Association of genetic polymorphism of PC‐1 gene (rs1044498 Lys121Gln) with insulin‐resistant type 2 diabetes mellitus in Punjabi Population of Pakistan

Abstract Background Insulin resistance (IR), known to reduce the response to insulin action, develops with obesity leading to type 2 diabetes mellitus (T2DM). The PC‐1 gene has been associated with dyslipidemia, polycystic ovarian disease and T2DM in different regions of the world. The objective of the present study was to investigate the genetic association of PC‐1 rs1044498 polymorphism with insulin resistance in type 2 diabetes in the Punjabi population of Pakistan. Methods This study was carried out on 161 healthy controls and 161 patients of T2DM with insulin resistance. Whole blood was collected for DNA extraction and molecular studies. PCR‐RFLP with AvaII was performed to determine the genotype in cases and controls. Chi‐square and Hardy Weinberg analyses were carried out. Statistical analysis was performed by SPSS software. Results The demographic data of cases and controls showed significant differences for different parameters like glucose, insulin, Homeostatic model assessment‐insulin resistance (HOMA‐IR) and lipid profiles (p < 0.000). Different statistical models revealed that all the dominant models were found associated in between alleles for disease risk (p < 0.001) while no association of PC‐1 rs1044498 (K121Q) polymorphism was found with insulin‐resistant parameters in T2DM cases. Conclusion Overall, the results indicate that the K121Q polymorphism was not found associated with insulin resistance in type 2 diabetes in a Pakistani Punjabi population. This is the first‐ever report about the genotype of PC‐1 gene in this population.

Unhealthy changes in lifestyle also lead to both insulin resistance and metabolic syndrome. An individual's risk of developing insulin resistance and metabolic syndrome is also determined by genetic factors (Brown & Walker, 2016). The vast majority of loci related to T2DM is primarily associated with insulin secretion and β-cell function with far fewer variants apparently influencing insulin resistance. There are number of reasons for this, not the least that insulin resistance is strongly linked to obesity, and dissecting out the role of common genetic variation in insulin resistance in the absence of obesity has been problematic (Brown & Walker, 2016).
Plasma membrane glycoprotein 1 (PC-1) is also known as Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1). It is located on the long arm of chromosome 6 (6q23.2) and encodes for a protein which is one of the factors determining the insulin sensitivity. It is a class-II transmembrane glycoprotein located both on the plasma membrane and in the endoplasmic reticulum (ER). Although, the role of PC-1 is not completely understood, it is capable of inhibiting the autophosphorylation of insulin receptor by interacting directly with the insulin receptor α-subunit, thus preventing subsequent downstream signaling (Johnson, 2006;Pappalardo et al., 2017).
The results of association studies of the K121Q polymorphism in various reports remain conflicting and further investigations are needed to elucidate its exact molecular mechanism. Previous studies documented the association of the Q allele of PC-1 (rs1044498) with insulin resistance (Gu et al., 2000;Pizzuti et al., 1990) while other studies demonstrated no association of this allele with the risk of disease (Rasmussen et al., 2000). From the South Asian subcontinent, very few reports have been conducted on the involvement of PC-1 risk alleles in disease (Prakash, Mittal, Awasthi, Agarwal, & Srivastava, 2013). Therefore, we investigated the relationship between the K121Q polymorphism and IR in T2DM patients of Punjabi population of Pakistan.

| Ethical statement
Prior to start of the study, institutional approval was obtained from Research and Ethical Board of University of Health Sciences, Lahore, Pakistan. For the collection of samples, guidelines for human subjects were followed (Puri, Suresh, Gogtay, & Thatte, 2009).

| Study population
A total of 161 patients who had been recently diagnosed with T2DM and 161 age and sex-related healthy controls were recruited during the period of August 2013 to August 2016 from the outdoor clinics of different teaching hospitals of Lahore which is the capital of the Punjab province of Pakistan.
Insulin resistance (IR) was confirmed by applying HOMA-IR equation according to the American Heart Association guidelines. A total of 5 ml blood sample was obtained from each participant for biochemical and molecular studies.

| DNA extraction and genotyping
After blood collection in EDTA tubes, genomic DNA was extracted from the whole blood by using a BioNeer Blood DNA extraction Kit (centrifugal column; Daejeon city, Korea) according to the manufacturer's specifications. Polymerase chain reaction (PCR) was carried out using primers (F-5'CTGTGTTCACTTTGGACATGTTG3'; R-5'GACGTTGGAAGATACCAGGTTG3') of PC-1 polymorphism (rs1044498), followed by the restriction fragment length polymorphism using AvaII (BioLab) restriction enzyme to genotype the alleles. The digested products were resolved on 2.5% agarose gel electrophoresis for band interpretation along with marker DNA (100 bp ladder).

| Statistical analysis
SPSS 21.0 was used for data analysis of the quantitative variables by performing Student's t test for continuous values and by the chi-squared test for nominal variables. The genotype distributions, which were expected to be in Hardy-Weinberg equilibrium, were confirmed using a goodness-of-fit chi-square test, | 3 of 6 ALBEGALI Et AL. and the differences in variables and genotype frequency distributions between the patients and controls were tested using a two-sided chi-squared test. The relationship between the PC-1 K121Q polymorphism and IR in T2DM was described by calculating odds ratios and 95% CIs through a binary logistic regression analysis. Statistical significance was considered if p ≤ 0.05.

| Population characteristics
A total of 161 cases of T2DM and 161 healthy controls comprised the population analyzed in our study. The demographics and clinical parameters of all participants are shown in Tables 1 and 2. The range of age of the participants was 45-65 years. The IR group comprised 82 (50.9%) male patients and 79 (49.1%) female patients. No difference in gender (p < 1.00) was observed between cases and controls.
An analysis of the body mass index in cases revealed 91 (56.5%) patients were overweight and one (0.3%) patient was obese (Table 1). The clinical parameters including waist circumference, HOMA-IR, and lipid profile were found to be significantly different in cases when compared to controls (p < 0.001) which explain the metabolic abnormalities among IR of T2DM patients (Table 2).
To investigate the genetic association between PC-1 K121Q polymorphism and IR, the genotype frequency distributions of

| DISCUSSION
The association between insulin resistance and PC-1 rs1044498 (K121Q) polymorphism was analyzed in our case-control investigation. This study is the first to examine PC-1 K121Q polymorphism in insulin-resistant T2DM patients from Pakistan. The functions of PC-1 protein remain to be fully elucidated, however, it has been demonstrated that overexpression of PC-1 suppresses activity of insulin receptor of tyrosine kinase and subsequently cellular signaling in various cells (Maddux & Goldfine, 2000).
In this study, the demographic data showed that there was a correlation of insulin resistance with other biochemical parameters like glucose, insulin, and serum lipid profiles which were significantly different in the cases and controls. A genetic association between the PC-1 K121Q polymorphism and insulin resistance in T2DM was not established in our study. Consequently, it may be proposed that PC-1 K121Q combined KK/QQ alleles are not risk alleles to arbitrate the disease risk in T2DM patients.
The PC-1 K121Q polymorphism is a functional missense alteration (Sortica et al., 2015) in which the 121Q variant binds insulin receptors more firmly than the 121K variant (Costanzo et al., 2001). Previous studies have documented an association of this polymorphism with insulin resistance, T2DM, and metabolic syndrome (Abate et al., 2003;Bhatti et al., 2010;Daoming, Chaoren, Xijun, & Junjun, 2006;Grarup et al., 2006;Marchenko et al., 2018;Vasudevan, Ismail, Ali, & Mansor, 2009). On the other hand, some other studies did not document the genetic association of PC-1 K121Q polymorphism with F I G U R E 1 Genotype of PC-1 alleles on agarose gel. A band size of 238 bp of PCR products for PC-1 K121Q polymorphism after running its PCR product in 2% agarose gel using 50-bp DNA marker T A B L E 3 Genotype models for disease association of PC-1 rs1044498 SNP genotype insulin resistance (Rasmussen et al., 2000). A study conducted in the China also showed that the genotype distribution of PC-1 K121Q polymorphism has no association with T2DM or any of metabolic syndrome characters (Daoming et al., 2006). These results are similar to the present study which have not established any association of PC-1 with risk of type 2 diabetes in the Pakistani Punjabi population.

Controls Cases
A meta-analysis on the correlation between PC-1 K121Q polymorphism and T2DM risks showed positive findings and established the genetic associations (Abate et al., 2005;Hamaguchi et al., 2004), that the exact molecular mechanism of risk factor for this gene is still unclear.
In conclusion, our study did not find any association between insulin resistance type 2 diabetes mellitus and PC-1 K121Q polymorphism. It is suggested that further studies on larger scale are to be carried out to explain the precise molecular mechanism of association between studied SNP and development of insulin resistance in the pathogenesis type 2 diabetes mellitus.