Role of alpha and gamma Klotho genes in the development of differentiated thyroid carcinoma on top of goiter

The present case control study was carried out in Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt. This study included 40 DTC patients (31 PTC and 9 FTC patients) in addition to 40 age- and sex-matched subjects diagnosed as goiter and enrolled as a control group. All subjects were selected from Mansoura Oncology Center, Mansoura University, Mansoura, Egypt between December 2019 and March 2021. Informed consents were obtained from all participants in the study. The study protocol was approved by Institutional Research Board (IRB) of Mansoura Faculty of Medicine (IRB code MDP.19.09.25).

The included patients were diagnosed with primary DTC by preoperative palpation, fine-needle aspiration cytology (FNAC), and US. The diagnosis was confirmed by intraoperative rapid pathological and postoperative pathological examination. For all patients, full medical history was taken and careful clinical examination, including size and consistency of thyroid gland, assessment of lymph node, and body mass index (BMI) was done.

Patients with other primary malignancies or who previously underwent hemi-thyroidectomy, anti-TC chemotherapy, radiotherapy, or radioactive iodine treatment before surgery were excluded from this study. All laboratory investigations, including complete blood count (CBC), serum total triiodothyronine (T3), total thyroxine (T4), and thyroid stimulating hormone (TSH) were done in Mansoura Oncology Center in a time frame of less than 3 months preoperatively. The thyroid gland was evaluated by US characteristics, including margins, calcifications, and echogenicity.

Thyroid tissue specimens were obtained at the time of surgery from patients and controls and dissected immediately after surgery. The tumor tissues were collected from the center of the suspected viable cancer tissue^[17]. The other part of the tissue samples was transported to the Pathology Department for confirmation of histopathological diagnosis and collection of other clinicopathological data. According to the American Joint Committee on Cancer (AJCC), 2010 recommendations, the TNM staging was accomplished (32 cases were stage I, three cases were stage II, one case was stage III, and four cases were stage IVA cancers).

To preserve RNA integrity, the fresh tissue samples were completely submerged as quickly as possible in an appropriate volume of RNAlater RNA Stabilization Reagent, approximately 10 μl of the reagent per 1 mg of tissue (Qiagen, Germany, Cat. No 76104). The samples were transported on ice and stored at 4°C for at least 24 hours, then stored at −80°C for subsequent total RNA extraction.

Referring to the modified Chomczyński and Sacchi's method^[18], total RNA was extracted from thyroid tissue samples, using QIAzol Lysis Reagent kit (Qiagen, Germany, Cat No 79306) according to the kit's protocol. The integrity of RNA was evaluated by loading RNA samples on agarose gel electrophoresis. The RNA concentration and purity of the samples were assessed using the NanoDrop 2000c Spectrophotometer (Thermo Scientific, USA). The purity of RNA was assessed in each sample via two optical density (OD) ratios (A260/A280 and A260/A230). The RNA samples with 1.8 to 2.0 A260/A280 ratios were used. The isolated RNA was then stored at −80°C for subsequent reverse transcription.

Complementary DNA (cDNA) was synthesized using COSMO cDNA synthesis kit (Willowfort, COSMO cDNA synthesis kit, Birmingham Research and Development Park, Birmingham, WF-10205002), in accordance with manufacturer's instructions. The cDNA was synthesized using the thermal cycler (Applied Biosystem, Waltham, Massachusetts, USA) with the following program of 5 min at 25°C, 15 min at 45°C, and 5 min at 85°C. The synthesized cDNA samples were stored at −20°C.

The RT-qPCR assays were carried out on the 7500 Real Time PCR System, Applied Biosystem, USA, with HERAPLUS SYBR® Green qPCR Master Mix (2X), Birmingham Research and Development Park, Birmingham, WF10308001, and gene-specific real-time qPCR primers. It was performed according to the method described by Freeman et al.^[19]. Each 20 μL reaction mix contained 10 μL of HERAPLUS SYBR® Green qPCR Master Mix (2X), 2 μL of the synthesized cDNA, 1 μL of forward primer, 1 μL of reverse primer, and the remaining 6 μL was RNase free water.

The primers for the α-Klotho and γ-Klotho genes were chosen from NCBI databases [http://www.ncbi.nlm.nih.gov/tools/primer-blast]. The primer sequences were checked for the melting temperature, product length, GC ratio, 3′ complementarity, and self-complementarity using Primer3 v.4.1.0 software [http://primer3.ut.ee/]. The forward primer sequence of the α-Klotho gene was 5′-CTGGATCACCATCGACAACCC-3′ and the reverse primer was 5′-GACACCTGACCTCCCTGAGT-3′ for a product size of 186 bp. The forward primer sequence of the γ-Klotho gene was 5′-GGCTCTGACTACCAGCGACT-3′ and the reverse primer was 5′-CACCAGCAGTAGCATCCACA-3′ for a product size of 125 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene, and its primers were selected based on published sequences^[20]. The forward primer sequence of the GAPDH gene was 5′-GTCAAGGCTGAGAACGGGAA-3′ and the reverse primer was 5′-AAATGAGCCCCAGCCTTCTC-3′ for a product size of 158 bp.

The real-time PCR reactions were performed with the following thermal cycling program of 2 min at 95°C, followed by 40 cycles of denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s. The analysis of melting curve of all the reactions was performed for evaluation of the specificity of the products.

Relative quantification (RQ) of mRNA expression was estimated using the comparative threshold method (ΔΔCt)^[21]. The data were presented as RQ of the target mRNA, normalized as regards the mRNA of the reference gene GAPDH and in respect to the control samples. The fold change was calculated using the equation RQ = 2^−ΔΔCt.

The collected data were introduced to a PC using Statistical Package for Social Science (SPSS) Version 25. Qualitative data were expressed as count and percent. Quantitative data were initially tested by Kolmogorov–Smirnov and Shapiro–Wilk's, then expressed as mean ± standard deviation (SD) for parametric numerical data or median and interquartile range (IQR) for nonparametric numerical data. Qualitative data were compared via Chi-square test (or Fisher's exact test), whereas quantitative data were compared via independent samples t-test or nonparametric Mann–Whitney U test. Receiver operating characteristic (ROC) curve analysis was used to determine the discrimination accuracy of the diagnostic test to distinguish between DTC and goiter^[22]. Comparisons of area under ROC curve (AUC) were performed. Logistic and ordinal regression analyses were used for prediction of risk factors, using generalized linear models. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated. The results were considered statistically significant if p-value ≤ 0.05 for any used test.

Routine demographic and laboratory data of the patients and controls are presented in Table 1. DTC patients had a statistically significantly lower BMI than the controls (p = 0.008). The serum TSH level was statistically significantly higher in patients with DTC than in controls (p = 0.016).

The α-Klotho gene expression was statistically significantly lower in DTC patients (median: 0.34; IQR: 0.22–0.54) compared to controls (median: 0.96; IQR: 0.83–1.19; p < 0.001). However, there was no statistically significant difference between the case (median: 0.95; IQR: 0.63–1.35) and the control groups (median: 1; IQR: 0.80–1.30; p = 0.643) regarding the γ-Klotho gene expression.

ROC curve analysis showed that the α-Klotho mRNA levels can discriminate between malignant and benign thyroid tissues by providing an AUC of 0.954 value (95% CI = 0.910–0.999; p < 0.001). However, ROC curve analysis of the γ-Klotho mRNA levels could not discriminate between malignant and benign thyroid tissues by providing AUC of only 0.530 (95% CI = 0.401–0.659; p = 0.644) (Figure 1).

Figure 1:

With a specificity of 97.5% and a sensitivity of 87.5%, the α-Klotho cutoff value of ≤ 0.68 showed a significant diagnostic accuracy of DTC. However, the γ-Klotho cutoff value of ≤ 0.93 did not show significant diagnostic accuracy of the tumor with a specificity of only 55.0% and a sensitivity of only 50.0%.

DTC patients were classified into high and low α-Klotho gene expression groups, according to the cutoff value of the α-Klotho mRNA level (≤0.68). Low expression of the α-Klotho gene was identified in 35 of the 40 samples of the tumor tissues. The low α-Klotho expression group had larger tumor size (p < 0.001) and multifocality tendency (p = 0.031). However, there was no significant correlation between the α-Klotho expression and the other studied variables (p > 0.05; Table 2).

There was a statistically significant inverse correlation between the α-Klotho gene expression and the age of the studied groups (p = 0.037), the stage of the DTC (p = 0.026), as well as the tumor size (p < 0.001). However, there were no statistically significant correlations between the α-Klotho gene expression and the other studied parameters (p > 0.05; Table 3).

There were no statistically significant correlations between the γ-Klotho gene expression and the studied parameters (p > 0.05; Table 4).

Logistic regression analysis revealed that low α-Klotho mRNA expression was demonstrated to be significant predictor for the likelihood of DTC on top of goiter (p = 0.001; Table 5). Univariate ordinal regression analysis was performed. It revealed that the female gender (p = 0.025), high-serum total T4 (p = 0.010), high-serum TSH (p = 0.024), and low α-Klotho mRNA expression (p < 0.001) could be significant predictors for the probability of higher stage of DTC.

Multivariate ordinal logistic regression was performed to ascertain the effects of the female gender, high-serum total T4, high-serum TSH and low α-Klotho mRNA expression on the likelihood of higher stage of DTC. Patients with higher α-Klotho mRNA expression had a lower odds (0.671) to exhibit higher stage of DTC (p = 0.037; Table 6). Higher α-Klotho mRNA level appeared as a protective factor against higher DTC stage.