Association of Nicotinamide- N -Methyltransferase Gene rs694539 Variant with Patients with Nonalcoholic Steatohepatitis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal hepatic steatosis in the absence of a history of alcohol use and with a prevalence of 15%-45% in developed nations. Nonalcoholic steatohepatitis (NASH) is an advanced stage of NAFLD with a pronounced major inflammatory component. The aim of this study was to investigate the possible role of nicotinamide-N-methyltransferase (NNMT) gene rs694539 variant in the development of NASH. Therefore, we analyzed 80 NASH patients and 183 healthy controls using a polymerase chain reaction-restriction fragment length polymorphism method developed in our laboratory. The NNMT rs694539 variant was found to be significantly associated with NASH (χ²=9.349, p=0.009). The individuals with the GG genotype had protection against NASH (χ²=3.793, p=0.051, odds ratio [OR]=0.580, 95% confidence interval [CI]=0.334-1.006), whereas the individuals with the AA genotype showed statistically significant increased risk for NASH (χ²=7.748, p=0.005, OR=7.338, 95% CI=1.448-37.190). Moreover, the G allele was protective against NASH (χ²=7.748, p=0.005, OR=0.136, and 95% CI=0.027-0.691). On the other hand, the A allele was a risk factor for NASH (χ²=3.793, p=0.051, OR=1.725, and 95% CI=0.994-2.996). Consequently, the rs694539 variant of NNMT gene is a genetic risk factor for developing NASH.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal hepatic steatosis in the absence of alcohol abuse with a prevalence of 15%-45% in the developed countries. Nonalcoholic steatohepatitis (NASH) is the most progressive form of NAFLD with a pronounced major inflammatory component (Farrell et al., 2012).

Hyperhomocysteinemia is a multifactorial trait that is determined by multiple genetic and environmental factors (Carr et al., 2009; Malinowska and Chmurzynska, 2009). Methylenetetrahydrofolate reductase (MTHFR; EC1.5.1.20) is one of the key enzymes of one-carbon metabolism. The 677T and 1298C alleles of MTHFR, associated with elevated plasma homosysteine levels (Frosst et al., 1995), were found to be associated with NASH (Sazci et al., 2008b). Another enzyme implicated in one-carbon metabolism is nicotinamide-N-methyltransferase (NNMT), which methylates nicotinamide and other pyridine compounds in a reaction that transfers the methyl group released from S-adenosyl-methionine (SAM). As a result, SAM is converted into S-adenosyl-homocysteine (SAH), which is then further converted into homocysteine (Aksoy et al., 1994). Human NNMT (EC 2.1.1.1), expressed in a number of tissues, is primarily a liver enzyme that exists as a tetramer (Sano et al., 1992).

The NNMT gene, located on chromosome 11q23.1, is 16.703 bp in length and has three exons and two introns. The rs694539 NNMT variant is found at 114133419 bp (G>A transition) (dbSNP), is a single nucleotide polymorphism (SNP) that is found to be significantly associated with hyperhomocysteinemia (Souto et al., 2005; Bromberg et al., 2012) which causes steatosis.

To examine whether the rs694539 variant of NNMT gene was associated with NASH, we analyzed the allele and genotype frequencies of the NNMT gene rs694539 variant in 80 NASH patients and 183 healthy controls in a case-control study of Turkish subjects of Caucasian origin.

Materials and Methods

Genotyping

Genomic DNA was isolated from EDTA-treated whole blood. The allele and genotypes of the rs694539 variant of NNMT gene were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method developed in our laboratory (Fig. 1). The PCR cycling conditions for rs694539 variant were as follows: Briefly, genomic DNA was denatured at 95°C for 5 min followed by 35 cycles of 95°C for 1 min, annealing at 55°C for 30 s, synthesis at 72°C for 1 min, and a final extension step of 72°C for 10 min. The amplified 187 bp fragment was digested with NlaIII restriction enzyme at 37°C overnight. Subsequently, the digested fragment was run on an 8% polyacrylamide gel electrophoresis (PAGE) for 30 min at 20 W; thereafter, it was stained with silver and scanned (Fig. 1).

FIG. 1.

(A) A schematic illustration of the nicotinamide-N-methyltransferase (NNMT) gene showing the location of NlaIII recognition sequences (5′.CATG.3′) relative to the primers annealing sites. The forward and reverse primers depicted on the gene after amplification can create a fragment of 187 bp of which there is an NlaIII site at position of 114133419. (B) Given next is the polyacrylamide gel electrophoresis (PAGE) of NNMT gene. The amplified 187 bp fragment was cut with the restriction endonuclease NlaIII and run on an 8% PAGE at 20 W for 30 min followed by silver staining. Lane M showing the marker (M); lane GG showing the GG genotype with one fragment of 187 bp; lane GA showing the GA genotype with three fragments of 187, 106, and 81 bp; and lane AA showing the AA genotype with two fragments of 106 and 81 bp.

Human subjects

The demographic data for subjects were recently published (Sazci et al., 2008a). The subjects were 80 patients with NASH (41 women and 39 men with an age range of 18-66) and 183 healthy controls (95 women and 88 men with an age range of 19-65), matched for age, sex, ethnicity, and geographical area. These subjects were recruited from the University of Kocaeli Hospital, Gastroenterology Clinic, from May 2002 to May 2010. The mean age and standard deviation were 44.73±5.15 years in the patients and 44.25±5.85 years in the controls. All of the patients and controls were unrelated Turkish-Caucasian subjects. Patients with histologically confirmed NASH who had elevated liver aminotransaminases or negative serologic markers of viral or autoimmune hepatitis with no use of alcohol were studied (Sazci et al., 2008a). Healthy controls did not have any known disease or symptoms. Informed consent was obtained from each individual included in the study. The institutional review board approved the study.

Liver biopsy specimens were treated the way in which was suggested by Brunt et al. (1999), Kleiner and Brunt (2012). All laboratory studies were performed within 6 weeks of liver biopsy (Sazci et al., 2008a).

Statistical analysis

The Hardy-Weinberg equilibrium was verified for all of the tested populations. The relative risk as odds ratio (OR) analysis was carried out with 2×2 cross-tabulation and a binary logistic regression model for the age and sex. All statistics were performed with the SPSS v17 software package. A value of p<0.05 was taken as significant.

Results and Discussion

We report for the first time that the rs694539 NNMT gene variant was significantly associated with NASH (χ²=9.349, p=0.009). The individuals with the GG genotype had protection against NASH (χ²=3.793, p=0.051, OR=0.580, and 95% confidence interval [CI]=0.334-1.006). However, the individuals with the AA genotype showed significant association with NASH (χ²=7.748, p=0.005, OR=7.338, and 95% CI=1.448-37.190). The G allele showed protection against NASH (χ²=7.748, p=0.005, OR=0.136, and 95% CI=0.027-0.691), while the A allele was a risk factor for NASH (χ²=3.793, p=0.051, OR=1.725, and 95% CI=0.994-2.996). After the stratification of the NASH patients according to gender, both female and male genders equally showed an association with NASH (χ²=6.659, p=0.036; χ²=6.089, p=0.048, respectively). Our controls and cases were in Hardy-Weinberg equilibrium (p=0.38, p=0.36, respectively).

The A allele frequency of the NNMT gene rs694539 variant was 14% in overall controls and 24% in overall cases (Table 1). Among the Spanish, it was 15% (Souto et al., 2005).

Table 1.

Genotype and Allele Frequencies of NNMT rs694539 Polymorphism in Overall Patients with NASH and Controls, Female and Male NASH Patients and Controls

Gene	Cases	Controls	χ²	p-Value	OR; 95% CI
NNMTA	80 (100.0)	183 (100.0)	9.349	0.009
GG	48 (60.0)	132 (72.1)	3.793	0.051	0.580 (0.334-1.006)
GA	26 (32.5)	49 (26.8)	0.895	0.344	1.317 (0.744-2.330)
AA	6 (7.5)	2 (1.1)	7.748	0.005	7.338 (1.448-37.190)
Allele frequency
G allele	122 (0.76)	313 (0.86)	7.748	0.005	0.136 (0.027-0.691)
A allele	38 (0.24)	53 (0.14)	3.793	0.051	1.725 (0.994-2.996)
HWE (exact)	0.36	0.38

Gene	Cases female	Controls female	χ²	p-Value	OR; 95% CI
NNMTA	41 (100.0)	95 (100.0)	6.659	0.036
GG	23 (56.1)	73 (76.8)	5.936	0.015	0.385 (0.0177-0.840)
GA	16 (39.0)	21 (22.1)	4.140	0.042	2.255 (1.021-4.983)
AA	2 (4.9)	1 (1.1)	1.943	0.163	4.821 (0.425-54.718)
Allele frequency
G allele	62 (0.76)	167 (0.88)	1.943	0.163	0.207 (0.018-2.355)
A allele	20 (0.24)	23 (0.12)	5.936	0.015	2.597 (1.191-5.662)
HWE (exact)	1.0	1.0

Gene	Cases male	Controls male	χ²	p-Value	OR; 95% CI
NNMTA	39 (100.0)	88 (100.0)	6.089	0.048
GG	25 (64.1)	59 (67.0)	0.105	0.746	0.878 (0.398-1.936)
GA	10 (25.6)	28 (31.8)	0.492	0.483	0.739 (0.317-1.724)
AA	4 (10.3)	1 (1.1)	5.943	0.015	9.943 (1.073-92.112)
Allele frequency
G allele	60 (0.77)	146 (0.83)	5.943	0.015	0.101 (0.011-0.932)
A allele	18 (0.23)	30 (0.17)	0.105	0.746	1.139 (0.517-2.513)
HWE (exact)	0.09	0.45

HWE, Hardy-Weinberg equilibrium; OR, odds ratio; CI, confidence interval; NASH, nonalcoholic steatohepatitis; NNMT, nicotinamide-N-methyltransferase.

The NNMT is predominantly expressed in the liver. The N-methylation of nicotinamide is known to be altered in hepatic cirrhosis (Cuomo et al., 1994). The abnormal expression of NNMT has been identified in hepatocellular carcinoma (HCC) (Kim et al., 2009). The human liver NNMT gene encodes a full-length monomeric 264-amino acid protein with a calculated molecular mass of 29,600 Da. A real-time reverse-transcription PCR analysis of HCC suggested that NNMT mRNA level appeared markedly altered in tumor samples compared with the surrounding healthy tissue. Moreover, NNMT expression was significantly associated with tumor stage (Kim et al., 2009).

In humans, the NNMT gene is highly polymorphic. Most of the SNPs are in the noncoding regions. The rs694539 variant of the NNMT gene in noncoding region affects the regulation of transcription (Zhang et al., 2007). Some SNPs in the NNMT 5′ flanking region may influence its transcriptional efficiency. There is also a report on the association between NNMT genetic variations and the alteration of cellular pathways (Saito et al., 2001).

NNMT is an important cytosolic methyltransferase, belonging to Phase II metabolizing enzymes. The enzyme catalyzes the N-methylation of nicotinamide, pyridines, and other structural analogs, playing a crucial role in the biotransformation and detoxification of many xenobiotic compounds. N-methylation is one method by which drugs and other xenobiotic compounds are metabolized by the liver, and the enzyme NNMT is responsible for this activity that uses S-adenosyl-l-methionine as the methyl donor. The NNMT reaction yields two products: S-adenosyl-l-homocysteine and N¹-methylnicotinamide. S-adenosyl-l-homocysteine is converted into homocysteine by S-adenosyl-l-homocysteine hydrolase. N¹-methylnicotinamide is mostly excreted into urine and partly further converted via catalysis by aldehyde oxidase to N¹-methyl-2-pyridone-5-carboxiamide and N¹-methyl-4-pyridone-5-carboxiamide, which are also excreted into urine. N-methylation has been proposed as a metabolic pathway for nicotinamide excretion, and NNMT is the only enzyme known to utilize nicotinamide as a methyl acceptor substrate. Therefore, NNMT could participate in the regulation of nicotinamide intracellular levels, modulating its excretion after N-methylation. Nicotinamide, the amide of nicotinic acid, is the precursor of the coenzyme β-nicotinamide adenine dinucleotide (NAD), an essential cofactor for several oxidoreductases, which participates in a wide range of biological processes, including energy supply, cellular resistance to stress or injury, and longevity (Williams et al., 2005). In addition, several enzymes that use NAD as a substrate can be inhibited by nicotinamide. Due to this type of product inhibition, the salvage and/or elimination of nicotinamide are crucial steps in NAD metabolism, and the enzyme NNMT could be involved in controlling these cellular events. NNMT activity may also play a role in regulating biological processes related to N¹-methylnicotinamide. It has recently become apparent that it possesses anti-inflammatory (Bryniarski et al., 2008), anti-thrombotic (Chlopicki et al., 2007), vasoprotective (Bartus et al., 2008), and gastroprotective (Brzozowski et al., 2008) properties. NNMT is highly expressed in the liver, where its activity displays a five-fold variation among individuals and has a bimodal frequency distribution (Cantoni et al., 1951). The enzyme activity may be regulated by a genetic polymorphysm. Such a polymorphism could have functional implications for individual differences in the metabolism and therapeutic effect of drugs (Aksoy et al., 1994) and in the formation of potentially toxic pyridine metabolites. Moreover, elevated NNMT activity was reported in many kinds of tumors. The up-regulation of this enzyme suggests a possible role of NNMT in cancer growth, migration, and metastasis (Sartini et al., 2006; Wu et al., 2008). However, the biological significance of alterations in NNMT activity in various pathological conditions remains largely unknown.

Transcription of the human NNMT gene produces a full-length mRNA transcript of 1579 bp. It was recently shown that NNMT expression in some thyroid cancer cells may be regulated by hepatocyte nuclear factor beta (HNF-1β). HNF-1β is able to function as a transcription activator of the NNMT promoter, binding to specific sites in the basal promoter region between nucleotides −148 and −162 relative to the translation initiation codon (Xu et al., 2005). It was also reported that in BHP 18-21 papillar thyroid cancer cells, the histone deacetylase inhibitor depsipeptide reduces NNMT mRNA level through down-regulation of transcription activator HNF-1β (Xu and Hershman, 2006). Elevated NNMT expression has also been correlated to activation of STAT3 in Hep-G2 liver cancer cells stimulated with IL-6 and in colorectal cancer tissues (Tomida et al., 2008).

In conclusion, the role of the rs694539 variant of the NNMT gene in NASH is unclear, through dysregulation of epigenetics and/or elevated homocysteine levels or/and dysregulation of the nicotinamide levels may be one of the causes of the disease. Our findings suggest that the rs694539 variant of NNMT gene is involved in the etiopathology of NASH in the Turkish patients.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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