Genetic association of MBL-2 gene polymorphisms with Filarial chyluria

Lymphatic filariasis has become a significant public health issue in North India. The association of polymorphisms in MBL2 gene with filarial chyluria (FC) is evaluated in the North Indian patients for the first time. Hence, a tertiary care hospital based case-control study was conducted in north India where FC is endemic. Therefore, 186 confirmed patients of FC as cases and 210 age-, sex- and residence-matched subjects as controls were enrolled for the study. Filarial etiology was confirmed using diethylcarbamazine (DEC)-provocation test, immune chromatographic test and IgG/IgM antibody test. MBL2 gene polymorphisms at codon 54 and -221 promoter region were genotyped by PCR followed by RFLP. Wild-type, heterozygous and homozygous mutant frequencies of MBL2 genotype at the codon 54 were 57.5%, 32.8% and 9.7% in the case group and 62.9%, 30.5% and 6.7%, in controls, respectively. The same at the -221 position were 51.1%, 44.1% and 4.8% in FC patients and 44.3%, 40.0% and 15.7% in controls, respectively. Thus, results no significant association between MBL2 polymorphism at codon 54 and FC. However, polymorphism at the -221 promoter region is linked with FC with a significant odd-ratio of 0.27 (confidence interval at 95% was 0.12-0.59; p<0.001). This preliminary finding is intriguing for further confirmation using a larger study with more patients.


Background:
Chyluria is an uncommon and delayed manifestation of lymphatic filariasis (LF) and is characterized by the presence of chyle in urine resulting from fistulous communications between the lymphatics and the urinary tract [1]. It is estimated that around 1,100 million people across the world are living in endemic regions for LF and exposed to risk of developing infection. Of the total global burden, India contributes about 40% and approximately 50% of the residents at risk of filarial infection [2]. The World Health Organization declared LF as a "neglected" tropical disease initiating "The Global Program for Elimination of Lymphatic Filariasis" and set the target for the year 2020 for elimination [3, 4]. Up to 10% of patients with filariasis develop chyluria. It is predicted that approximately 90% cases globally and more than 99% filarial cases in our country are due to the infection of the parasite Wuchereria bancrofti [5]. The factors that predispose a person to develop LF have not been investigated. It is still unknown as to why one member of a family develops LF while another is spared. As the chances of mosquito bite remains the same in endemic areas, the answer to predilection for LF probably lies in genetic studies. Unfortunately, genetic studies on LF are rare. 807 ©Biomedical Informatics (2019) Mannose-binding lectin (MBL) protein is first-line of defense which is encoded by MBL2 gene that has a key role in activation of complement system and opsonization of pathogenic virus, bacteria and parasites. MBL2 gene is located on chromosome 10q21.1. There are six single-nucleotide polymorphisms (SNPs) in MBL2 gene out of which three SNPs are located in structural region of exon 1 [codon 54 (Gly-Asp), codon 57 (Gly-Glu) and codon 52 (Arg-Cys)], while the rest of the SNPs are in the promoter region [-550 (G>C), -221 (G>C) and +4(C>T

Estimation of sample size:
The sample size was estimated on the basis of reported prevalence of genetic polymorphism of MBL2 -221 XX type in exposed control group (general population) as 16%, OR = 2.0, Zα =1.96 for significant level 0.05 and Zβ =0.84 for 80% power. Based on the above assumptions, 186 patients and 210 healthy controls were enrolled.

Genotyping of MBL2 gene:
Genomic DNA was obtained from whole blood by using commercially available DNA extraction kit (Quick-g DNA TM , USA) as per manufacturer's protocol. 100 ng of DNA was used as template for subsequent PCR reactions. Genotyping of MBL2 gene at site codon54: rs1800450 and -221:rs7096206 was done by polymerase chain reaction followed by restriction fragment length polymorphism with specific primer pairs and restriction enzymes. For codon 54: primers used for amplification were; forward 5'GTAGGACAGAGGGCATGCTC3' reverse 5'CAGGCAGTTTCCTCTGGAAGG3' with a product size of 329 base pair. PCR product was successively amplified at 94ºC for 5minutes, 94ºC for 40-seconds, 58ºC for 40-seconds, 72ºC for 40seconds and final extension at 72ºC for 7 minutes. For promoter site -221: primers used for amplification were; with a product size of 608 base pair [13,14]. PCR product was successively amplified at 95ºC for 5-minute, 95ºC for 30-sec, 58ºC for 20-sec, 72ºC for 45-sec and final extension at 72ºC for 7 minutes. PCR products were further digested by restriction enzyme Ban1and 808 ©Biomedical Informatics (2019) Btg1 (NEB Inc., USA), respectively. The digestion reaction system was kept at 37°C overnight. Finally, the digested PCR products were separated by 2% agarose gel electrophoresis. Gel imaging processing system was used to observe the electrophoresis results of the digested PCR products to determine the genotype.

Statistical analysis
Genotype data for control group were analyzed for fitness in the Hardy-Weinberg equilibrium using the online calculator available at http://ihg.

Results:
A total of 396 demographically-matched subjects were enrolled, of which 186 were FC cases and 210 were controls. The mean age was 37.70±0.81 years (range 18-60 years) for FC patients and 35.98±0.77 years (range 18-60 years) for controls. There were no significant differences in mean age, sex and residence distribution between FC patients and controls (p>0.05), suggesting that matching was adequate ( Table 1). The clinical and biochemical parameters were documented only in cases ( Table 2).
The frequency of genotype AA, AB and BB at codon54 in FC patients was found to be 107 (57.5%), 61 (32.8%) and 18 (9.7%), while in controls it was 132 (62.9%), 64 (30.5%) and 14 (6.7%), respectively. No significant association was observed between case and control group for variation at codon-54 and susceptibility to FC. Although the rare allele frequency in cases was higher than controls but it failed to attain statistical significance. In addition, allelic frequencies of MBL2 at codon54 showed no significant differences between the two groups (p>0.05).

Discussion:
The population of endemic region is exposed to mosquitoes but only some acquire infection. The population in our study was from an area that is endemic to filariasis. We planned the present study to find out the genetic factors that might influence the risk for an individual to develop FC. In spite of the fact that FC is a common public health problem affecting several regions of the world, it has 809 ©Biomedical Informatics (2019) been neglected by researchers. Unlike other diseases, genetic aspects of this disease have not been looked into seriously by researchers. MBL, a serum protein synthesized by liver that plays a key role in innate immunity. MBL binds to surface of oligosaccharides of bacteria, viruses and parasites and induces the compliment system via interaction with MBL-associated serine protease, and facilitates phagocytosis and opsonization of pathogens We noticed that FC group has lower frequency of AA genotypes of MBL2 gene codon 54 (57.5% versus 62.9%), and higher frequency of AB and BB genotype (32.8% versus 30.5%) and (9.7% versus 6.7%) when compared with control group. While rare allele frequency in FC group was higher than controls but it failed to attain statistical significance. In control group, we found that prevalence of AB and BB genotypes of MBL2 codon 54 polymorphism was 30.5% and 6.7%, respectively, and distribution of YX and XX genotypes of MBL2 promoter (-221) was 40% and 15.7%, respectively. In study from south India, the frequency of MBL2 polymorphisms (-221) in healthy controls were 33% and 16%, respectively, which is fairly similar to our observation [9]. Existing literature suggests a minor or no roles of promoter variants in diseases; only promoter variants -221 and structural variant codon 54 are clinically important [19]. The prevalence of codon 52 and 57 mutants is rare in North Indian population. In a study, the reported gene frequencies of codon 54, 52 and 57 were 15%, 5% and 2%, respectively, in HIV-1 infected patients [20]. Another study carried out on 213 subjects of systemic lupus erythromatous in the province of Odisha in India reported only three patients to be heterozygous for codon 52 and one for codon 57 [17]. We did not analyze the impact of other genes. More gene polymorphism site and large sample size will be analyzed in the future. Serum analysis of MBL levels was not performed. Thus, we were unable to assess whether serum MBL levels altered during infection. Our study is a preliminary study; there is need for more studies to confirm our findings. However, the strength of our study is that we have explored a condition on the literature is scanty. Our study will help in understanding the patho physiological basis of this important but neglected disease that afflicts millions of people.

Conclusions:
We report the significant association of MBL2-221 promoter polymorphism with the susceptibility to FC infection. However, this is not true for the polymorphism at codon 54. These 810 ©Biomedical Informatics (2019) observations are interesting requiring further confirmation using a larger study with more patients.