Association of Kaphaja and Kapha-Pittaja Prakriti and methylenetetrahydrofolate reductase C677T allele with type 2 diabetes.

BACKGROUND AND OBJECTIVES: Type 2 diabetes is a multifactorial disorder that results from the interaction between genetic predisposition and environmental factors. Different Prakriti (body constitution) individuals have different susceptibility for the diseases, and this Prakriti is determined by both genetic and environmental factor. This study was undertaken to determine the association status of Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C with type 2 diabetes and Prakriti.
MATERIALS AND METHODS: After informed consent, 54 patients with type 2 diabetes and 56 individuals as normal controls were analyzed. Their constitution and pathological data were collected and MTHFR C677T and A1298C genotypes were determined.
RESULTS: Kapha/Kapha-Pittaja Prakriti were associated and found to be strong risk factors (Chi-square test = 39.67, P < 0.00001, odds ratio [OR] = 16.133, 95% confidence interval [CI] = 6.32-41.20) for type 2 diabetes. MTHFR C677T was associated (Chi-square test = 7.743, P = 0.02) with type 2 diabetes where the major CC genotype was found to be a risk for type 2 diabetes (OR = 3.78, 95% CI = 1.14-12.45). A1298C was not associated with type 2 diabetes (Chi-square test = 2.264, P = 0.322). None of the Prakriti was associated with C677T and A1298C variants. INTERPRETATION AND
CONCLUSION: In the present study, an extremely strong association between Prakriti (Kaphaja/Kapha-Pittaja) and type 2 diabetes (P < 0.00001) was detected. The present study gives a strong clue for the association of Prakriti (body constitutional) and clinical phenotype.

Introduction

According to Ayurveda Dosha (bio humors) are one of the factors; others are Dhatu (bodu tissue) and Mala (metabolic waste) responsible for the state of health or diseases. Among these Vata, Pitta and Kapha are the Sharirika Dosha (body humors).[1] Vata governs movements, Pitta is concerned with functions of digestion, metabolism and energy production and Kapha governs physical structure, fluid balance and immune response of the body.[2]

The natural constitution of the body (Prakriti) is determined by these three Dosha at the time of fertilization. One or more of these three Dosha may dominate in an individual resulting into seven types of body constitutions (Prakriti) i.e. Vata, Pitta, Kapha, Vata-Pitta, Vata-Kapha, Kapha-Pitta and Vata-Pitta-Kapha. This classification of an individual is based on their physical, physiological and psychological characteristics. Different Prakriti individuals have susceptibility for particular disease. As per Sushruta Samhita, Sharira Sthana 4/74, Prakriti of an individual does not change except when the end of life is approaching.

Prakriti, the inherent characteristic of an individual refers to the genetically determined physical and mental makeup and is determined by (a) sperms and ovum; (b) forming condition of the uterus; (c) food and regimens of the mother during pregnancy; and (d) nature of predominant Mahabhuta of the fetus. The fetus gets afflicted with one or more of the two Dosha, which are dominantly associated with the above-mentioned factors. Dosha dominating the sperms and ovum during the time of conception determine Prakriti of the individual. Food and regimens of the mother, which aggravates Dosha at that time, also determine the physical constitution. The Dosha that ultimately emerge as dominant factors actually determine Prakriti.[3] Therefore, the knowledge of Prakriti may be important for prevention and better management of diseases.

Type 2 diabetes is a multifactorial disorder that results from the interaction between genetic predisposition and environmental factors. The risk for type 2 diabetes is increased when there is a positive family history of the disease. The genes that predispose to type 2 diabetes are poorly identified, but recent genome-wide association studies have identified several genes that contribute smaller risks to type 2 diabetes (relative risk, 1.1–1.5).[4] The extrinsic factors include advanced age, obesity, diet, sedentary lifestyle and increasing urbanization. The incidence of the disease is continuously increasing in developing countries like India. The World Health Organization (2003) has predicted that by 2030 the number of adults with diabetes will be almost doubled worldwide, from 171 million in 2000 to 366 million in 2030.[5] Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in folate metabolism. It catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for the transformation of homocysteine to methionine through a transmethylation pathway. The gene encoding MTHFR is located on chromosome 1 at 1p36.6.[6] Reduction in the activity of MTHFR caused due to C677T change is known to be risks for various diseases. The T allele is also known to be associated with the elevated levels of plasma homocysteine which is also individually risks for many diseases.[7] MTHFR polymorphisms have been linked to type 2 diabetes and micro/macrovascular complications.[8] Numerous studies have demonstrated associations between MTHFR polymorphisms (C677T and A1298C) and risk of type 2 diabetes.[89]

MTHFR C677T and A1298C genetic variants have been found to be associated with type 2 diabetes both Indian as well as in other populations in the world.[ref10ref11ref12ref13] The results are variable. These variants lead to an increase in homocysteine level and are a risk for various conditions including vascular diseases, ischemic stroke which in turn may increase the risk for type 2 diabetes.[1415]

The present study also aims to determine the association of Kapha Prakriti with type 2 diabetes.[1617] It also aims to determine whether the Kaphaja Prakriti have an association with MTHFR C677T and A1298C variants or not.

Aims and objectives of the study

The objectives of the study are (1) to determine whether MTHFRC677T and A1298C alleles are associated with Madhumeha (type 2 diabetes) or not, (2) to determine whether Madhumeha (type 2 diabetes) is associated with Prakriti or not, (3) to determine whether MTHFRC677T and A1298C alleles are associated with Prakriti or not.

Materials and Methods

The study was approved by the Ethical Committee of the Institute, faculty of Ayurveda, BHU, Varanasi, and informed consent was taken from each of the participants. A total of 54 type 2 diabetes patients (36 males and 18 females) and 56 normal controls (34 males and 22 females) (matched on geographic origin ethnicity, gender, age, environmental conditions and socioeconomic status) were registered for the study. The age of cases and controls were between 25 and 70 years and the median age of cases and controls was 54 and 44 years. The individuals were registered from the outpatient department and inpatient department and clinical laboratory of the hospital of faculty of Ayurveda, BHU, Varanasi. Detailed clinical examination and medical history were recorded. Additional investigations including plasma glucose level, lipid profile, hemoglobin A1c and urine investigations were also performed. Prakriti of each of the individuals was determined according to questionnaire-based scoring (Cronbach’s alpha reliability of the selected questions after Prakriti assessment). Peripheral blood (3–5 ml) was collected for DNA isolation. MTHFR C677T, as well as A1298C genotypes of each of the participants, were determined by PCR followed by restriction digestion using the protocol as described in Kumari et al.[15] Statistical analysis was performed using computer programs quatpsy.org for calculation of Chi-square test and P value and hutchon.net for calculating odds ratio.

The selection of cases was done on the basis of fulfillment of diagnostic criteria of Madhumeha (type 2 diabetes).

Inclusion and exclusion criteria

Inclusion criteria

The age of patients between 25 and 75 years

Patients who fulfilled the criteria of diagnostic features of Madhumeha (type 2 diabetes)

Both male and female patients.

Exclusion criteria

Patients with type 1 diabetes

Patients with type 2 diabetes who were insulin dependent

Patients above the age of 75 years.

Results

Association analyses have been performed between different groups as follows:

Association analysis between Prakriti-type and type 2 diabetes

Association analysis was performed between various Prakriti - types and type 2 diabetes, where an extremely strong association between Kaphaja/Kapha-Pittaja Prakriti and type 2 diabetes was observed [Table 1].

Table 1

Frequencies of K/KP and non-K/KP in type 2 diabetes cases and controls

Prakriti	Cases (%) (n=54)	Controls (%) (n=56)
K/KP	44 (81.48)	12 (21.43)
Non-K/KP	10 (18.52)	44 (78.57)
χ2	39.67	Reference
P (df=1)	<0.00001	Reference
K/KP versus non-K/KP or	16.133	Reference
95% CI	6.32-41.20	Reference

CI: Confidence interval, K/KP: Kapha/Kapha-Pittaja

Association analysis between methylenetetrahydrofolate reductase C677T and A1298C variants and type 2 diabetes

The present study revealed a significant association of MTHFR C677T single-nucleotide polymorphisms (SNP) with type 2 diabetes (Chi-square test = 7.743, P = 0.02) where its major genotype “CC” is associated (and not the minor allele) with type 2 diabetes (odds ratio [OR] =3.78, 95% Confidence interval = 1.14–12.45) in the population studied [Table 2]. Whereas, MTHFR A1298C was not associated with type 2 diabetes (Chi-square test = 2.264, P = 0.322) [Table 3].

Table 2

Allelic and genotypic frequencies of methylenetetrahydrofolate reductase C677T in type 2 diabetes cases and controls

Frequencies	T2D cases	Controls
Allele	108	112
C	103 (95.37)	99 (88.39)
T	5 (4.62)	13 (11.60)
χ2	3.563	Reference
P (df=1)	0.059	Reference
Genotype	54	56
CC	50 (92.6)	43 (76.78)
CT	3 (5.55)	13 (23.21)
TT	1 (1.85)	0 (0.00)
χ2	7.743	Reference
P (df=2)	0.02*	Reference
CC versus (CT + TT)
OR	3.78	Reference
95% CI	1.14-12.45	Reference

OR: Odds ratio, CI: Confidence interval, T2D: Type 2 diabetes

Table 3

Allelic and genotypic frequencies of methylenetetrahydrofolate reductase A1298C in cases and controls

Frequencies	Cases	Controls
Allele	106	102
A	41 (38.68)	49 (48.04)
C	65 (61.32)	53 (51.96)
χ2	1.855	-
P (df=1)	0.173	-
Genotype	53	51
AA	9 (16.98)	15 (29.41)
AC	23 (43.39)	19 (37.25)
CC	21 (32.00)	17 (33.33)
χ2	2.264	-
P (df=2)	0.322	-

Association analysis between methylenetetrahydrofolate reductase C677T and A1298C variants and Prakriti-type

No association was found between any of the Prakriti - types and MTHFR C677T as well as A1298C variants [Table 4].

Table 4

Allelic and genotypic frequencies of methylenetetrahydrofolate reductase C677T and A1298C in cases and controls of two Prakriti groups

Frequencies	Case		Control
	K/KP	Non-K/KP	K/KP	Non-K/KP
C677T
CC	41	9	10	33
CT	2	1	2	11
TT	1	0	0	0
χ2	0.673	-	0.367	-
P (df=2)	0.714	-	0.544	-
A1298C
AA	8	0	4	11
AC	19	5	4	15
CC	13	6	3	14
χ2	3.333	-	0.388	-
P (df=2)	0.189	-	0.824	-

Discussion

Methylenetetrahydrofolate reductase C677T and A1298C variants and type 2 diabetes

Being a multifactorial disease, type 2 diabetes is caused due to more than one genetic risk factors together with the effect of extrinsic factors. So far, there have been several genetic risk factors identified for the type 2 diabetes and still being unravelled.

The frequency of T allele of MTHFR C677T observed in type 2 diabetes cases was lower than that of normal controls. Although this difference was not significant (P = 0.059). Other studies on Indian populations have also documented the lower frequency of this allele.[14151617] The frequency of MTHFR gene mutation is quite variable in different geographic and ethnic groups. The genotype frequency of MTHFR C677CC described in the different populations fluctuates between 40% and 49% as in type 2 diabetes cases of Turkey (49%), Tunisia (45%), Brazil (46%) and China (44%).[8161718] In the present study, the CC frequency was 92.6%.

There are a very few studies on MTHFR and type 2 diabetes.[1819] Variable results are found for the association of MTHFR C677T with type 2 diabetes. This variability could be due to population-specific genetic variations and effect of their environment. Most of the studies have shown T allele as a risk for type 2 diabetes in Caucasian populations[20] and found a significant association between MTHFR C677T polymorphism and type 2 diabetes in Moroccan population with a significant difference in the T allele frequency between the diabetic and control groups (26.06% vs. 33.20%, respectively). However, this group also did not find an association between A1298C polymorphism and type 2 diabetes. Lunegova et al. studied among ethical Kirghizes that the frequency of CT and TT genotypes was significantly higher in patients with insulin resistance than in controls (P = 0.027).[21] 677T allele was also associated with obesity, hypertriglyceridemia and low level of high-density lipoprotein cholesterol. A study from Taiwanian population has shown that T is not a risk to type 2 diabetes. No significance difference in the distribution of MTHFR genotypes between healthy and type 2 diabetes individuals is found in Taiwanese patients.[11] Besides, no significant associations between lipid/glucose metabolic indices with MTHFR genotypes among diabetic patients are observed. Whereas similar to results; a study from Indian population has shown that CC is a risk for type 2 diabetes.[19] This suggests that probably CC has different effects depending on other genetic modifiers and extrinsic factors.

Prasher B and others observed that individuals from the three (Vata, Pitta and Kapha) most contrasting constitutional types exhibited striking differences with respect to biochemical and hematological parameters and at genome-wide expression levels.[22] They also reported that biochemical profiles such as liver function tests and lipid profiles and hematological parameters like hemoglobin levels exhibited differences between Prakriti types. Thus, they concluded that Ayurveda-based method of phenotypic classification of extreme constitutional types may be utilized to uncover genes that may contribute to physical, physiological and psychological differences in normal individuals and their susceptibility to the diseases.

MTHFR A1298C shows variable results in different studies. In the present study, it was not associated with type 2 diabetes.

Prakriti and type 2 diabetes

In the present study, an extremely strong association between Prakriti (Kapha/Kapha-Pittaja) and type 2 diabetes (P < 0.00001) was detected. This is the first study at the molecular level that confirms the Kaphaja Prakriti individuals are highly prone for diabetes. There is one more report confirming the association of Prakriti with clinical phenotypes (cardiovascular risk factors, inflammatory markers, and insulin resistance).[23] Mahalle et al. and the present study gives a strong clue for the association of Prakriti (body constitutional type) and clinical phenotype.[24] This will strongly help in the prevention and management of type 2 diabetes. This could be because these variants may not contribute to the constitution of Prakriti type, at least in this population. It is further required to test the association of Prakriti and genetic make-up at the whole genome level.

Conclusions

In present study, the major ‘C’ allele showed an association with type 2 diabetes in the present Indian population studied. However, no significant association was found between another SNP MTHFR A1298C and type 2 diabetes in this study. MTHFR variants did not show association with the Prakriti in the present study.

Limitations and further scope

The analysis on larger sample size from other Indian cohorts on type 2 diabetes is required to confirm the association findings. The inclusion of samples from other cohorts of Indian populations should also be incorporated in further studies on larger scale.

The present study will provide a way to further analyze the association status of MTHFR SNPs with type 2 diabetes as well as Prakriti.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.