Association of leptin receptor gene polymorphisms with post-transplant diabetes mellitus: Short report and literature review

Background: Post-transplant diabetes mellitus (PTDM) is a common and important metabolic complication after renal transplantation. Although genetic variants of the leptin ( LEP ) and leptin receptor ( LEPR ) gene have been reported to be associated with insulin resistance and diabetes mellitus, few studies have examined these variants in patients with post-transplant diabetes mellitus (PTDM). In this study, we investigated the association between LEP and LEPR polymorphisms and PTDM in renal transplant recipients. We also reviewed the literature on the genetic variants associated with development of PTDM. Methods: A total of 301 patients who received renal transplants and had no history of diabetes were included in this study. We analyzed the associations between development of PTDM and the five single nucleotide polymorphisms (SNPs) in LEP (rs1322837 and rs2167270) and LEPR (rs8179183, rs1137100, and rs1137101). Results: PTDM developed in 48 of the 301 patients studied (15.9%). Patients with PTDM had significantly higher allele frequency of the LEPR rs1137100*G allele and rs1137101*G allele. After adjustment for age, gender, and tacrolimus usage, rs113700 and rs 1137101 in LEPR showed significant association with the development of PTDM. Conclusions: LEPR polymorphisms were significantly associated with PTDM in renal transplant recipients. These data suggest that SNPs of LEPR may be associated with the pathogenesis of PTDM and may act as genetic markers for the development of PTDM.


Introduction
The development of post-transplant diabetes mellitus (PTDM) is a devastating metabolic complication after renal transplantation [1]. It affects 2-50% of renal transplant recipients and is associated with graft failure, cardiovascular complications, infection, and mortality [2,3]. As in type 2 diabetes mellitus (T2DM), decreased insulin secretion, increased insulin resistance, or a combination of both are believed to be involved in PTDM [3,4]. Although various risk factors such as older age, obesity, hepatitis C infection, and type of immunosuppressive regimen are well established, they do not fully account for the development of PTDM [3]. Recently, many studies have been conducted to analyze genetic polymorphisms as markers for PTDM [5,6]. These studies have suggested that the development of PTDM is related to the genotypes of several genes, such as adiponectin (ADIPOQ), transcription factor 7-like 2 (TCF7L2), potassium voltage-gated channel subfamily Q member 1 (KCNQ1), and C-C motif ligand 5 (CCL5), which are involved in insulin resistance and sensitivity [7][8][9][10]. Leptin (LEP) and leptin receptor (LEPR) gene polymorphisms have been reported to be associated with insulin resistance, obesity, and diabetes mellitus [11,12]. However, only a few studies have evaluated the clinical impact of LEP and LEPR polymorphisms on the development of PTDM [13].
In this study, we ascertained whether LEP and LEPR polymorphisms are associated with PTDM in Korean patients who underwent renal transplantation. We also reviewed literature that investigated associations between gene polymorphisms and PTDM in renal transplant recipients.

Methods
A total of 301 renal transplant recipients were recruited at three transplant centers in the Republic of Korea (Kyung Hee University Medical Center, Kyung Hee University Hospital at Gangdong, and Inje University Busan Paik Hospital) from 2000 to 2009. Patients were excluded when they had a history of diabetes or impaired fasting glucose (fasting glucose level 100-125 mg/dL) before transplantation. All study procedures complied with the ethical guidelines of the 1975 Declaration of Helsinki, as revised in 2000. This study was approved by the ethics review committees of all three transplant centers and written informed consent was obtained from each subject.
PTDM was diagnosed based on American Diabetes Association guidelines [14]. SNPs were selected in LEP and LEPR using the NCBI dsSNP database, version 131 (http:// www.ncbi.nlm.nih.gov/SNP) and the database of the International Hapmap Project (http://www.hapmap. org/index.html). Two LEP (rs1322837 and rs2167270) and three LEPR SNPs (rs8179183, rs1137100, and rs1137101) were ultimately selected and used to genotype the patients. Blood samples were collected from each subject and then stored at -20℃. Genomic DNA was isolated from blood samples with a commercially available Qiagen DNA extraction kit (Qiagen, Tokyo,Japan). SNP genotyping was conducted by direct sequencing. Genomic DNA was amplified with specific primers for two LEP and three SNPs ( Table 1). The amplified products were sequenced with an ABI PRISM 3730XL analyzer (PE Applied Biosystems, Foster City, Calif, USA), and sequence data were analyzed with SeqManII software (DNASTAR Inc., Madison, Wisc., USA).

Results
The overall incidence of PTDM in the study population was 17.9% (54 of 301 patients). Table 1 shows the baseline characteristics of the study population according to the development of PTDM (PTDM vs. non-PTDM group). The mean follow-up duration for all 301 patients was 87.9 months. Patients in the PTDM group were significantly older than those in the non-PTDM group (45.17 ± 9.26 vs. 38.58 ± 11.20 years, respectively; p <0.001). Patients in the PTDM group used tacrolimus more frequently than did those in the non-PTDM group (p = 0.044).
Allele frequencies are shown in Table 2. The PTDM group had a significantly higher allele frequency compared to the non-PTDM group for the rs1137100*G allele (OR = 1.924; 95% CI: 1.024-1.192; p = 0.025) and the rs1137101*G allele (OR = 1.131; 95% CI: 1.045-1.1225; p = 0.019). The effect of genotype on development of PTDM remained significant even when adjusting for age, gender, and tacrolimus usage ( Table 3). We nest, tested whether the LEPR haplotype was associated with PTDM. To demonstrate pair-wise linkage disequilibrium (LD), we analyzed three SNPs and found that they were in LD. The D' values between rs8179183 and rs1137100, and between rs8179183 and rs1137101, between rs1137100 and rs1137101 were 0.572, 0.655, and 0.876, respectively. The r 2 values between SNPs were also calculated.

Discussion
This is the first study to evaluate the genetic association of LEPR and PTDM in renal transplant recipients. Our study demonstrated that two SNPs in LEPR (rs1137100 and rs1137101) were significantly associated with the development of PTDM in Korean renal transplant patients. LEP is a hormone which is synthesized and secreted by adipose tissue, and is known to be important in regulating several neuropeptides and energy homeostasis. It has been reported to play an important role in regulation of body weight, fat metabolism, and glucose uptake [11,15]. Patients with T2DM showed decreased LEP expression in adipose tissue, and had lower serum LEP levels [16]. LEP modulates insulin secretion and action via LEPRs present in the hypothalamus, pancreatic cells, adipose tissue, and muscles [17]. In previous studies, LEPR polymorphisms were reported to be associated with diabetes, insulin resistance, metabolic syndrome, and obesity [18][19][20]. A hypothetical explanation for these results is that polymorphisms of the LEPR result in dysfunction of the LEP-associated signaling pathway and inhibition of the favorable effects of LEP [21].
In addition to the present study, our group previously reported that polymorphisms in AGT, CCL5, IL17E, IL17RA, IL17RB, IL1B, IL2, IL4,  IL7R, MMP2, TLR4, and TLR6 were significantly associated with the development of PTDM in Korean renal transplant recipients [10,[22][23][24][25]. Considering the clinical impact of each gene, our previous data suggests that impaired insulin secretion, decreased insulin sensitivity, inflammation of islet β-cells, and activation of the innate immune system may play essential roles in the pathogenesis of PTDM.
In the last decade, numerous other genetic studies for PTDM have been conducted in renal transplant recipients, and nearly 50 loci have been established as suspected loci (  [32] reported that polymorphisms in IRS1 and HNF4 increased the risk of PTDM in a Hispanic population. Elens et al. [37] showed that PPARα and POR polymorphisms are significantly associated with PTDM in renal transplant patients treated with tacrolimus. The CYP4F2 gene, which is known to be the main gene involved in creation of 20-hydroxyeicosatetraenoic acid, was also reported as an independent risk factor for PTDM [30]. These genes are associated with decreased insulin secretion through β-cell impairment   However, most of these studies are underpowered and were conducted in relatively small populations. To overcome these limitations, alternative approaches such as genome-wide association studies (GWAS) and meta-analyses are performed. McCaughan et al. [2] performed GWAS with secondary validation. They reported 26 SNPs that were associated with PTDM, and the association was validated for 8 SNPs. These SNPs were associated with apoptosis of beta cells, and the authors suggested that beta cell dysfunction and death play a crucial role in the pathogenesis of PTDM. Benson et al. [6] conducted a comprehensive meta-analysis of 18 polymorphisms in 12 genes which were reported to be genetic markers of PTDM. Of these various polymorphisms, CDKAL1 rs10946398, KCNQ1 rs2237892, and TCF7L2 rs7903146 were significantly associated with PTDM (p < 0.05).
In conclusion, we demonstrated a significant association between LEPR polymorphisms and the development of PTDM in Korean renal transplant patients. Considering our present results and the above Yang et al. [32] mentioned studies, genetic susceptibility plays an essential role in the pathogenesis of PTDM. Discovery of precise genetic polymorphisms that impact the development of PTDM is important to understanding its mechanisms and contributing to early diagnosis and proper management of the condition. Prospective, large-scale investigations that include assessment of the biological effects of gene polymorphisms are needed.