Frequency of I148M Polymorphism of the PNPLA3 Gene Associated with a Risk of Steatosis and Liver Fibrosis in Residents of the Republic of Sakha (Yakutia)

1 Department of Infectious Diseases, Phthisiology and Dermatovenerology of the North-Eastern Federal University, Institute of Medicine, Yakutsk, Russia 2 Department of Surgery of the North-Eastern Federal University, Institute of Medicine, Yakutsk, Russia 3 Department of Normal and Pathologycal Physiology of the North-Eastern Federal University, Institute of Medicine, Yakutsk, Russia 4 Department of Pediatrics and Pediatric Surgery of the of the North-Eastern Federal University, Institute of Medicine, Yakutsk, Russia 5 Research Center of the North-Eastern Federal University, Institute of Medicine, Yakutsk, Russia. 6 Research Laboratory "Molecular Medicine and Human Genetics" of the NorthEastern Federal University, Institute of Medicine, Yakutsk, Russia. 7 Department of Public Health and Nursing Norwegian Norwegian university of science and technology, Norway Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 14 July 2021 doi:10.20944/preprints202107.0330.v1


Introduction
Hereditary diseases and congenital malformations make a significant contribution to the morbidity, disability and mortality of people, being not only a medical, but also a social problem.
The successes of modern genetics are associated with the study of multifactorial diseases. The study of genetic polymorphism was crucial in determining the causes of hereditary changes. Genetic polymorphism is a change in the genome of a population in two or more variants (alleles) with a frequency of at least 1%. Single nucleotide polymorphism (SNP) are the most common type of genetic polymorphism. SNP can significantly affect the expression activity of genes and the level of protein production (mRNA stability, transcription rate, changes in the structure of transcription factor binding sites). Researching the role of SNP in the occurrence of diseases is an important and urgent task, as it allows to predict the risk of pathology, its severity and outcome.
Purpose of the study: study of the frequency of I148M polymorphism of the PNPLA3 gene in residents of the Republic of Sakha (Yakutia), associated with a high risk of steatosis and liver fibrosis. The study showed that the frequency of the mutant allele of functional polymorphism I148M of the PNPLA3 gene in the Yakut population was higher than in other known world populations.  Inclusion in the study control group (n=1425) required that all individuals belong to the Yakut, Even or Russian ethnic group, were healthy and unrelated.

Blood samples
Patients with non-alcoholic fatty liver disease (NAFLD) (n = 97), chronic hepatitis B and chronic hepatitis C (n = 39) were included in the study. All of them are living in the above areas. Verification of NAFLD, chronic hepatitis B and chronic hepatitis C was carried out using clinical data, laboratory studies, ultrasound studies and serological blood tests (ELISA). Note: Group 1-1 -patients with signs of NAFLD (n = 120); Group 2-2patients with signs of diffuse changes in the liver parenchyma without NAFLD (n = 120); Group 3-3 -patients with signs of diffuse changes in the pancreatic parenchyma (n = 395). All individuals provided informed consent before the study was performed.
Blood samples were collected in sterile VACUETTE ethylenediaminetetraacetic acid tubes (Greiner Bio-One, Austria).

Genotyping methods
Genomic DNA was isolated from peripheral blood by phenol-chloroform extraction, ExtractDNA commercial kit (Evrogen, Russia) and kits for isolating genomic DNA from blood using magnetic particles "NucleoMag 96 Blood" (Macherey-Nagel, Germany). The purified DNA was eluted in Milli-Q™ water.
The concentration and purity of extracted DNA was determined by measuring the optical density using Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA).

Statistics
Statistical analysis was performed using the software STATISTICA 7.0 (StatSoft Inc.

Results and discussion
Several authors have identified the association of NAFLD with the PNPLA3 gene and confirmed in different ethnic groups as the cause of the disease [1,2,3]. So, in a study by Romeo et al. (2008) [5] identified the association of the PNPLA3 gene with the development of NAFLD among Americans.
The most significant polymorphism in the PNPLA3 gene is I148M (rs738409). The I148M polymorphism is the replacement of the cytosine nucleotide with guanine, which leads to a change in the amino acid isoleucine to methionine at position 148. This replacement leads to a disruption of the mechanism of lipid metabolism in the liver. This polymorphism is associated with susceptibility to NAFLD and affects the histological picture and the development of fibrosis in children and adolescents with obesity [3]. The frequency of the studied polymorphism in various world populations is presented in Table 2.  Table 3.   (Table 4).  Notes: 1 G -guanine (guanine), C -cytosine (cytosine); 2 χ 2 -"chi" square Hardy-Weinberg equilibrium; 3 OR (odds ratio) -odds ratio; 4 CI (confidence interval) -confidence interval.
It is known that the frequency of allelic variants in a population may vary depending on ethnicity. In the present study, it was found that in the Yakut population the carriage of the GG genotype (49%) of the PNPLA3 gene I148M polymorphism predominates.
We analyzed the frequency distribution of genotypes and alleles of the  (Table 6). Notes: G -guanine (guanine), C -cytosine (cytosine).
For the first time, the association of the polymorphic variant I148M of the PNPLA3 gene with fatty liver infiltration was established in 2008 [4]. Since then, many studies have been conducted to establish the association of this polymorphism with NAFLD [1,3,13,14,15,16]. The association remained significant even despite adjustments for body mass index, diabetic status, alcohol use, and the absence of dyslipidemia [17,18,19]. In addition, it was found that this polymorphism is associated with increased serum levels of aminotransferase [4]. In a study by Seko et al. [19] it was found that the GG genotype is a predictor of the development of hepatocellular carcinoma in the Japanese population. In a previous study, it was shown that homozygous carriers of the GG genotype were characterized by an increased risk of steatohepatitis  [22]. In a study by Lee et al. (2014) [7] it was shown that the frequency of the G allele (31.6%) in the group of patients with NAFLD was significantly higher than in the control group (20.1%). It was also found that the frequency of GC + GG genotypes among NAFLD patients was significantly higher in patients with advanced fibrosis.
In this study, we performed genotyping of patients with NAFLD of Yakut nationality. The results of the comparison of the I148M polymorphism of the PNPLA3 gene in a group of relatively healthy individuals and patients with NAFLD of Yakut nationality are presented in Table 7. Notes: 1 G -guanine (guanine), C -cytosine (cytosine); 2 χ2 -"chi"square; 3 OR (odds ratio) -odds ratio; 4 CI (confidence interval) -confidence interval When conducting a comparative frequency analysis, there were no statistically significant differences between the control group and the group with NAFLD patients (p = 0.82).
In a study by Jiang et al. (2014) [23] it was found that the frequency of the G allele in the Chinese population (Qingdao, China) has significant differences between the group with patients with chronic hepatitis B (31.9%) and the group with conditionally healthy individuals (21.9% ). Carriers of the GG genotype of  Table 8. Ezzikouri [30 ] Notes: G -guanine (guanine), C -cytosine (cytosine) In this study, we performed a comparative frequency analysis of the distribution of genotypes and alleles of the I148M polymorphism of the PNPLA3 gene in the control group and the group of patients with chronic hepatitis B and C. In this analysis, we did not reveal significantly significant differences (p = 0.45) ( Table 9). Notes: 1 G -guanine (guanine), C -cytosine (cytosine); 2 χ 2 -"chi" square; 3 OR (odds ratio) -odds ratio; 4 CI (confidence interval) -confidence interval.
Thus, in the present study, it was found that the frequency of the mutant allele of functional polymorphism I148M of the PNPLA3 gene is higher than in other known world populations. A normally functioning PNPLA3 gene protein regulates the activity of triglyceride hydrolase and lysophosphatidic acid acyltransferase.
I148M polymorphism leads to the replacement of the amino acid isoleucine with methionine at position 148, which does not affect the orientation of the catalytic dyad, but the longer side chain of methionine limits the substrate access to the catalytic serine at position 47 [31]. The size of the access site to the substrate is significantly reduced in the presence of the mutant G allele, which leads to limited access of palmitic acid to the catalytic dyad [32]. In a study by Kumari et al. (2012) [33] it was found that the P PNPLA3 I148M polymorphism induces an increase in lipogenic activity, which leads to an increase in the synthesis of triglycerides in the liver. Pollorphism I148M of the PNPLA3 gene has three effects on the metabolism of triglycerides in the liver: increased synthesis of fatty acids and triglycerides; violation of the hydrolysis of triglycerides; a decrease in the level of polyunsaturated fatty acids [34]. Therefore, it can be assumed that the high frequency of the mutant G allele of the I148M polymorphism of the PNPLA3 gene in the Yakut population may be one of the reasons for the violation of the mechanism of lipid metabolism in the liver.
It is now recognized that the prevalence dynamics of NAFLD is an epidemic.
The studies revealed the presence of regional fluctuations in the level of detection of NAFLD. Apparently, this disease has genetic determinism, that is, it depends on ethnic factors. The PNPLA3 gene I148M polymorphism is associated with susceptibility to NAFLD, chronic hepatitis B, chronic hepatitis C. The presence of a mutant allele affects the histological picture, the development of steatosis, and liver fibrosis. It is likely that due to the high frequency of the mutant G allele of the I148M polymorphism of the PNPLA3 gene, the Yakut population will be more susceptible to the above pathological conditions. However, in this study, we did not establish an association between the control group and groups with sick liver diseases (NAFLD, chronic hepatitis B, chronic hepatitis C).
In view of the fact that the G14 allele of PNPLA3 gene polymorphism I148M is associated with an increased risk of developing liver diseases and is a marker of the progression of the pathological process, it is necessary to conduct a thorough and detailed scientific study of this association among the indigenous population of the Republic of Sakha (Yakutia). The establishment of this polymorphism as a marker indicating the risk of adverse development of the disease will allow for effective preventive and therapeutic measures.

Conclusion
The study showed that the frequency of the mutant allele of functional polymorphism I148M of the PNPLA3 gene in the Yakut population was high, as in other world populations.
Therefore, it can be assumed that the high frequency of the mutant G allele of the I148M polymorphism of the PNPLA3 gene in the Yakut population may be the cause of the hereditary pathology of lipid metabolism in the liver. It is likely that due to the high frequency of the mutant G allele of the I148M polymorphism of the PNPLA3 gene, the Yakut population has a high predisposition to the development of steatosis, liver fibrosis, especially in combination with chronic viral hepatitis B, C, D.

Acknowledgments
We thank all patients and blood sample donors who have contributed to this study. This study, "A multivariate study of the health status of the indigenous and newcomers of the Republic of Sakha (Yakutia) in order to optimize regional programs to improve the quality of life of the republic's residents, taking into account territorial, ethnic characteristics in the context of modern socio-economic development", was supported by the Russian Foundation for Basic Research (grant# 18-415-140001 р_а, grant#18-05-60035_Arctica).

Ethics Statement
All written informed consent forms signed by the participants or the guardians of the underage participants involved in our study were obtained before beginning the testing procedures. This study was approved by the local Biomedical Ethics Committee of North-Eastern Federal University, Yakutsk, Russia (Yakutsk, Protocol No 11, September 18, 2017).