we evaluated whether the genetic variation in genes coding (Dectin-1) PRRs involved in Candida recognition affects susceptibility to RVVC. This was the first attempt to study this issue in the Iranian population. Our results showed that no obvious polymorphism in Dectin-1 is associated with increased susceptibility to RVVC. Although Dectin-1 plays a significant role in Candida defense by TLR2 and
|Polymorphism||Group||WT||HET||HOM||In HWE (yes/ no)||P|
|DECTIN-1 Tyr238Stop||Controls (N = 25)VVC/RVVC (N =25)||25(100%)25 (100%)||0 (0%)0 (0%)||0 (0%)0 (0%)||YesYes||11|
|Abbreviations: WT (wild type); HET (Heterozygous); HOM (Homozygous); *P<0.05 were considered statistically significant|
TLR4, which recognize mannan and mannose-binding receptors on the Candida cell wall, functional consequences of Dectin-1 deficiency were demonstrated to include impaired induction of both innate and adaptive Th17 immune responses . Based on several reports, Dectin-1 polymorphism affects the immunological response and is a predisposing factor for Candida infections in rats. In addition, Dectin-1 plays a crucial role in production of pro-inflammatory cytokines with Toll-like receptors in animal experiments [17, 18].
Interestingly, the previous investigation showed that mutation in genes coding Dectin-1 in murine model causes an increased susceptibility to some opportunistic fungi, while they were not susceptible to candidiasis . However, other researchers believed that individuals with this mutation are susceptible to chronic mucocutaneous candidiasis . In another study, Plantinga et al. showed a link between candidemia and Dectin-1 Y238X polymorphism in cancer patients . However, limited numbers of studies on the relationship between PRR defects and RVVC have demonstrated that genetic diversity in TLRs and CLRs can influence predisposition to RVVC [9, 20]. Moreover, Diana et al. in a study performed in 2014 showed that SNPs in PRRs (TLR1, TLR4, Dectin-1, and CARD9) did not affect susceptibility to RVVC; however, TLR2 increased approximately threetimes . It seems that variation in TLR2 is caused by decreased induction of mucosal antifungal host defense. In the current study, the correlation between RVVC and mutation in Dectin-1 was investigated and heterozygous and homozygous Dectin-1 polymorphisms were not detected, which implies low incidence of Dectin-1 Y238X polymorphisms in the Iranian population. Nonetheless, it was not possible to correlate the presence of Dectin-1 Y238X polymorphism with RVVC etiology and history of familial RVVC.
Other genetic mechanisms might play a role in the mucosal defense. In this study, only Dectin-1 Y238X polymorphisms were assessed, whereas the presence of other mutations and epigenetic factors might be responsible. Therefore, further clinical investigations with wide population screenings are required to elucidate the relationship between RVVC and Dectin-1 Y238X polymorphism.
- Rathod SD, Klausner JD, Krupp K, Reingold AL, Madhivanan P. Epidemiologic features of Vulvovaginal Candidiasis among reproductive-age women in India. Infect Dis Obstet Gynecol. 2012; 2012:859071.
- Jaeger M, Plantinga TS, Joosten LA, Kullberg BJ, Netea MG. Genetic basis for recurrent vulvo-vaginal candidiasis. Curr Infect Dis Rep. 2013; 15(2):136-42.
- Rosentul DC, Delsing CE, Jaeger M, Plantinga TS, Oosting M, Costantini I. Gene polymorphisms in pattern recognition receptors and susceptibility to idiopathic recurrent vulvovaginal candidiasis. Front Microbiol. 2014; 5:483.
- Corrigan EM, Clancy RL, Dunkley ML, Eyers FM, Beagley KW. Cellular immunity in recurrent vulvovaginal candidiasis. Clin Exp Immunol. 1998; 111(3):574-8.
- Mahmoudi Rad M, Zafarghandi AS, Amel Zabihi M, Tavallaee M, Mirdamadi Y. Identification of Candida species associated with vulvovaginal candidiasis by multiplex PCR. Infect Dis Obstet Gynecol. 2012; 2012:872169.
- Plantinga TS, Johnson MD, Scott WK, Joosten LA, van der Meer JW, Perfect JR. Human genetic susceptibility to Candida infections. Med Mycol. 2012; 50(8):785-94.
- Usluogullari B, Gumus I, Gunduz E, Kaygusuz I, Simavli S, Acar M. The role of Human Dectin-1 Y238X Gene Polymorphism in recurrent vulvovaginal candidiasis infections. Mol Biol Rep. 2014; 41(10):6763-8.
- Rosentul DC, Plantinga TS, Oosting M, Scott WK, Velez Edwards DR, Smith PB. Genetic variation in the dectin-1/CARD9 recognition pathway and susceptibility to candidemia. J Infect Dis. 2011; 204(7):1138-45.
- Ferwerda B, Ferwerda G, Plantinga TS, Willment JA, van Spriel AB, Venselaar H. Human dectin-1 deficiency and mucocutaneous fungal infections. N Engl J Med. 2009; 361(18):1760-7.
- Drummond RA, Saijo S, Iwakura Y, Brown GD. The role of Syk/CARD9 coupled C-type lectins in antifungal immunity. Eur J Immunol. 2011; 41(2):276-81.
- Plantinga TS, van der Velden WJ, Ferwerda B, van Spriel AB, Adema G, Feuth T. Early stop polymorphism in human DECTIN-1 is associated with increased candida colonization in hematopoietic stem cell transplant recipients. Clin Infect Dis. 2009; 49(5):724-32.
- Yazdanparast SA, Khodavaisy S, Fakhim H, Shokohi T, Haghani I, Nabili M. Molecular characterization of highly susceptible candida africana from Vulvovaginal Candidiasis. Mycopathologia. 2015; 180(5-6):317-23.
- Cunha C, Di Ianni M, Bozza S, Giovannini G, Zagarella S, Zelante T. Dectin-1 Y238X polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient- and donor-dependent mechanisms of antifungal immunity. Blood. 2010; 116(24):5394-402.
- Carvalho A, Marques A, Maciel P, Rodrigues F. Study of disease-relevant polymorphisms in the TLR4 and TLR9 genes: a novel method applied to the analysis of the Portuguese population. Mol Cell Probes. 2007; 21(4):316-20.
- Antachopoulos C, Walsh TJ, Roilides E. Fungal infections in primary immunodeficiencies. Eur J Pediatr. 2007; 166(11):1099-117.
- Taylor PR, Tsoni SV, Willment JA, Dennehy KM, Rosas M, Findon H. Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat Immunol. 2007; 8(1):31-8.
- Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM. Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J Exp Med. 2003; 197(9):1107-17.
- Ferwerda G, Meyer-Wentrup F, Kullberg BJ, Netea MG, Adema GJ. Dectin-1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages. Cell Microbiol. 2008; 10(10):2058-66.
- Saijo S, Fujikado N, Furuta T, Chung SH, Kotaki H, Seki K. Dectin-1 is required for host defense against Pneumocystis carinii but not against Candida albicans. Nat Immunol. 2007; 8(1):39-46.
- De Luca A, Carvalho A, Cunha C, Iannitti RG, Pitzurra L, Giovannini G. IL-22 and IDO1 affect immunity and tolerance to murine and human vaginal candidiasis. PLoS Pathog. 2013; 9(7):e1003486.