Introduction

Niemann-Pick C1–like 1 (NPC1L1) protein, a newly identified sterol influx transporter, located at the apical membrane of the enterocyte, which may actively facilitate the uptake of cholesterol by promoting the passage of sterols across the brush border membrane of the enterocyte (1,2). The protein has been characterized by the presence of a signal peptide, 13 putative transmembrane regions, a conserved NPC1 domain and a sterol sensing domain (SSD). Although expressed within several tissues in the body, the expression is predominant in liver and small intestine (3, 4, and 5). In rodents, NPC1L1 is highly expressed on the surface of jejunal absorptive cells whereas in humans the expression is more in the hepatoma cells in the liver (6). The protein plays a key role in cholesterol uptake and intracellular cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (7). A fact further strengthened by the observation of Gracio-Calvo et al., 2005 (8) that the protein was the direct molecular target of ezetimibe, a drug that inhibits cholesterol absorption. Recently Temel et al., 2007 (9) have suggested the presence of NPC1L1 on the canalicular membrane of hepatocytes which may modulate biliary cholesterol excretion. Thus this protein is actively involved in the cholesterol homeostasis pathway (10, 11).

Single nucleotide polymorphisms (SNPs), together with copy number variation, are the primary source of variability in the human genome. As amino acid substitutions currently account for approximately half of the known gene lesions responsible for human inherited disease, study of nsSNPs are important in delineating the etiology of many such disorders (12, 13). These SNPs may lead to changes in protein confirmation and may be associated with altered response to drug treatment, susceptibility to disease, and other phenotypic variations (14). The study of population data for the distribution of NPC1L1 has lead to the identification of six nsSNPs (non-synonymous single nucleotide polymorphism). Among the 6 identified nsSNPs, M510I (rs1438384) shows decrease in the stability of the protein as analysed in silico by MuPro (15) and StructureSNP (16) softwares. The M510I polymorphism is the result of a nucleotide change G to A at position 2993 of the cDNA sequence in exon 2, and it results in the substitution of isoleucine for methionine at amino acid 510 of the NPC1L1 protein. The SNP has already been studied in Caucasian, Asian and African American populations by sequencing as given in NCBI database. Till date, no data is available on Indian population. Thus, a reproducible and cost-effective PCR-RFLP based assay was developed to study the distribution of this SNP as well as its association with plasma lipid levels and further implication in development of atherosclerosis.

Material and Methods

High molecular weight genomic DNA was extracted from 3.0 ml of the blood samples collected from 150 normal healthy individuals in the age group 20-50 years with informed consent. The DNA was isolated by methodology as given by Lahiri et al., 1991 (17).

Primer designing

The primers for the PCR were designed by using the software GENE RUNNER Version 3.05. The selected primers are listed in the table 1.

Figure 1

Table 1. The Primer Pair selected by GENE RUNNER Version 3.05

PCR Reaction Optimization

RFLP Analysis

Measurement of plasma lipids

Results and Discussion

An efficient PCR reaction not only generates product of requisite size but also should utilize the primers completely. Thus a minimal difference in their melting temperatures (Tm) is favourable (20) and in the case of all primer pairs selected as per Table No.1 the Tm difference is of 1ºC. Both primers and target sequence affect this efficiency. Moreover as nucleic acids have tendency to fold into conformations (secondary structures), which have high negative free energy at room temperatures. Thus the stability of these template secondary structures depends largely on their free energy and melting temperatures(Tm) and is extremely important for designing primers (21, 22, & 23). Keeping these guidelines in mind, the four primer pairs were scrutinized. The second primer pair was finally selected as it had least number of secondary structures (table 2); with no hair pin loop, no bulge loops and just 1 internal loop in sense strand not at the temperature range during PCR. Although dimers were observed both in sense and antisense primer strand but at temperatures far below the experimental temperature range. Moreover the primers so designed were unique i.e. they targeted and amplified only the specific gene in the genomic DNA as given by the Blast (24) (Table No.1). The second primer pair gave minimum and unique hits.

Figure 2

Table 2: Structural Analysis of the primer pair selected

The PCR reaction was set at three different annealing temperatures i.e.59.5°C, 60.5 °C and 61.5 °C. The results are shown in the figure 1. The annealing temperature of 61.5 °C proved to be the most stringent, giving optimum results at which no spurious amplification were observed in the PCR products as compared to other temperatures (Figure 1).

Figure 3

Figure 1: A 1.5% Agarose gel depicting NPC1L1 PCR product of size 437 bp at different annealing temperatures (Lane A- 61.5°C, Lane B- 60.5° , Lane C- 59.5°C & Lane M -Marker).

The optimization of amount of Taq polymerase and number of PCR cycles was then carried out. The amount of Taq polymerase was tested in the range 0.25 to2 U per reaction. As little as 0.5 U enzymes could be used without a decrease in the yield of PCR product (data not shown). PCR was performed for 25, 30 and 35, cycles, including the initial cycle. The PCR products could be detected after 25 cycles, maximal PCR product was obtained with 30 cycles of PCR amplification without the production of nonspecific amplification and complete utilization of primers (data not shown). The product was purified and sequenced to confirm the region amplified. The figure 2 depicts the sequence of the PCR product as obtained from Bangalore Genei and viewed in CHROMAS.

Figure 4

Figure 2: Sequence of PCR product amplified as obtained from Bangalore Genei and viewed in CHROMAS.

The amplified product was subjected to digestion by Bcc1 restriction enzyme. After digestion of the 437 bp fragment obtained by PCR, the three possible genotypes were distinguishable: homozygous GG (437 bp), heterozygous GA (437, 278 and 159 bp), and homozygous AA (278 and 159 bp).The table 3 and the gel picture (figure 3) below depicts the band pattern for different genotypes.

Figure 5

Table 3. The Band Pattern obtained after digestion with enzyme

Figure 6

Figure 3. Determination of the M510I genotype by PCR amplification and restriction analysis.

The genotype frequency distribution for the M510I polymorphism is shown in table 3 with heterozygous AG having maximum percentage of 43.3%, followed by homozygous AA (32.6%) and GG (24%). The difference in allele frequency distribution between different population groups was observed. The “G”allele present in Indian Population showed the least frequency of 0.46 (95% CI) and Caucasian Population samples the maximum at 1.000. For “A” allele it is 0.54 (95% CI) in Indian Population (table 5). The data was then analyzed for χ2 value to study difference in allele distribution amongst the Indian population and other populations of the world studied so far (table 6). Statistically, highly significant differences were observed on comparison with these populations. Even the population labelled as Asian on closer scrutiny was found to be a mixed group constituted of populations of mongoloid origin like the Chinese, Malaysians etc. This group also showed the allele distribution to be highly significantly different from the North Western Indian Population under study.

Figure 7

Table 4. NPC1L1 Genotype Distribution amongst North Western Indian population.

Figure 8

Table 5. NPC1L1 allele frequency distribution amongst different populations

Figure 9

Table 6. Comparison between North Western Indian population and other populations of the world.

Contribution of the rs1468384 NPC1L1 gene variants on plasma Lipid levels

The individuals under study were divided into two group’s i.e. lower plasma lipid group and higher plasma lipid group. The characteristics of the individuals in the two groups are shown in the table 7, below. The LDLc levels of the individuals in the low lipid group ranged from 10-149mg/dl (mean ± S.D, 136.96 ± 2.396) and those in high lipid group ranged from 150-300 mg/dl (mean ± S.D, 161.30±2.44). HDLc levels were significantly higher in low lipid group ranging between 40-65 mg/dl (mean ± S.D, 58.51±1.32) and lower in higher plasma lipid group (31.24±1.079). The Lipid levels among two groups were compared by Student’s t-test which depicted significant probability values.

Figure 10

Table 7. Characteristics of individuals in the low plasma lipid and high plasma lipid groups.

Statistical analysis was also carried out in order to determine whether this non synonymous sequence variant (rs1468384) in the NPC1L1 gene affect plasma lipid levels. Deviations in the distribution of genetic variant in either the low plasma lipid group or the high plasma lipid group were assessed by Chi square test. The table 8 shows that this nsSNP is significantly associated with the plasma lipid levels.

Figure 11

Table 8. Comparison of Allele frequency distribution amongst lower and higher plasma lipid groups.

The above analysis depicts that this nsSNP has a significant association with plasma lipid levels. Thus it can be hypothesized that this mutation has an implication on the function of NPC1L1gene as cholesterol transporter and may affect its role in the intestinal uptake of cholesterol. This study can provide us a lead to determine the affect of this nsSNP on cholesterol homeostasis pathway and its association with some complex disease states as atherosclerosis resulting due to any interruption in the cholesterol pathway.

Acknowledgement