A GENETIC ANALYSIS OF PNPLA3 I148M VARIANT IN ULTRASOUND PROVEN STAGES OF LIVER DISEASES

 Nonalcoholic Fatty liver disease (NAFLD) is the range of disorders caused by accumulation of fats in the liver globally. It is commonly perceived in people whose are bulky and obese. The starting phases of NAFLD have insignificant quantity of the fats in liver and sometimes it can causes severe liver impairment, together with cirrhosis. Domain containing patatin like phospholipase 3(PNPLA 3) gene exhibits metabolic (anabolic or catabolic) events in lipid metabolism. PNPLA3 gene and its variant rs G are being declared amongst candidate disease markers and hepatic lipid content. The influence of numerical and physical characteristics of PNPLA3 Isoleucine to methionine substitution on liver will be analyzed as it will reveal the part of the PNPLA 3 mutation in progress of nonalcoholic fatty liver diseases. For this purpose, a questionnaire based data and blood samples will be collected from patients and healthy controls for DNA extraction and PCR based genotyping of rs58542926 variant. About 250 adults having NAFLD will be taken from gastroenterology clinic at the Holy Family Hospital Islamabad between August 2018 and December 2018. Genomic DNA will be isolated from WBC through DNA Kit in EDTA. The outcome of this study will contribute in understanding function of the PNPLA3 in the hepatic disorder development.  The study will help to estimate C/G allele frequency of rs738409, identification of risk allele and its association with disease phenotypes in Pakistani population.

                                                                                                                   

INTRODUCTION

               The medical course of severe fatty liver disease is inconstant and the genetic aspects play an important part in defining inter individual predisposition to incurable fatty liver impairment. A number of metabolic complications especially diabetes, cardiovascular disease, metabolic syndrome, and obesity could contribute to a NAFLD. Many genetic factors are being considered responsible for NAFLD development. Due to the mutation of these hereditary components, it can cause permanent liver damage or inflammation of the liver. Despite the fact that case-control studies based on initial hypotheses have identified heritable constituent related to  development of liver lesions, the heritable component of nonalcoholic liver disease have persisted doubtful until now (Dongiovanni et al., 2013).

               Early genomic findings have identified rs738409, an impulsive method of primary heritable factor of liver fat concentration. (Yuan et al., 2008). This polymorphism is the simple Cytosine to guanine substitution which encodes for amino acid isoleucine by varying methionine at position 148 of PNPLA3. Central objective of research is to offer insights into current knowledge about part of PNPLA3 isoleucine to methionine polymorphism in progression of the fatty liver disease (Romeo et al., 2008).

               Single nucleotide polymorphism of I to M in PNPLA3 is related to development of the NAFLD. It is associated with hepatic fats build up or accumulation which causes damage similar to that caused by hepatic impairment from alcohol. NAFLD happens in the individuals who are not consuming many alcohols. In the certain cases, NAFLD causes liver burns and liver damage caused by prolonged liver injury (Guerrero et al., 2009).

               The PNPLA3 variant associated with NAFLD encoding isoleucine for substitution of methionine at position 148. Protein studies suggest that mutated amino acid causes greater than before lipogenesis and lesser lipolysis of the fats in liver. Findings and research are underway to define that genetic factors are associated with progression of NAFLD. Epidemiologic, family and twin studies give indication of the factor of heritability in fatty liver disease (Sookoian, and Pirola, 2011).

               In 2008 two genome independent association linked protein polymorphism rs738409 PNPLA3 (I148M) with liver fat content.  In particular, an analysis of all the genome association variations of the non-synonymous sequences in  Heart Study established strong link between the hepatic fat content and single nucleotide polymorphism of isoleucine to methionine in PNPLA3 (Guerrero et al., 2009). The variant type 3 gene PNPLA3 develops the fatty acids droplets of hepatocytes. It produces low density lipoprotein and is main factor of the inter individual differences and the related ethnic origin regarding content of liver fat. Mutation to PNPLA3 causes changes in lipid catabolism of deterioration, makeover of lipid droplets and VLDL secretion. More prominently in the individuals of  stressors of the variant gene I148M different diagnosis are  seen such as  abdominal pain due to excessive fats,  hepatitis due to  severe viral infection  and obesity  triggers liver diseases and impairment.

PNPLA 3 gene which encodes 481  amino acids, having  molecular weight of about 53 kDa acid protein in humans expressed mainly in the membranes present inside the liver cells. In humans, PNPLA3 is the protein located in endoplasmic reticulum and expressed on the exterior of the lipid droplets. Its maximal expression is found in the hepatic stellate cells and the hepatocytes.  22q13.31 their cytogenetic location, in which q is the long arm of the chromosome 22 and its position 13.31, 43923739-43947568 are base pairs on chromosome 22.  PNPLA3 has triglyceride activity and retinyl palmitate esterase. Wild-type of the PNPLA3 contains isoleucine at 148 position shows lipolysis action against fats (Li et al., 2013). 

                    The 148M mutation causes essential amino acid substitution by the catalytic domain, such as reduced PNPLA3 enzyme activity for lipids, leading to the development of non-alcoholic steato hepatitis. (Pirazzi et al., 2014). The worldwide occurrence of NAFLD is described up to 25.24% whereas in Asian countries it is present between 15–45%.  World Gastroenterology Organization Global Guidelines 2014 reported that NAFLD occurrence proportion in Pakistan is about 18% and increasing with time rapidly. In this research we are going to genotype Pakistani subjects diagnosed with simple steatosis, NASH, and advanced liver complications resulting either from metabolic complications or from Hepatitis B or C infection with following objective. The main objectives of this study:

Ø  Genotyping of PNPLA3 variant rs738409 C>G in metabolic and infectious liver disease patients and healthy controls.

Ø  Identification of the risk allele of variant rs 738409 C>G and its association with metabolic and infectious liver disease phenotypes.

REVIEW OF LITERATURE

               NAFLD has some metabolic risks and association with hyperglycemia, obesity, dyslipidemia and T2D with the prevalence of about 25.23%. In west it is about 30 -35 % by the leading cause of death, it is mainly due to sluggish lifestyle and higher usage of fats in the diet without any exercise. Many studies of genome association polymorphisms have been indicated some genes that showed susceptibility to the NAFLD including PNPLA3 and TM6SF2. According to the signature of the W.WRUCK PNPLA3 gene had contributed to the progression of many metabolic complications especially NASH, fibrosis, cirrhosis and then HCC. He explained models of the selected patients who had differentiated pluripotent cells in the liver. These cells were stimulated by 18 carbon monounsaturated oleic acid that provided the main concept of mechanisms which was involved in the etiology of NAFLD (Wruck et al., 2017).

               NAFLD is a prolonged liver disorder whose occurrence rate is increasing very rapidly and has extended to 25.24% worldwide. It has been observed that in early stages the disease is gentle or benign and normally causes no harm but when it progresses to worsen stages such as NASH, fibrosis, cirrhosis and HCC it can lead to the severe liver damage or even death of the patient. Early genome related studies  and research has provide the evidence that  Genetic components, environment stress and increasing body are the complex processes that play important role in the development of the NAFLD. Researchers mainly focused on genetic susceptibility by the particular focus has been given to background family history of this disease and influence of hereditary factors in pathogenesis of this disease, together with they also discussed and  used many  genetic analysis  to identify the  lean or thin people who are having NAFLD  (Rotman et al., 2010).

               Single nucleotide polymorphisms (SNP) through genomic investigations and studies have been identified to relate with high fats in the liver fat or higher hepatic enzyme associated with the NAFLD possibly. For the determination of histological severity of this disease and its relationship with SNPs researchers have been performed many experiments. For this purpose in the genome enzyme association research about 1117 selected patients of NAFLD including 894 adults and 223 children were enrolled in the clinical Research and National Institute of Health Clinical Center where NAFLD patients’ genomes were sequenced for the 6 SNPs. These 6 SNPs had association with hepatic fats or liver. Out of these 6 SNPs, 3 on the chromosome number 22 showed their susceptibility for NAFLD. One SNP rs738409 cytosine to Guanine mutation which is non-synonymous coder of the PNPLA3 protein which coded for isoleucine to Methionine substitution at position 148, was identified as the marker of NAFLD and steatosis with the P value (P5 0.03), the site of inflammation with P value (2.5 2.5 1024), Denk M’s organs with the P5 value of (0.015) and also NAFLD activity score (NAS) with the p value of (0.004). Same relationship has been shown by the remaining 2 SNPs locating on the same 22 chromosome (Wruck et al., 2017).

               Remaining 3 SNPs out of 6 SNPs, on the chromosome no 10 close to CHUK (conserved helix loop helix ubiquitous kinase) gene has been shown their association for fibrosis with the P value less than 0.010. No SNPs to be associated with NAFLD or histological severity were found in children but multivariate analysis of P value 0.045 showed that rs 738409 SNP was found in adolescent at phase of liver biopsy. In this research it had been concluded that steatosis and histological severity had allelic SNP rs738409 G and children would  perceived the disease and had the rs738409 G SNP in case of early onset of NAFLD. They have been demonstrated the relationship between the previously unknown SNP locus on the chromosome number 10 and also fibrosis severity in NAFLD. (Wruck et al., 2017).

               NAFLD end stages conditions are dangerous especially cirrhosis fibrosis and HCC. When fat synthesis, its consumption and its secretion are imbalance, it would lead to liver fat accumulation and also it is the source of fatty acids present in liver. Many studies provide the evidence which supports the concept that too much liver TGs are derived due to increased insulin resistance in the adipose tissues .because insulin resistance causes lipolysis in these tissues likewise due to more consumption of fats in diet or hyperinsulinemia liver synthesizes more fats. In fact, the main contributor to NAFLD, metabolic disorders and CVD is the Insulin resistance because liver is aggravated for insulin resistance by the steatosis itself. Secretion of hepatocellular TAGs across VLDLs and their use in mitochondria reduced due to damage in VLDLs and mitochondria has been involved in the liver fats accumulation. (Sookoian, and Pirola, 2011).

               This study would carried out sampling out and target population will be included in the areas of Rawalpindi and Islamabad (hospitals).  Sampling will be made on the basis of the next obese child, obese adult men / women and obese men / women. The data will be collected through the use of these techniques, such as the questionnaire, interview, surveys, documents and files. The genome DNA will be extracted from EDTA blood using the mini blood kit. The PNPLA3 polymorphism rs 738409 will be determined by real-time PCR. PCR is a way to amplify a specific part of the DNA. PCR can generate many copies of a template DNA fragment within PCR reaction repeats the next steps upto 20 to 30 times.