Significant ACE Gene Haplotype Stipulates Development of Type 2 Diabetes in Women with Gestational Diabetes Mellitus

Introduction: Angiotensin-converting enzyme (ACE) plays a key role in glucose and insulin regulation, and in the onset of diabetes. ACE gene polymorphisms A240T, C1237T, G2350A and I/D located in the promoter, coding and non-coding regions have been studied in both type-2 diabetes and gestational diabetes mellitus (GDM). However, their impact on the development of type-2 diabetes post GDM remains unknown, especially in under-represented population. digestion through restriction endonuclease enzymes. Results: Study results suggest a significant association of ACE genes SNPs A240T, C1237T, G2350A and I/D haplotype with GDM cases progressing to type-2 diabetes later in life (P =.02). Individuals possessing haplotype “CAAI ” derived from these SNPs had a 3.65 fold increased risk of type-2 diabetes development in GDM cases later in life relative to other haplotypes. Conclusion: Due to its pivotal role in the pathogenesis of both diseases, the current finding might be of future therapeutic value. Larger-scale studies are required to confirm this novel finding in multi-ethnic populations.


INTRODUCTION
Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase enzyme encoded by the ACE gene. This gene is located on chromosome 17q23 and contains 26 exons and 25 introns [1,2]. ACE plays a key role in circulating the renin-angiotensin system (RAS) pathway that regulates blood pressure and electrolyte balance [2]. ACE converts angiotensin-1 (ANG I), an inactive 10 amino-acid peptide to the biologically active angiotensin-II (8 amino acids) [2]. Several studies show that the Alu Insertion /Deletion (ACE I/D) polymorphism present on intron 16 of the ACE gene is associated with high concentrations of ACE levels in plasma [2], as well as with gestational diabetes mellitus (GDM), type-2 diabetes, and diabetes-related complications [3][4][5][6][7]. Previous studies involving ACE polymorphisms have provided contradictory results when assessing the effect on GDM [3,4], type-2 diabetes, and type-2 diabetes complications in different populations [5][6][7]. ACE plays an important role in developing diabetic complications in type-2 diabetes cases [6,7], however, study involving the role of ACE polymorphisms with progression to type-2 diabetes post GDM has not yet been reported to the best of our knowledge.
GDM is defined as any degree of glucose intolerance with onset or first recognition during pregnancy [8]. Women diagnosed with GDM are at an increased risk of developing type-2 diabetes in later life (usually within 10-15 years), with incidence increasing markedly in the first 5 years and plateauing after 10 years [8]. The rates of progression of type-2 diabetes post GDM ranges from 2.6-70% over follow-up periods of 6 weeks to 28 years [8]. A revision by Bellamy et al. assessed 20 cohort studies and over 600,000 women diagnosed with type-2 diabetes; of these, 30,000 were diagnosed with GDM [9]. This finding suggests that, women with a GDM diagnosis had at least a sevenfold increase in the risk of type-2 diabetes incidence in later life when compared to those with normo-glycemic pregnancies [9]. This association, together with ethnicity and a family history of type-2 diabetes ratify similar risks in developing both GDM and type-2 diabetes. Accumulative studies also indicate that a similar pathogenesis and genetic variants associations exist among GDM and type-2 diabetes patients [10][11][12][13]. Many studies showed that SNPs present on $ CDKAL1, genes which are strongly associated with type 2 diabetes are significantly associated with a higher risk of GDM too [10][11][12][13].
Patho-physiology of GDM is similar to type 2 diabetes pathophysiology [14]. Women undergo major physiological changes during pregnancy that allow the fetus to survive and thrive in the intrauterine environment. One important physiological change that occurs in normal pregnancies is an increase in insulin resistance throughout pregnancy [14]. By the second trimester of pregnancy, women's insulin sensitivity declines to one third that of their nonpregnant state. This increased insulin resistance facilitates continuous glucose transfer to the fetus, and when insulin resistance is accompanied by pancreatic β-cell insufficiency, GDM may develop [15]. Peripheral insulin resistances followed by an insulin-secretory defect are some of the pathophysiological changes of GDM similar to those observed in type 2 diabetes [13].
Studies have shown that genetic networking affects disease susceptibility [16][17][18][19]. There are reports suggesting that due to interactions between several loci, an increase in significant genetic susceptibility between ACE predispositions facilitates the onset of hypertension and coronary artery disease [20][21][22]. For example, a haplotype construction among ACE gene polymorphisms has shown synergistic effects on hypertension and coronary artery disease; however, no such association was observed in some ACE gene polymorphisms individually with hypertension [20,22]. On the other hand, detecting gene-to-gene interactions remains a challenge due to a large number of possible SNP haplotype configurations [16][17][18][19][20][21][22].
A linkage disequilibrium (LD) block difference of haplotypes varies with populations. For example, in Africans haplotypes have been shorter and more diverse, while Europeans reported longer and fewer [18][19][20][21][22]. This suggests that different LD of the SNPs located in the ACE gene may vary with geographically distributed populations [20][21][22]. Consequently, this article evaluated ACE polymorphism networking with type-2 diabetes progression in GDM cases from North India.

Analysis of ACE gene polymorphisms A-240-T (ACE 4), C-1237-T (ACE 6), A-2350-G (ACE 8),
and I/D, were achieved as per the methodology specified by Zhu et al. [22]. ACE SNPs listed in Table 2 were amplified using PCR, followed by digestion of PCR products following manufacturer protocol (see Table 2). The digested products were viewed under UV visualization after running them on the agarose gel stained with ethidium bromide. SNPs ACE 6 and ACE 8 were amplified in a 15 ul reaction containing 25 ng of genomic DNA, 0.3 mM of each primer, 7.5 ul of 2X PCR master-mix (Fermentas, USA), with MgCl 2 concentrations (Table 3). Cycling conditions were 95°C for 15 min; followed by 35 cycles of 94°C for 30s, annealing at the appropriate temperature (TA) (see Table 3) for 30s, and 72°C for 30s. Finally, a 10-minute extension at 72°C.
For ACE 4, ACE 5 amplification was conducted, followed by nested PCR of ACE 4 taking ACE 5 PCR product as a template. The following procedure was used to amplify ACE 4: ACE 5 amplified by its primers (Table 3); in a 50 ul -ml reaction containing 100 ng of genomic DNA, 0.3mM of each primer, 25 ul of 2 X PCR master mix (Fermentas, USA), 2.5 mM MgCl 2 and 10% glycerol. Cycling conditions for touchdown PCR for ACE 5 were 95°C for 1 min 30s; then 10 cycles of 95°C for 1 min, 65°C (0.5°C/ cycle) for 1 min, and 72°C for 3 min; and then 30 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 3 mins. A 1:25 dilution of the resulting products was then used as a template to amplify the region containing ACE 4 in a 25 ul reaction containing 0.3 mM of each primer, 1 mM MgCl 2 , and 12.5 ul of 2X PCR master-mix (Fermentas, USA). Cycling conditions were 95°C for 1 min 30s; then 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 3 mins; this was followed by 72°C for 10 mins (Table 3). For I/D polymorphism, the primers listed in Table 3 were used to amplify the region of Alu insertion or deletion. PCR amplification was performed in a 15 ul reaction tube containing 25 ng of DNA, 0.45 mM of each primer, 7.5 ul of 2 X PCR master-mix (Fermentas, USA), and 3 mM MgCl 2 . The cycling conditions were 95°C for 15 mins; then 35 cycles of 94°C for 1 min, 62°C for 45s, and 72°C for 1 min, respectively. A final run was performed at 72°C for 10 mins. All samples sequence analysis was done using ABI3730XI (Applied Biosystem, Texas, USA), which confirmed our genotyping results.  Table 1 Only 1 value ≥ threshold values mentioned in Table 1 Fasting ≥ 126 mg /dl or PP^ after 2 hrs ≥ 200 mg/dl ; Two or more values≥ threshold values mentioned in Table 1 Fasting ≥ 126 mg /dl or PP^ after 2 hrs ≥ 200 mg/dl ; Only 1 value ≥ threshold values mentioned in Table 1 Other    The linkage disequilibrium mapping of four ACE polymorphisms is shown in Fig. 1. Haplotype analyses of ACE gene polymorphisms were carried out by combining them in two, three and four loci respectively. Of 64 haplotypes combinations observed, only 13 haplotypes were significant. Only polymorphisms with high linkage disequilibrium (LD` values = 0.9 and r 2 values = 0.8) were considered. Table 4

DISCUSSION AND CONCLUSION
It is widely accepted that GDM is one of the strongest predictors for the long-term development of type-2 diabetes in at-risk women [8][9][10][11][12][13]. In order to prevent progress of GDM to chronic diabetic state later in life it is necessary to screen women with GDM for those high-risk genetic factors. This allows the timely introduction of measures to delay or prevent diabetes onset. ACE is a zinc metalloprotease that is widely distributed on the surface of epithelial and endothelial cells [22]. The ACE gene is one of the most intensely studied genes because of the key role it plays in the RAS. This study therefore calculated the haplotype configurations of four polymorphisms in the coding and non-coding region of the ACE gene in order to assess the future progression of GDM to type 2 diabetes in 224 North-Indian women.
Our results indicate that haplotypes D-1 D-2, D-4 in two loci, DD-2, DD-3, DD-5, DD-6 in three loci, and DDI-2 in four loci were protective haplotypes, i.e. GDM cases comprising these haplotypes are not susceptible to future onset of type 2 diabetes. GDM cases having haplotype combinations D-3, DD-1, DD-4 and DDI-1 were susceptible to type-2 diabetes post GDM. Of these, haplotype DDI-1 in GDM cases posed the highest risk for type 2 diabetes development than other haplotypes (Table 4). This is the first study to demonstrate the role of haplotype configurations of ACE gene polymorphisms with regard to development of type-2 diabetes post GDM. Considering the critical role of ACE gene interactions during GDM and the risk this poses for type-2 diabetes onset in life, these findings could therefore be of potential therapeutic value in the future. A confirmatory larger scale population with different ethnicities studies is recommended to validate the results of the present study. Although specific predictive factors for the development of type-2 diabetes post-GDM can be identified in the index pregnancy, women with a history of GDM and ACE DDI-1 haplotype, as a group, are worthy of long-term follow-up to ameliorate type 2 diabetes risk.