Development of amplification refractory mutation system polymerase chain reaction-based assay for genotyping the single nucleotide polymorphisms of MED12 gene: An economical approach for case–control investigations of uterine leiomyoma

INTRODUCTION

Uterine leiomyomas (ULs), also known as fibroids or myomas, are benign tumors composed of smooth muscle tissue that originates from the uterine myometrium. They represent the most frequently diagnosed tumors in gynecology worldwide. As women near menopause, lifestyle factors and the consumption of contaminated foods could heighten the likelihood of developing these tumors, frequently leading to infertility without an obvious cause. Numerous studies have highlighted the importance of diet and nutrition in influencing various gynecological disorders, including uterine fibroids.^[1]

It is estimated that between 15% and 30% of women in their reproductive years have ULs, with this percentage increasing to 70–80% by the time they reach 50.^[2,3] In India, findings from the 2015 to 2016 National Family Health Survey-4 indicate that the prevalence of ULs (30–70%) is closely associated with various demographic, geographic, and ethnic factors. These variations suggest a complex and multifaceted origin of the condition, with the potential for even greater prevalence if root causes remain unaddressed.^[4] Shekhar et al.^[5] reported that about 6% of Indian women of reproductive age undergo hysterectomy, most commonly due to heavy menstrual bleeding or pain (56%) and the presence of fibroids or cysts (20%). Further research confirms that symptomatic fibroids are a leading reason for hysterectomy among women aged 30–49.^[5-7] According to Prusty et al.,^[8] approximately 0.2–6.3% of women between the ages of 15 and 49 have undergone hysterectomy in 21 of the 36 Indian states and Union territories. This trend suggests that India is emerging as a significant region for the occurrence of ULs, with an increasing number of hysterectomies among women of reproductive age. Studies indicate that a significant percentage – approximately 70–80% – of uterine fibroids contain particular somatic mutations in the MED12 gene, a trend seen in different populations such as Caucasians from Finland, individuals in the northern United States, and South Africans.^[9-11] This evidence highlights the necessity for genetic data that are specific to populations or ethnicities to enhance our comprehension of the fundamental mechanisms of the disease. The MED12 gene, a component of the mediator complex composed of 26 transcription-regulating subunits, facilitates the interaction between DNA regulatory regions and ribo nucleic acid (RNA) polymerase II, thereby playing a vital role in the control of gene transcription.^[12] Studies have identified frequent heterozygous missense and frameshift mutations in exons 1 and 2 of the MED12 gene, marking them as common genetic alterations associated with fibroid formation.^[13] In addition, somatic mutations such as rs199469672 have been detected specifically in exon 2; however, the frequency and impact of MED12 mutations on fibroid development among the North Indian population remain largely unexamined.

In this study, we introduced a novel approach using Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) to screen for a somatic mutation (rs199469672) in MED12 codon 131 G>A in blood samples.

MATERIAL AND METHODS

RESULTS

The distribution of genotypes and allele frequencies for the MED12 variant

Utilizing the ARMS-PCR assay previously discussed, a band size of 293 bp is designated as the standard marker for genotyping the 131 G>A variant in the MED12 gene. We identified all three genotypes among our sampled individuals, which included both control groups and those with UL. For homozygous conditions, we successfully amplified either the GG or AA genotypes, while in the case of heterozygous conditions, we observed PCR amplification for both the G and A genotypes in distinct allele-specific reactions [Figure 1].

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Figure 1:

Agarose gel showing that sample 1 is homozygous mutant (one band with 171 bp) and samples 2, 3, 4, and 5 were heterozygous (two bands; 293 bp and 171 bp) for MED12 gene in the case of uterine fibroid. bp: Base pair.

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The UL patients and controls’ genotype and allele frequency distribution are summarized in Table 1. Specifically, the genotypic distribution of GA/AA versus GG and allele frequencies were examined. In both groups, the frequencies of genotypes showed a significant deviation from hardy-weinberg equilibrium (P = 0.02). The results related to the genetic variations of the (rs199469672) polymorphism were obtained through allele-specific ARMS-PCR, which utilized three primers for each sample. No significant differences in allele frequencies or genotype distributions were observed between the UL patients and the control group. Although the odds ratio for disease risk associated with the T allele was lower compared to the G allele (odds ratio = 0.7, 95% confidence interval: 0.45–1.17), this variation was not statistically significant (P = 0.18) [Table 2].

Table 2: The frequency distribution of (rs199469672) polymorphism.

Genetic composition	Control (n=80)	Case (n=80)	OR	P-value
Genotypes
GG	38 (47.5%)	30 (37.7%)	1
GA	40 (50%)	45 (56.3%)	1.4 (0.7–2.7)	0.27
AA	2 (2.5%)	5 (6.2%)	3.1 (0.6–17.4)	0.19
Alleles
G	116 (72.5%)	105 (65.6%)	1
A	44 (27.5%)	55 (34.4%)	1.38 (0.88–2.2)	0.184

OR: Odds ratio

DISCUSSION

ULs, which are the most common non-cancerous tumors composed of smooth muscle in the uterus, affect a large number of women. Research suggests that around 70% of people with UL possess certain mutations in the MED12 gene, a crucial component in the mediator complex that is vital for the formation of these tumors. Specifically, mutations in exon-2 of the MED12 gene appear to alter the function of the MED12 protein, either by enhancing or reducing its activity. In our previous research, we identified the Med12c.131G>A mutation as a key pathogenic variation in tissue samples from North India, which was confirmed using sequencing techniques.

Genome-wide association studies (GWAS) are commonly used to explore the genetic basis of complex diseases. However, the findings from GWAS need to be confirmed across various ethnic populations using advanced genotyping methods for single nucleotide polymorphism (SNP) analysis. The identified SNPs are typically analyzed using established techniques such as Dot blot, Denaturing gradient gel electrophoresis (DGGE), Restriction fragment length polymorphism (RFLP), and TaqMan assay, among others.^[14] RFLP was the original method used to identify SNPs before PCR was developed. Nonetheless, RFLP has various drawbacks, including the expensive nature of restriction enzymes and the fact that the process is both time-consuming and labor-intensive. In addition, incomplete digestion of the amplified product in RFLP could lead to inaccurate genotyping results. In contrast, ARMS-PCR has become the preferred genotyping technique due to its simplicity and cost-effectiveness.^[15] An allele-specific PCR method relies on the concept that DNA polymerase will only extend primers if the 3' end matches the target sequence.^[16] In our research using the ARMS-PCR method, we successfully identified all three potential genotypes for this variant (rs199469672) Med12c.131G>A in the MED12 gene among leiomyoma individuals. ARMS-PCR has been shown to be an economical and quick genotyping technique, particularly for identifying UL-related SNPs in low-income nations. The accessibility and use of such SNP screening methods will play a crucial role in the future for forecasting or addressing complex diseases in developing countries.

In our analysis of 80 blood samples, the polymorphism was not detected. However, it was found in 12 out of 25 tissue samples through sequencing. This suggests that the polymorphism might be more prevalent or easier to detect in tissue samples compared to blood, possibly due to tissue-specific gene expression or other biological factors that influence its presence. Further research focusing on tissue types, sample processing methods, or the genetic context in which this polymorphism occurs could offer additional insights into the observed differences.^[17] In fact, mutations in exon-2 appear to lead to alterations in the function of the MED12 protein, either enhancing or diminishing its activity. Our previous laboratory study indicated that Med12c.131G>A as a hotspot pathogenic mutation found in North Indian tissue samples through sequencing analysis.^[18]

We did not find any MED12 gene polymorphism in 160 screened blood samples indicates that MED12 polymorphism could be tissue specific. However, it was identified in 12 out of 25 tissue samples through sequencing. This suggests that the polymorphism might be more prevalent or easier to detect in tissue samples compared to blood samples, potentially due to tissue-specific expression or other biological factors influencing its presence. Further investigation into the tissue types, the methods of sample processing, or even the specific genetic context in which this polymorphism is found could provide more insights into the observed discrepancy.

CONCLUSION

The somatic mutation (rs199469672) in the MED12 gene appears to be tissue-specific, likely occurring in a subset of cells within certain tissues. The absence of the mutation in blood samples supports the idea that this is a somatic event, possibly associated with a localized condition such as a tumor or other tissue-specific disease. Further studies involving larger and more diverse tissue samples, as well as methods to isolate and identify somatic mutations in blood (such as circulating tumor DNA), would be necessary to better understand the full scope of this mutation and its potential implications in disease.