All participants in the current study were subjected to full history taking, physical examination, and laboratory investigations for fasting serum glucose level, HbA1c, fasting C-peptide, and serum pancreatic amylase. Liver and kidney function and a complete blood count (CBC) were also done. Finally, KLRC3 gene expression was done using the real-time reverse transcriptase polymerase chain reaction (RT-PCR). Fresh peripheral blood samples were withdrawn from the patients in all groups under strict aseptic conditions in vacutainer EDTA tubes. Samples were processed within few hours of collection. Purification of total cellular RNA from human whole blood was done using miRNeasy Mini Kit (Qiagen, USA).15 RNA samples were stored at -80 oC until processed. cDNA was synthesized from total RNA using QuantiTect Reverse Transcription Kit.16 KLRC3 gene expression was analyzed by real-time quantitative PCR using real-time cycler Rotor gene Q® (Qiagen, USA) using QuantiTect® SYBR® Green PCR kit.17

The gene expression level was normalized with the values of the housekeeping control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

The results of real-time RT-PCR were represented by the parameter Ct (threshold cycle), Ct is the amplification cycle at which the signal crosses a detection threshold. The difference in Ct values between KLRC3 and GAPDH reactions was given by Delta Ct (∆ Ct).

Amplification plot curve was used to calculate Ct for both KLRC3 and GAPDH. A melting curve was done to increase sensitivity and specificity of the test [Figure 1 and Figure 2].

Statistical analysis was performed using unpaired chi-square, F-test (ANOVA), Mann-Whitney, and Kruskal-Wallis test. Results were considered significant if the p-value was less than 0.050. Statistical analysis was carried out using SPSS Statistics (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp).

Results

Patients in the two diabetic groups included 15 males and 15 females, and the control group included 10 males and 10 females [Table 1]. Patients were aged between 7–13 years. The mean age of group I, II and the control group was 10.6±1.7, 9.7±1.9, and 10.4±2.0 years, respectively. The majority of patients were children (below the age of puberty) and only eight patients were >12 years old.

The three studied groups were matched for age, sex, and family history of diabetes. The most frequent symptoms were polyuria in both groups I and II (100.0% and 73.3%, respectively), followed by polydipsia, and polyphagia [Table 2]. Only five patients presented with fever and influenza-like symptoms before the onset of DKA. Results of routine blood tests were comparable in the three studied groups.

There was no significant difference between groups I and II regarding fasting blood sugar levels. Mean values of HbA1c and C-peptide were higher in group I (p = 0.003 and p = 0.046, respectively). Levels of serum pancreatic amylase were not statistically different in the three groups [Table 3].

KLRC3 gene expression was compared between the two groups and against the control group [Figure 3 and Table 4].

The mean expression levels of KLRC3 receptor for groups I, II, and control group were 0.32±0.33, 0.06±0.06, and 0.66±0.49, respectively. The level was significantly lowest in the group of patients who presented with DKA; group II (p = 0.008).

Discussion

We found significant downregulation of the KLRC3 gene in patients with diabetes compared to healthy controls. Moreover, KLRC3 showed more downregulation in patients who presented with DKA.

NK cells could be involved in one or several steps of the immune-mediated attack that leads to T1DM.8 Downregulation of the KLRC3 gene may result in abnormal NKG2E receptor expression and, consequently, abnormal NK cells function. NKG2E forms heterodimeric complexes with CD94. These complexes interact with HLA-E, a major histocompatibility complex (MHC) class Ib protein, which is involved in pathogenesis of T1DM.9

In a previous study, KLRC3 was found to be downregulated in patients with T1DM and fulminant type 1 diabetes compared to normal controls.10 Fulminant type 1 diabetes is a clinical subtype of T1DM characterized by rapid onset of hyperglycemic symptoms, DKA at the time of diagnosis, and a fasting serum C-peptide level < 0.3 ng/mL. However, when comparing T1DM to the fulminant type 1 diabetes, there was no significant difference in KLRC3 gene expression. They also found no significant differences in the expression of the other NKG2 family genes that code for NK cell receptors as KLRC2 (NKG2C), KLRC1 (NKG2A), and KLRK1 (NKG2D) when comparing diabetic patients to normal controls suggesting a more important role of the KLRC3 gene.

A more recent study found decreased numbers of NK cell subsets (CD56bright and CD56dim) and an impaired function of NK cells in patients suffering from TIDM.18 There was a slightly decreased expression of NKG2D, an activating receptor belonging to the NKG2 family. The decreased expression was found in all patients with T1DM relative to control subjects, independent of the duration of disease.

The incidence of T1DM peaks in puberty.1,2 However, we did not find any positive correlation between KLRC3 expression and older age approaching puberty.

In our study, the most prevalent symptom in the diabetes groups was polyuria. A previous report found that polydipsia was the most prevalent symptom in their cases;19 however, patients with T1DM usually have both symptoms at presentation.

HbA1c was lower in patients with DKA at presentation. Although their fasting blood sugar was higher, this low HbA1c could be explained by their shorter duration of the disease compared to patients who presented with classical symptoms. However, we did not recognize any correlation between KLRC3 expression and HbA1c.

C-peptide was significantly lower in patients with DKA at presentation. C-peptide measurement assesses beta cell reserve and, hence, endogenous insulin secretion.20 This lower level of C-peptide indicates the more acute onset of the disease. However, there was no correlation between KLRC3 expression and C-peptide.

Serum pancreatic amylase was not affected in either diabetic group reflecting a normal exocrine pancreatic function. Previous studies19,20 have reported higher levels of serum pancreatic amylase in patients presenting with DKA. This result can be explained by temporary exocrine dysfunction, which resolved after insulin treatment and/or lymphocytic infiltration of the exocrine pancreas.

Our study was limited by its single-center design and small patient number.

Conclusion

The KLRC3 gene was downregulated in patients with T1DM. Greater downregulation was observed in patients with DKA compared to those who presented with classical symptoms. Expression of KLRC3 in T1DM might play a role in the pathogenesis of T1DM and be a predictor of its severity.

Disclosure

The authors declared no conflicts of interest. No funding was received for this study.

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