ABSTRACT
Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases. It has been reported that specific variants of patatin-like phospholipase domain-containing 3 (PNPLA3) gene, notably SNPs rs738409 and rs2294918 are associated with high risks of liver disease. PNPLA3 rs738409 polymorphism is the main determinant of fatty liver and affects development and progression of NAFLD. rs2294918 is another SNP localised in PNPLA3 gene, it is associated with reduced expression of the PNPLA3 protein, lowering the effect of the rs738409:G variant on predisposition to steatosis and liver damage. The frequencies of alleles, genotypes, haplotypes and diplotypes (combinations of genotypes at two loci) of polymorphic variants of the PNPLA3 gene (rs2294918 and rs738409) were studied in the cohort of Yakuts (n = 150) living in the Republic of Sakha (Yakutia). Genotyping of PNPLA3 (rs738409 and rs2294918) was performed by PCR-PDRF method. The single nucleotide polymorphism rs738409 (I148M) of the PNPLA3 gene in the Yakut population is characterised by a high frequency of the risk allele G (72%). Analysis of the distribution frequency of the rs2294918 polymorphism genotypes showed that the allele G was predominant in 89.3% of individuals of the studied group of Yakuts. In this study, we identified two major diplotypes [GG][GG] and [CG][GG]. The high frequency of the mutant rs738409: G variant in Yakuts may be an adaptation of the organism to low temperatures. The study of the adiponutringene may be an important key to understanding the mechanisms of adaptation to low temperatures and metabolic processes in the indigenous population of the North.
KEYWORDS:
Introduction
Yakutia is the coldest region of Russia, which occupies the major part of the country’s northeast. The climate of Yakutia is sharply continental with an amplitude of air temperature fluctuations exceeding 100°C (in winter −60°C and in summer reaching +40°C). Over centuries, indigenous people have adapted to living in such harsh climatic conditions [Citation1]. But in the modern era, the majority of people are living in warm houses, dress in warm clothes and their bodies are minimally exposed to the cold. In addition to that, the development of agriculture and road communications has changed the diet of the indigenous people of Yakutia. Just a few generations ago, the basis of their diet was primarily natural products rich in protein and lipids (meat, fish, dairy products). However, these days, the basis of the diet is mostly carbohydrate-rich products, such as potatoes, pasta and flour products, rice, buckwheat, etc.
Until recently, the indigenous people considered traditional fatty food to be beneficial for the body and keeping warm, but at the moment, this food has become the main cause of various metabolic diseases, including type 2 diabetes mellitus, atherosclerosis, non-alcoholic fatty liver disease (NAFLD) and others [Citation2].
NAFLD is characterised by changes in liver tissue due to excessive deposition of fatty droplets in hepatocytes. If before industrialisation period and the use of modern approaches of keeping heat in the body, under the exposure to the cold, this accumulated fat was converted into energy to generate heat, today it leads to various pathological changes in the liver.
Many foreign and domestic researchers indicate that the rs738409 polymorphism of the PNPLA3 gene is the main determinant of liver fat and affects the development and progression of NAFLD [Citation3]. Variant G (rs738409) of the PNPLA3 gene leads to the accumulation of triglycerides in hepatocytes. The rs2294918 polymorphism of the PNPLA3 gene reduces the expression of the PNPLA3 protein, decreasing the effect of variant G (rs738409) on the predisposition to steatosis and liver damage [Citation4].
Protein PNPLA3 belongs to the family of patatin-like phospholipases. It has hydrolase activity against triglycerides, catalyses the conversion of lysophosphatidic acid to phosphatidic acid. A mutation in the gene leads to the substitution of isoleucine for methionine at position 148 of the amino acid sequence, which leads to the loss of these functions and the accumulation of triglycerides and retinol palmitate in the liver [Citation5].
We have previously found a high frequency of the G allele (rs738409) of the PNPLA3 gene in the Yakut population (73%) [Citation6]. The aforementioned adaptation mechanisms to cold, such as the accumulation of fat in the liver, probably left their mark on the Yakut gene pool, in particular, in the genes that influence metabolism. In this regard, the purpose of our study was to study the frequency distribution of alleles, genotypes, haplotypes and diplotypes of polymorphic variants of the PNPLA3 gene (rs2294918 and rs738409) in Yakuts.
Materials and methods
Genotyping of the rs2294918 and rs738409 polymorphisms of the PNPLA3 gene was carried out in the laboratory of hereditary pathology of the Department of Molecular Genetics of the Yakutsk Scientific Center for Complex Medical Problems (YSC KMP). For the study, DNA samples of 150 healthy volunteers from the collection of biomaterials of the YSC KMP using the UNU “Genome of Yakutia” (reg. No. USU_507512) were used. This study involved participants who filled out a questionnaire indicating their genealogical information and voluntarily signed an informed consent to conduct a genetic study. The questionnaire was approved by the Local Committee on Biomedical Ethics at the YSC CMP. The inclusion criteria for the study were the absence of liver damage from chronic viral hepatitis; all subjects were excluded: autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hereditary haemochromatosis, Wilson-Konovalov disease; no alcohol abuse (>30 g/l). All participants in the study were ethnically Yakuts and lived on the territory of the Republic of Sakha (Yakutia).
The research protocol was in accordance with the ethical principles of the 1975 Declaration of Helsinki and was carried out in accordance with the recommendations of the Ethics Committee of the YSC KMP. Informed consent was obtained from each study participant.
DNA isolation from peripheral blood was carried out using a commercial kit from Newteryx, LLC (Yakutsk, Russia). Single nucleotide polymorphisms were determined by polymerase chain reaction (PCR) with subsequent analysis of restriction fragment length polymorphism (RFLP). The amplification of the SNP containing regions of PNPLA3 gene was performed using following primer pairs manufactured by Biotech-Industry, LLC (Moscow, Russia). rs738409 – forward primer: 5”-TGGGCCTGAAGTCCGAGGGT-3‘ and reverse primer: 5’-CCGACACCAGTGCCCTGCAG-3‘ (product length: 333 bp), rs2294918 – forward primer: 5’-CCTCTAAGCCAACTTCCTCC-3‘ and reverse primer: 5’-CCTCAAGTGACTCACAGACTC-3” (product length: 271 bp). The PCR reaction mixture contained 1 μL of forward and reverse primer, 10 pmol/μL; Dream Taq PCR master mix – 12.5 μL; deionised water 9.5 μL and sample DNA with the concentration of 100 μg/mL – 1 μL. The total reaction volume was 25 μL. The reaction mixture for RFLP with a total volume of 20 μL consisted of PCR product – 7 μL, deionised water – 10.9 μL, restriction buffer – 2 μL and 2 units of either BstF5 I restriction endonuclease (rs738409) or Ama87I (rs2294918). Interpretation of genotyping results was done based on different lengths of restriction fragments. The following band distribution was observed for the rs738409 polymorphism: CC genotype – 200 and 133 bp; CG genotype – 333, 200 and 133 bp; GG genotype – 333 bp, and for the rs2294918 polymorphism: AA genotype – 271 bp; AG genotype – 271, 160 and 111 bp; GG genotype – 160 and 111 bp.
PCR products were detected by 2% agarose gel electrophoresis with the addition of ethidium bromide, in a standard tris-acetate buffer in the electric field of ~20 V/cm for 30 minutes. RFLP products were visualised in a 4% agarose gel stained with ethidium bromide for 1 hour.
The correspondence of the distributions of genotypes to the expected values at the Hardy-Weinberg equilibrium and comparison of the frequencies of allelic variants/genotypes were performed using the χ2 test (chi-square). Statistical processing of the obtained data was done using Microsoft Excel 2010.
The haplotype frequency was determined using the EM algorithm. Linkage disequilibrium (LD) between SNP pairs was calculated using Lewontin’s D“ coefficient and Pearson’s r2 coefficient. Blocks of linkage disequilibrium were determined using the ‘Solid spine LD’ algorithm (D” > 0.75).
To compare the frequencies of alleles and genotypes of PNPLA3 rs2294918 and rs738409 of the studied Yakut population with those of other nationalities, we used data from the 1000 Genomes project open database (1000 Genomes Project Consortium, 2010) [Citation7]. Also, Haploview software (v4.2) [Citation8] was used to assess PNPLA3 haplotypes and frequencies based on genotyping data and to test the association between alleles and haplotypes of the PNPLA3 gene.
Results and discussions
The PNPLA3 gene in the Yakut population according to the rs738409 polymorphism is characterised by the highest frequency (72%) of the risk G allele compared to other previously studied groups (). According to the 1000 Genomes project data [Citation7], the highest frequency of the G allele was observed in populations of Central and South America (Peruvians – 71.8%, Mexicans – 55.5%, Colombians – 41%). Europeans have an average allele G frequency of 22.6%. Among Asians, the high frequency of the allele G is in the Japanese (41.8%). The owners of the lowest frequency of the allele G are Africans, on average 11.8%. In the population sample of Yakuts for rs738409, due to the shift of genotypes towards the homozygous GG genotype, a deviation from the Hardy-Weinberg equilibrium was revealed, which may be evidence of the accumulation of this genotype as an adaptation mechanism to a cold climate.
Table 1. Frequency of allele variants and missense mutations of the PNPLA3 gene in the Yakut population and in the populations of the 1000 Genomes project.
The analysis of frequency distribution of rs2294918 polymorphism genotypes according to the 1000 Genomes project [Citation7] revealed that the G allele is predominant in many human populations throughout the world. So, in the studied group of Yakuts, it was about 89.3%; in African populations – 91.3%, in the populations of Central and South America (Colombians, Mexicans, Peruvians and Puerto Ricans) – 78.8%, in the East Asian populations (Chinese, Japanese and Vietnamese) – 81.8%, in Europeans (Finns, British, Iberians, Tuscans and Utah residents of northern and Western European descent) – 62.9%, in South Asians (Indians and Pakistanis) – 77.2%.
The variant of PNPLA3 rs2294918 bearing the protective allele A, which suppresses the negative effect of rs738409, in Yakuts occurred only in 10.7% of the studied individuals. According to the 1000 Genomes project [Citation7], the protective allele A (rs2294918) is more common in European populations (32.3%). In representatives of the Negroid population from Barbados (ACB), Puerto Ricans (PUR) and the Indian population of Telugu from England (ITU), the frequencies of the risk allele G in SNP rs738409 and the protective allele A in SNP rs2294918 were close in value (13, 13%; 32, 31% and 25, 23% respectively). At the same time, in the populations of East Asia and America, the frequency of the risk allele G (rs738409) was significantly higher than the frequency of the protective allele A (rs2294918), while in the populations of Western Europe, a higher frequency of the protective A allele (rs2294918) was observed. The analysis of possible variants of haplotypes is performed according to .
Table 2. Possible haplotypes at two loci of the PNPLA3 gene.
A weak linkage disequilibrium (LD) was observed between two SNPs (D” = 0.096; r 2 = 0.003 in Yakuts) (). In other samples, strong linkage was observed D” = 1, r 2 = 0.015 in Africans, D” = 0.98, r 2 = 0.242 for Americans, D‘ = 1, r 2 = 0.12 for East Asians, D’ = 1, r 2 = 0.172 for Europeans and D” = 1, r 2 = 0.097 for southern Asians.
Figure 1. Linkage mismatch in the PNPLA3 gene.
The analysis of genotype frequency distribution in studied population of Yakuts demonstrated the predominance of the GG genotype (57.3%). Genotypes AA and AG carrying the protective allele A are more common in European populations (13.1% and 47.9%, respectively) ().
Table 3. The distribution of genotypes of PNPLA3 rs738409 and rs2294918 polymorphisms in Yakuts and some other ethnical groups according to the 1000 Genomes project.
The frequency distribution of PNPLA3 gene haplotypes for two SNPs (rs738409, rs2294918) based on all detected variants is presented in . We identified two major haplotypes, whose frequency was >0.1. One of the most common haplotypes carried variant G (148 M), the other carried variant C (148I), and both carried the same variant G (434E). In other words, the more common two haplotypes carry the G (434E) allele, while the A (434K) protective allele does not occur in the found main haplotypes. The protective allele A (434K) is shared by both rare haplotypes. Haplotype G-A (148 M-434K) was found only in Yakuts and Mexicans (6.9% and 1.1%, respectively).
Table 4. Frequency of I148M–E434K haplotypes in the studied group of Yakuts and in the populations from the 1000 Genomes project.
The diplotype frequency distribution for the two SNPs (rs738409–rs2294918) of the PNPLA3 gene based on all detected variants is presented in . Eight out of nine possible diplotype variants were found. The Yakuts often have two diplotypes [GG] [GG] and [CG] [GG]. Both carry allele G (rs738409) (45.3% and 28%) and do not carry the protective allele A (rs2294918). The same distribution of diplotype frequencies among Peruvians (52.9% and 24.7%), Mexicans (32.8% and 23.4%) and Japanese (22.1% and 32.7%). Diplotypes carrying the protective allele A (rs2294918) are found with a low frequency (). The [GG] [AA] and [CG] [AA] diplotypes were absent in all 25 populations, with the exception of the [GG] [AA] diplotype found in the Yakut population (1.3%). Among the seven detected diplotypes in the Yoruba tribe, [CC] [GG] was more common (63.9%). This diplotype does not carry the pathological allele G (rs738409) and does not carry the protective allele A (rs2294918).
Table 5. Distribution of diplotypes for two SNP markers of the PNPLA3 gene in the Yakut population and in the populations of the 1000 Genomes project.
NAFLD is one of the most common chronic liver diseases in the world, although accurate data on its prevalence and incidence do not exist due to its asymptomatic course and different diagnostic criteria. In Russia, according to the results of the DIREG 1 study (2007), the prevalence of NAFLD in outpatients was 27%, while 80.3% had steatosis, 16.8% had steatohepatitis, and 2.9% had liver cirrhosis. The results of the D1REG 2 study (2015) showed that the prevalence of NAFLD was already 37.3% [9]. There are no data on the prevalence of NAFLD in Yakutia. The rs738409 polymorphism of the PNPLA3 gene is a major determinant of fatty liver and predisposes to a wide spectrum of liver damage in NAFLD. Many researchers have concluded that the rs738409: G variant can increase the development of non-alcoholic fatty liver disease while increasing serum ALT levels [Citation2,Citation4,Citation5]. In their studies, Donati, Motta, Pingitore, et al. (2016) found that carriers of the allele A (rs2294918) have lower levels of the PNPLA3 protein in the liver (P < 0.05), thereby, this allele prevents the negative effects of the allele G (rs738409) [Citation4].
Thus, in all samples of African origin, among the detected diplotypes, [CC] [GG] is more common, which does not carry the pathological allele G (rs738409) and does not carry the protective allele A (rs2294918). An interesting fact is the absence of the [GG] [AA] and [CG] [AA] diplotypes in all studied 25 world populations, except for the Yakuts in whom we found the [GG] [AA] diplotype with a frequency of 1.3%. This diplotype may be found in the Yakut population due to the high prevalence of carriers of the homozygous GG variant (rs738409).
In the studied group of Yakuts, two diplotypes [GG] [GG] and [CG] [GG] were more common. These diplotypes carry the mutant allele G (rs738409) and do not carry allele A (rs2294918), which has a weakening effect on 148 M [Citation4], which in turn promotes the accumulation of triglycerides in hepatocytes [Citation5]. Perhaps in the Yakuts, the accumulation of fat in the liver in the past did not lead to NAFLD, since the accumulated fat was quickly converted into energy to generate heat. In modern realities, this diplotype has its detrimental effect by increasing the frequency of metabolic diseases, including NAFLD.
Conclusion
The current study of the PNPLA3 gene in Yakut population has demonstrated that the frequency of the mutant allele of the PNPLA3 rs738409 was higher compared to other known world populations. According to the PNPLA3 rs2294918 polymorphism, which suppresses the negative effect of rs738409, the occurrence of protective allele A in the Yakut population was only 10.7%. Adiponutrin, which is the product of the PNPLA3 gene, normally regulates the activity of triglyceride hydrolase and lysophosphatidic acid acyltransferase. This results in the accumulation of triglycerides in liver cells, but reduces the release of very low-density lipoproteins (VLDL) into the circulatory system. Low lipid concentrations in the blood can reduce lipid deposition on the walls of blood vessels. There is a natural reaction of the organism to the cold, the so-called “peripheral vasoconstriction”, which consists in keeping the internal body temperature by constricting the blood vessels. Therefore, it can be assumed that the high frequency of the mutant rs738409: G variant in Yakuts may be an adaptation of the organism to the cold climatic conditions. The study of the adiponutrin gene may be a key to understanding the mechanisms of adaptation to low temperatures and metabolic processes in the indigenous population of the North.
Acknowledgments
This study was carried out within the project “Physiological and biochemical mechanisms of adaptation of plants, animals, humans to the conditions of the Arctic/Subarctic and the development of biological products based on natural northern raw materials that increase the efficiency of the adaptation process and the level of human health in extreme environmental conditions” (No. 0297-2021-0025 registration number АААА-А21-121012190035-9) and also was part of the research “Study of the genetic structure and burden of hereditary pathology of populations of the Republic of Sakha (Yakutia)” held by Yakutsk Scientific Center for Complex Medical Problems.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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