Frequency Distribution of Mannose Binding Lectin-2 and Vitamin D Receptor Gene Variants: Putative Markers for Tuberculosis

Genetic polymorphism in Mannose Binding Lectin-2 (MBL-2) and Vitamin D Receptor (VDR) is known to influence the susceptibility to tuberculosis. The objective of the present study was to evaluate the frequency distribution of the MBL-2 promoter and structural polymorphism (−550 H/L, −221 Y/X, and +4 P/Q; R52C, G54D, and G57F) and VDR polymorphism (FokI, BsmI, TaqI, and ApaI) in healthy individuals of Indian population and comparative analysis with the global population. In Indian population, the frequency of VDR mutant alleles “f” for FokI, “b” for BsmI, “t” for TaqI, and “a” for ApaI was 25%, 54%, 30%, and 61%, respectively. The allelic frequency of MBL-2 promoter polymorphism −550 H/L was H versus L: 32% versus 68%, −221 Y/X was Y versus X: 68% versus 32%, and +4 P/Q was P versus Q: 78% versus 22%. Mutant allelic frequencies of the MBL-2 exon 1 D, B, and C allele were 6%, 11%, and 3%, respectively. Comparative analysis with global populations showed a noteworthy difference for MBL-2 and VDR polymorphism frequency distribution, indicating the ethnic variability of Indians. The study signifies the differential distribution of susceptibility genes in Indian population, which can influence the understanding of the pathophysiology of tuberculosis in Indian population.


Introduction
Mycobacterium tuberculosis is one of the most ancient and life-threatening pathogens for mankind. More than onethird of the world's population harbours the tubercle bacilli asymptomatically. However, only 5-10% of the infected individuals develop the disease [1]. In 2012, one-third of the global deaths occurring due to tuberculosis were reported in India and South Africa [2]. The interindividual variation in disease susceptibility and progression is a consequence of the varied extent of host response to M. tuberculosis. Genetic polymorphism in Mannose Binding Lectin-2 (MBL-2), a central player in the innate immune response and Vitamin D Receptor (VDR), an immunomodulator has been found to influence the susceptibility to tuberculosis [3,4].
The MBL-2, a pattern recognition receptor of the innate immune system [5], acts as the first line of defense against infectious agents including M. tuberculosis. The MBL-2 enhances the opsonization and facilitates phagocytosis of infectious agents. Variations in MBL-2 gene influences tuberculosis susceptibility and the reports of MBL-2 gene involvement have been contradictory [6,7]. Low levels of MBL-2 have been associated with protection against tuberculosis [8][9][10] while others have reported its association with tuberculosis susceptibility [11][12][13]. The lower levels of MBL-2 have been attributed to the structural gene variants in the first exon of the gene: MBL-2 D (C>T transition, Arg52Cys), B (G>A transition, Gly54Asp), and C (G>A transition, Gly57Glu) are collectively referred to as O allele while wild type is referred to as A allele [14,15]. These results in the amino acid substitution in collagen like domain, significantly decreasing the functional MBL-2 serum levels [16]. The Single Nucleotide Polymorphisms (SNPs) in the promoter region, MBL-2 H/L (C>G transition, −550 bp in promoter region), Y/X (G>C transition, −221 bp in promoter region), and P/Q (C>T transition, +4 bp in 5 UTR region) [17], influence the MBL-2 transcription thus modulating the MBL-2 protein levels in serum. The role of 1,25-dihydroxyvitamin D3, an active metabolite of vitamin D in the calcium metabolism regulation, is well established. Vitamin D3 plays a key role of an immunomodulatory hormone, whose actions are mediated through binding to a nuclear receptor, Vitamin D Receptor (VDR). Vitamin D3 activates macrophages enhancing the phagocytosis and thus restricts the M. tuberculosis proliferation [18]. The polymorphism in the VDR gene may influence the VDR activity, subsequently associating it with increased susceptibility to tuberculosis and various clinical outcomes [3,19]. Several polymorphisms have been identified in VDR genes recognized by restriction endonucleases. These include FokI in the coding region and BsmI, ApaI, and TaqI in the 3 untranslated region. The FokI polymorphism (F/f, C>T) in exon 2 results in VDR length differentiation by three amino acids producing a less active 3 amino acid-elongated VDR when encoded by the "f" allele [20]. BsmI polymorphism (B/b, T>G) and ApaI polymorphism (A/a, T>G) occur in the intron separating exons 8 and 9. TaqI polymorphism (T/t, T>C) in exon 9, a synonymous SNP coding for isoleucine, enhances the VDR mRNA stability [21].
The aim of the present study was to estimate the allele and the genotype frequency distribution for MBL-2 and VDR gene polymorphism in Indian population and comparative analysis of the observed data with global population reported previously to get a glimpse of ethnic variability among different populations across the world.

Study Population.
The unrelated disease-free healthy subjects' samples were recruited at B. V. Patel PERD Centre, Ahmedabad, Gujarat. The age of healthy subjects was in the range of 19-50 years with male/female ratio of 5.4. The control individuals had no known history of tuberculosis. Healthy group comprised of individuals from western and northern parts of the country. Populations within Indian subcontinent are heterogeneous and represent elements of several ancestries. However, ancestry lineages are no longer demarcated in present geographical regions. Separate methods were used for the genotyping studies for MBL-2 and VDR. Samples which did not meet particular quality parameters for analysis were not included. This resulted in variation of sample size for all the polymorphisms studied. The MBL-2 polymorphisms were studied by sequencing of samples in 96-well plate; two of the samples failed to amplify. Genomic DNA of healthy subjects was extracted using phenol-chloroform extraction method [22]. Institutional ethical clearance and written informed consent of the blood donors were obtained prior to blood collection from the individuals.

2.2.
Genotyping. The six SNPs in the MBL-2 gene promoter and exon 1 (GenBank accession: rs11003125, rs7096206, rs7095891, rs5030737, rs1800450, and rs1800451) were genotyped by directly sequencing the 964 bp region amplified from genomic DNA samples of 94 disease-free healthy subjects. A preamplified PCR product of amplicon size 964 bp bracketing the region surrounding the polymorphic sites was sent for direct sequencing to Macrogen Inc., Korea, using a pair of specific primers (mentioned in Table 1).
The PCR amplification was performed for the VDR gene (FokI, BsmI, TaqI, and ApaI) polymorphism in a volume of 50 L in presence of 5 L of 10x PCR buffer provided with the Taq polymerase, 1.5 mM MgCl 2 , 0.2 mM dNTP mix, 0.2 M of each primer (mentioned in Table 1), 1-2 L of 1 U/ L Taq polymerase, and 280 ng of genomic DNA and subjected to thermal cycler at annealing temperatures mentioned in Table 1. The amplified products were electrophoresed and visualized in ethidium bromide stained 1.5-2% agarose gels. The amplified PCR products were subjected to restriction fragment length polymorphism (RFLP) using respective restriction enzymes. The genotypes were assigned in accordance with the number of bands obtained after the digestion for each of the four polymorphisms (for details see Table 2).

Statistical
Analysis. MBL-2 and VDR alleles and genotype frequencies in disease-free healthy subjects were calculated by direct counting. The Hardy-Weinberg equilibrium (HWE) was determined. Chi-square test was performed to compare the allelic frequencies of different populations and p values were calculated by unconditional logistic regression and p < 0.05 was considered to be significant. Linkage disequilibrium and haplotype analysis was performed by  The LD plot construction and haplotype frequencies were calculated using the same software [23]. The standardized disequilibrium coefficient (D ) analysis between the MBL-2 SNPs was also performed using the LD plot function of this software.

Results
In the present study, MBL-2 promoter polymorphism (−550 H/L, −221 Y/X, and +4 P/Q) and exon 1 polymorphism (R52C: D allele, G54D: B allele, and G57F: C allele) were analyzed in 94 healthy subjects of Indian population shown in Table 3. All polymorphism with the exception of R52C were in Hardy-Weinberg equilibrium. The genotype and the allele frequency of MBL-2 polymorphism are represented in Table 3. The allelic frequency of MBL-2 promoter polymorphism −550 H/L was H versus L: 32% versus 68%, −221 Y/X was Y versus X: 68% versus 32%, and +4 P/Q was P versus Q: 78% versus 22%. The MBL-2 exon 1 polymorphism mutant allelic frequency obtained for D, B, and C allele was 6%, 11%, and 3%, respectively. The observed genotype and allele frequencies in Indian population were compared with the previously reported different populations worldwide by using chi-square tests to elucidate the differences in the distribution of MBL-2 structural variant alleles (D, B, and C) and promoter variants ( Figure 1). The observations for MBL-2 structural polymorphisms were similar to the findings reported in South Indian population [11], Europeans [24,25], Denmark [4], and Brazilian population [26] ( > 0.05) (Figure 1). A significant difference was observed between the Indian (present study) and East African (Kenya) population for B ( = 0.002) and C ( < 0.0001) structural variants [24]. In comparison to MBL-2 promoter polymorphism distribution, a statistical significant genotype frequency distribution difference between the Indians (present study) and Chinese Han population ( < 0.01) was observed [10]. The sequence data generated for 964 bp MBL-2 promoter region and exon 1 was analyzed for the patterns wherein the presence of a polymorphism at one position would be consistently associated with polymorphism at one or more other positions. The examination of pairwise linkage disequilibrium (LD) between the MBL-2 variants was performed by construction of LD plot which revealed the presence of a single haplotype block (Figure 2). Seven putative haplotypes spanning the length of the sequenced region have been identified with a frequency of more than 1% (CCCCG = 29.4%, GGCCG = 23.8%, CGTCG = 21.4%, CGCCA = 11.7%, GGCTG = 5.9%, CGCCG = 5%, and GCCCG =1.9%) [27]. The distribution of VDR genotypes and allele frequencies of FokI, BsmI, TaqI, and ApaI in Indian population is shown in Table 4. The allelic frequency of "f," "b," "t," and "a" alleles was 25%, 54%, 30%, and 61% obtained in Indian population. The genotype frequency of FokI and TaqI was in agreement with Hardy-Weinberg equilibrium. The observed VDR polymorphism genotype frequency distribution was compared individually with the different populations worldwide by using 2 tests (Figure 3). There was a statistically significant difference between Indians (present study) and the West Africans in FokI, BsmI, and ApaI polymorphism ( < 0.01) but nonsignificant difference in TaqI polymorphism ( = 0.142) [28]. Japanese population differs significantly from Indians (present study) in TaqI and ApaI ( < 0.05) and FokI ( < 0.001) polymorphism [29]. VDR polymorphism frequency distribution differs significantly from the Korean population in TaqI and BsmI ( < 0.01) but is similar in ApaI and FokI ( > 0.05) polymorphism [30]. The frequency of the VDR genotypes in the present study also differs from that of studies conducted in North Indian and East Indian populations [31,32]. Upon comparative analysis, there was a significant difference in our data from East Indians in TaqI, FokI, and BsmI polymorphism ( < 0.01) and from North Indians in ApaI and FokI except TaqI polymorphism ( < 0.01). The frequency observed was however similar to the Turkish population for VDR FokI, BsmI, and TaqI polymorphism [33]. There was a statistically significant difference between Indians (present study) and the Europeans (Finnish, French, Austrians, and Swedes) ( < 0.01), as well as with Asians (Thais and Chinese) ( < 0.05), for VDR FokI, ApaI, and TaqI polymorphism ( Figure 3) [29,31]. This demonstrates the genetic diversity within Indian population and also between different populations globally.

Discussion
The innate immunity is the first line of defense against infectious microorganisms. There are several key players of the innate immune system that interact, coordinate, and act against these infectious agents. Genetic variations in these key players can influence their mechanism of action leading to variable immune response. MBL-2 and VDR are among these key players which influence the susceptibility to M. tuberculosis infection and disease development. The present study involved the frequency distribution analyses of MBL-2 and VDR polymorphisms, two molecules known to play a key role in tuberculosis susceptibility. The study also included haplotype study for MBL-2 polymorphisms and evaluation of global frequency distribution disparity among different populations worldwide. Frequency distribution of genotype and alleles of VDR and MBL-2 gene varies among different ethnic population, which may lead to variable susceptibility to the infection. For VDR FokI "f" allele, the frequency varies from 43% in Finland to 21% in West Africa. The occurrence of "f" allele  [28, 30-33, 37, 38].
was much higher in Caucasian population. The VDR TaqI "t" allele frequency was found to be higher in Caucasians than Asian and African populations.  [34]. A study conducted in Gujarati Indians living in London showed strong association of ff genotype with pulmonary tuberculosis [36]. Therefore, we are not able to draw any conclusion.
In summary, a lot of studies have been conducted through the years to understand the role of Vitamin D Receptor polymorphism in susceptibility to infectious diseases. Unfortunately, the results have been conflicting and still the role of VDR in tuberculosis susceptibility is not clear: the polymorphism determines susceptibility to the development of clinical disease or susceptibility to infection. Therefore the use of VDR polymorphism as marker for tuberculosis susceptibility is under debate. MBL-2 variant alleles have been associated with lower serum MBL levels and contrasting associations for MBL-2 variant alleles and mycobacterial infection have been reported in different populations. VDR and MBL-2 polymorphisms' global and regional distribution, widely studied in several populations worldwide, varies significantly in each population, thus making it unique for the tuberculosis susceptibility studies. The differences in genotype and allele frequencies observed between populations could have been result of different selective pressures such as differences in diet, climate, latitude, or exposure to pathogens leading to adaptations. The efforts should be placed on a molecular understanding of the pathogenic processes, allowing a clear insight of genetic influences on the infectious diseases.
The population based studies are important to reveal the population specific component to tuberculosis susceptibility which can prove effective in incorporating this into treatment and prevention strategies specifically. The study needs to be validated in larger samples to get a clearer picture of frequency distribution of MBL-2 and VDR genetic variants and their association to tuberculosis susceptibility in different parts of India.

Ethical Approval
Institutional ethical clearance was obtained.

Consent
Written informed consent of the blood donors was obtained prior to blood collection from the individuals.