Nicotinamide N-methyltransferase Gene Research Articles

Nicotinamide N-methyltransferase (NNMT): a novel therapeutic target for metabolic syndrome.

Metabolic syndrome (MetS) represents a constellation of metabolic abnormalities, typified by obesity, hypertension, hyperglycemia, and hyperlipidemia. It stems from intricate dysregulations in metabolic pathways governing energy and substrate metabolism. While comprehending the precise etiological mechanisms of MetS remains challenging, evidence underscores the pivotal roles of aberrations in lipid metabolism and insulin resistance (IR) in its pathogenesis. Notably, nicotinamide N-methyltransferase (NNMT) has recently surfaced as a promising therapeutic target for addressing MetS. Single nucleotide variants in the NNMT gene are significantly correlated with disturbances in energy metabolism, obesity, type 2 diabetes (T2D), hyperlipidemia, and hypertension. Elevated NNMT gene expression is notably observed in the liver and white adipose tissue (WAT) of individuals with diabetic mice, obesity, and rats afflicted with MetS. Knockdown of NNMT elicits heightened energy expenditure in adipose and hepatic tissues, mitigates lipid accumulation, and enhances insulin sensitivity. NNMT catalyzes the methylation of nicotinamide (NAM) using S-adenosyl-methionine (SAM) as the donor methyl group, resulting in the formation of S-adenosyl-l-homocysteine (SAH) and methylnicotinamide (MNAM). This enzymatic process results in the depletion of NAM, a precursor of nicotinamide adenine dinucleotide (NAD+), and the generation of SAH, a precursor of homocysteine (Hcy). Consequently, this cascade leads to reduced NAD+ levels and elevated Hcy levels, implicating NNMT in the pathogenesis of MetS. Moreover, experimental studies employing RNA interference (RNAi) strategies and small molecule inhibitors targeting NNMT have underscored its potential as a therapeutic target for preventing or treating MetS-related diseases. Nonetheless, the precise mechanistic underpinnings remain elusive, and as of yet, clinical trials focusing on NNMT have not been documented. Therefore, further investigations are warranted to elucidate the intricate roles of NNMT in MetS and to develop targeted therapeutic interventions.

Systematic pan-cancer analysis of the nicotinamide n-methyltransferase in human cancer.

In several tumors, Nicotinamide N-Methyltransferase (NNMT) was identified as a bridge between methylation metabolism and tumorigenesis and was associated with a poor prognosis. This research aims is to study the prognostic value of NNMT in cancer, its relationship with DNA methylation, and the immune microenvironment. On the basis of the Cancer Genome Atlas and the Cancer Cell Line Encyclopedia, Genotype Tissue-Expression, cBioPortal, Cellminer, Gene Expression Profiling Interactive Analysis, Human Protein Atlas and Clinical Proteomic Tumor Analysis Consortium, we used a series of bioinformatics strategies to investigate the potential carcinogenicity of NNMT, including the relationship between NNMT expression and prognosis, tumor mutational burden, microsatellite instability, and sensitivity analysis of anticancer drugs. The GeneMANIA, STRING, and BioGRID databases were examined for protein-protein interactions, and Gene Ontology and the Kyoto Encyclopedia of Genes were used to infer the signal pathway. The results indicated that NNMT was significantly expressed in several tumor tissues compared to the matching non-tumor tissues. Increased NNMT expression was linked to reduced OS, DSS, and DFI. In addition, there was a link between NNMT expression and TMB and MSI in 18 cancer types, and between NNMT expression and DNA methylation in 23 cancer types. Further study of NNMT gene alteration data revealed that deletion was the most prevalent form of NNMT mutation, and that there was a significant negative association between NNMT expression and mismatch repair genes. In addition, there was a strong positive connection between NNMT and immune infiltration in 28 types of tumors, and the immune cells that infiltrated the tumors displayed a characteristic NNMT pattern. According to the enrichment study, cell migration, cell motility, and cell adhesion were highly enriched in biological processes, and NNMT may be associated with the PI3K-Akt signaling pathway. By downregulating gene methylation or impacting the immunological microenvironment widely, NNMT may drive carcinogenesis and cause a poor prognosis. Our research showed that NNMT could be used as a biomarker of tumor immune infiltration and poor prognosis, thus providing a unique strategy for cancer therapy.

Lipopolysaccharide affects energy metabolism and elevates nicotinamide N-methyltransferase level in human aortic endothelial cells (HAEC).

This study aimed to investigate the putative role of nicotinamide N-methyltransferase in the metabolic response of human aortic endothelial cells. This enzyme catalyses S-adenosylmethionine-mediated methylation of nicotinamide to methylnicotinamide. This reaction is accompanied by the reduction of the intracellular nicotinamide and S-adenosylmethionine content. This may affect NAD+ synthesis and various processes of methylation, including epigenetic modifications of chromatin. Particularly high activity of nicotinamide N-methyltransferase is detected in liver, many neoplasms as well as in various cells in stressful conditions. The elevated nicotinamide N-methyltransferase content was also found in endothelial cells treated with statins. Although the exogenous methylnicotinamide has been postulated to induce a vasodilatory response, the specific metabolic role of nicotinamide N-methyltransferase in vascular endothelium is still unclear. Treatment of endothelial cells with bacterial lipopolysaccharide evokes several metabolic and functional consequences which built a multifaceted physiological response of endothelium to bacterial infection. Among the spectrum of biochemical changes substantially elevated protein level of nicotinamide N-methyltransferase was particularly intriguing. Here it has been shown that silencing of the nicotinamide N-methyltransferase gene influences several changes which are observed in cells treated with lipopolysaccharide. They include altered energy metabolism and rearrangement of the mitochondrial network. A complete explanation of the mechanisms behind the protective consequences of the nicotinamide N-methyltransferase deficiency in cells treated with lipopolysaccharide needs further investigation.

Nicotinamide N-Methyltransferase inhibits HBV replication by suppressing NR5A1 expression invitro

Chronic hepatitis B virus (HBV) infection can lead to fibrosis, liver cirrhosis, and primary hepatocellular carcinoma. Investigating host factors that regulate HBV replication helps to identify antiviral targets. In the current study, we identified Nicotinamide N-Methyltransferase gene (NNMT) as a novel factor that regulates HBV transcription. NNMT is up-regulated at both the mRNA and protein levels in HepG2.2.15 cells compared to HepG2 cells. Overexpression of NNMT reduces HBV replication in several cell models, while knockdown of NNMT enhances HBV DNA levels. Mechanistically, NNMT suppresses HBV DNA replication by inhibiting HBV RNA transcription. The region required for the inhibitory effect of NNMT was narrowed to nt 1672–1708 in enhancer II by luciferase assays. On the other hand, ChIP assays and EMSA results showed that NNMT does not bind to this region substantially, either directly or indirectly. Next, a collection of hepatic nuclear receptor transcription factors was screened to determine whether they were affected by NNMT overexpression. NR5A1, a positive regulator of HBV replication, decreased significantly after NNMT overexpression. Collectively, the findings of this study shed light on the regulation of HBV transcription.

Nicotinamide N-Methyltransferase in Head and Neck Tumors: A Comprehensive Review.

The head and neck tumors (HNT) are a heterogeneous group of diseases ranging from benign to malignant lesions, with distinctive molecular and clinical behaviors. Several studies have highlighted the presence of an altered metabolic phenotype in HNT, such as the upregulation of nicotinamide N-methyltransferase (NNMT). However, its biological effects have not been completely disclosed and the role of NNMT in cancer cell metabolism remains unclear. Therefore, this comprehensive review aims to evaluate the available literature regarding the biological, diagnostic, and prognostic role of NNMT in HNT. NNMT was shown to be significantly overexpressed in all of the evaluated HNT types. Moreover, its upregulation has been correlated with cancer cell migration and adverse clinical outcomes, such as high-pathological stage, lymph node metastasis, and locoregional recurrences. However, in oral squamous cell carcinoma (OSCC) these associations are still debated, and several studies have failed to demonstrate the prognostic significance of NNMT. The shRNA-mediated gene silencing efficiently suppressed the NNMT gene expression and exhibited a clear inhibitory effect on cell proliferation, promoting the expression of apoptosis-related proteins and modulating the cell cycle. NNMT could represent a new molecular biomarker and a new target of molecular-based therapy, although further studies on larger patient cohorts are needed to explore its biological role in HNT.

Nicotinamide N-Methyltransferase: An Emerging Protagonist in Cancer Macro(r)evolution.

Nicotinamide N-methyltransferase (NNMT) has progressed from being considered merely a Phase II metabolic enzyme to one with a central role in cell function and energy metabolism. Over the last three decades, a significant body of evidence has accumulated which clearly demonstrates a central role for NNMT in cancer survival, metastasis, and drug resistance. In this review, we discuss the evidence supporting a role for NNMT in the progression of the cancer phenotype and how it achieves this by driving the activity of pro-oncogenic NAD+-consuming enzymes. We also describe how increased NNMT activity supports the Warburg effect and how it promotes oncogenic changes in gene expression. We discuss the regulation of NNMT activity in cancer cells by both post-translational modification of the enzyme and transcription factor binding to the NNMT gene, and describe for the first time three long non-coding RNAs which may play a role in the regulation of NNMT transcription. We complete the review by discussing the development of novel anti-cancer therapeutics which target NNMT and provide insight into how NNMT-based therapies may be best employed clinically.

The association between nicotinamide N-methyltransferase gene polymorphisms and primary hypertension in Chinese Han Population

<abstract> Although no direct evidence shows that Nicotinamide N-methyltransferase (NNMT) is involved in hypertension to date, many reports have shown that NNMT plays important roles in pathogenesis of metabolic syndrome and in various cardiovascular diseases. Here we explored the genetic association between <italic>NNMT</italic> polymorphisms and primary hypertension. 308 primary hypertension cases (aged &lt; 60 years, blood pressure ≥ 140/90 mmHg or taking antihypertensive therapies) and 315 controls (aged ≥ 60 years and without any diagnosed diseases) were recruited from unrelated Chinese Han ethnicity volunteers. Then a case-control study was carried out to explore the genetic association between <italic>NNMT</italic> polymorphism and primary hypertension. A significantly associated SNP (rs1941404) was found in <italic>NNMT</italic> gene (P &lt; 0.0125). At this locus, the risks for minor homozygote CC carriers being of hypertension were highly significantly higher than those for the TT + CT carriers (OR = 2.120, OR_adjusted = 2.573, P = 0.000, P_adjusted = 0.001 and statistical power = 0.997), but the differences between the genotypes CT + CC and TT (dominant genetic model) were not statistically significant (P = 0.321, P_adjusted = 0.230 and statistical power = 0.200). In conclusion, rs1941404 in <italic>NNMT</italic> gene sequence is significantly associated with primary hypertension under a recessive inheritance mode. At this locus, the minor homozygote CC carriers are susceptible population and the TT and CT carriers are non-susceptible population. </abstract>

Genetic Nicotinamide N-Methyltransferase Deficiency Improves Insulin Sensitivity in DIO Mice

Nicotinamide N-methyltransferase (NNMT) is expressed in most tissues including muscle, adipose tissue, liver and digestive organs. Recent research suggests the involvement of NNMT in the pathogenesis of obesity, insulin resistance and related metabolic disease. Antisense oligonucleotide (ASO) knockdown in high-fat diet (HFD)-fed mice led to reduced weight gain, relative fat mass, plasma insulin levels and improved glucose tolerance. We tested the hypothesis, that genetic NNMT deletion protects mice on HFD and Western diet (WD) against obesity and explored the metabolic effects of NNMT. Using NNMT ASO treatment and a NNMT knockout mouse (NNMT-/-), we investigate the effects of NNMT deletion on energy metabolism, glucose homeostasis and the development of obesity. We also examined data from a human study concerning NNMT expression and 1-methylnicotinamide (MNA) levels regarding weight reduction. In WD-fed mice, NNMT ASO treatment improved acute glucose tolerance, reduced weight gain and fat mass increase and, consequently, lowered plasma insulin levels. NNMT-/- mice exhibited virtually no MNA but threefold higher nicotinamide levels. Whereas NNMT-/- male and female mice on normal chow revealed no metabolic phenotype, NNMT-/- males on HFD showed improved glucose infusion rates, nearly complete insulin-mediated suppression of endogenous glucose production and an enhanced glucose uptake during a hyperinsulinemic-euglycemic clamp. Furthermore, NNMT-/- females on WD showed reduced weight gain, less fat mass and lower plasma insulin levels compared to controls. While NNMT gene expression in human fat biopsies increased over weight loss, corresponding plasma MNA levels significantly declined after weight reduction, suggesting that other mechanisms rather than adipose NNMT expression modulate circulating MNA levels during weight reduction. In conclusion, we observed an improvement of basal metabolic parameters and insulin sensitivity in NNMT-deficient mouse models. Disclosure S. Brachs: Research Support; Self; Sanofi-Aventis Deutschland GmbH. J. Polack: None. M. Brachs: Research Support; Spouse/Partner; Sanofi. K. Jahn-Hofmann: None. R. Elvert: Employee; Self; Sanofi. A. Pfenninger: None. F. Bärenz: None. D. Margerie: Employee; Self; Sanofi-Aventis Deutschland GmbH. K. Mai: None. A. Kannt: Employee; Self; Sanofi. Stock/Shareholder; Self; Sanofi. J. Spranger: Research Support; Self; Sanofi R&amp;D.

An Epigenetic Signature in Adipose Tissue Is Linked to Nicotinamide N-Methyltransferase Gene Expression.

The enzyme nicotinamide N-methyltransferase (NNMT) is a major methyltransferase in adipose tissue. We hypothesized an epigenetic signature in association with NNMT gene expression in adipose tissue. The global human methylome was analyzed in visceral adipose tissue (VAT) from morbidly obese patients using the Infinium Human Methylation 450 BeadChip array (discovery cohort: n=11). The findings were confirmed in two additional independent cohorts (cohort 1: n=60; BMI 20-60 kg m-2 and cohort 2: n=40; BMI > 40 kg m-2 ) and validated after weight loss (using microarray data). Among the genes associated with the largest methylation fold change were genes related to metabolic processes, proliferation, inflammation, and extracellular matrix remodeling, such as COL23A1, PLEC1, FBXO21, STEAP3, RGS12, IGDCC3, FOXK2, and ORAI2. In fact, the results showed 577 differentially methylated CpG sites (DMCpGs) associated with the NNMT expression levels, with low methylation levels paralleling high NNMT expression. The expression of FBXO21 and FOXK2 was specifically modified after weight loss concomitantly with a decrease in NNMT expression and inflammation-related genes. Interestingly, the adipose tissue NNMT gene expression correlated with markers of adipose tissue inflammation. The expression of NNMT in VAT is associated with a specific methylome signature involving genes linked to adipose tissue metabolic pathophysiology.

Metabolomics study on the association between nicotinamide N-methyltransferase gene polymorphisms and type 2 diabetes

Numerous reports have demonstrated that activities of nicotinamide N-methyltransferase (NNMT) are significantly associated with type 2 diabetes (T2D), and more than 200 single nucleotide polymorphisms (SNPs) have been identified across NNMT gene to date. Although two SNPs (rs694539 and rs1941404) have been found significantly associated with a variety of noninfectious chronic diseases, the association between NNMT gene polymorphisms and T2D has not been reported yet. In this paper, 558 T2D patients and 442 healthy controls were recruited from Chinese Han population. After a case-control study on the association between the two SNPs (rs694539 and rs1941404) and T2D, we found that the rs1941404 is significantly associated with T2D and the CC carriers at this locus are T2D susceptible population. The following metabolomics study showed that the rs1941404 variation is able to affect the metabolic pathways of tryptophan, tyrosine, and arginine, which may partly explain why the rs1941404 variation is significantly associated with T2D.

Association between Nicotinamide N-Methyltransferase Gene Polymorphisms and Obesity in Chinese Han Male College Students.

Some reports have shown that nicotinamide N-methyltransferase (NNMT) is associated with the body mass index (BMI) and energy metabolism. Here we explored the association between NNMT gene polymorphisms and obesity. The subjects were recruited from male Chinese Han college student. 289 of them (19 ≤ body fat percentage (BF%)) were selected as the high body fat group (HBFG), 494 of them (3 ≤ BF% < 13.5) were selected as the low body fat group (LBFG), and then a case-control study (fat versus thin) was carried out to explore the association between the NNMT gene polymorphism and the body composition using tagSNPs method. A tagSNP (rs10891644) in NNMT gene was found significantly associated with the body composition (P < 0.0026). At this locus, the BF% for the genotype GT, TT, and GG were 14.56 ± 8.35, 13.47 ± 8.11, and 12.42 ± 7.50, respectively, and the differences between the GT and the GG + TT were highly significant (P < 0.01); the ORadjusted value of the GT versus (GG + TT) was 1.716 (Padjusted = 0.002, 95% CI = 1.240–2.235). Therefore, the variation of the tagSNP, rs10891644, is significantly associated with obesity and the GT carriers are the susceptible population.

Associations of nicotinamide N-methyltransferase gene single nucleotide polymorphisms with sport performance and relative maximal oxygen uptake

ABSTRACTTo observe the associations between single nucleotide polymorphisms (SNPs) of nicotinamide N-methyltransferase (NNMT) gene and sport performance and to analyse genotype associations of the associated SNPs with sport performance and relative maximal oxygen uptake ().Participants were selected from 685 Chinese Han male college students. The completion times of a 1000-m run and a 50-m run were used to reflect sport performance, respectively. Nineteen tagSNPs were genotyped with Polymerase chain reaction–ligase detection reaction. Relative was directly determined with a cardiopulmonary function analyser.A significant association was found between rs2256292 and 1000-m run performance, but no significant association was found between any tagSNPs and 50-m run performance. The genotype associations of rs2256292 with 1000-m run performance and with relative were both significant under the recessive model (CC vs. CG + GG).No tagSNP in NNMT is significantly associated with 50-m run performance but rs2256292 is significantly associated with 1000-m run performance. The genotype associations of rs2256292 with sport performance are significant under recessive model, and a higher relative may be the physiological reason for minor homozygote CC carriers being of the better 1000-m runners.

Abstract 1693: Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases

Abstract Background/Purpose: Nicotinamide N-methyltransferase (NNMT) is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. It is overexpressed in different cancers and has been shown to enhance the migratory and invasive activity of cancer cells. Additionally, NNMT is an important regulator of the methylation potential in cells by consumption of S-adenosyl methionine (SAM), an important substrate for methyltransferases. By this mechanism NNMT reduces methylation of proteins such as certain histones leading to epigenetic remodeling and induced expression of tumor-promoting genes (Ulanovskaya et al, Nat. Chem. Biol. 2013). In clear cell renal cell carcinoma (ccRCC), the dominant histological subtype of renal cell cancer, NNMT is expressed at high levels compared to normal kidney tissue and could represent a novel therapeutic target, especially for the treatment of metastatic disease which is marked by a five-year survival rate of below 10% with currently available therapies. Methods: We analyzed NNMT in a ccRCC patient cohort from The Cancer Genome Atlas (TCGA) concerning mRNA expression (n = 463), somatic variations (n = 410) and association with clinical parameters and patient survival. In an independent collection of ccRCC tissues (n = 64), corresponding normal kidney tissues and metastatic tissues from different organs (n = 129), NNMT mRNA and protein expression were quantified via Real-Time PCR and immunohistochemical staining of tissue microarrays, respectively. Results: In both cohorts, NNMT mRNA expression was significantly upregulated in tumor compared to non-tumor tissue. Somatic mutations and copy number alterations in the NNMT gene locus were observed in only a small number of patients. NNMT mRNA expression correlated weakly with tumor stage and grading in both cohorts, but not with the presence of lymph node or distant metastases. In Kaplan-Meier analyses high NNMT expression was associated with worse overall and cancer-specific survival. In ccRCC metastases from different organ sites, NNMT mRNA and protein expression were also induced compared to normal kidney tissue and high expression was again associated with worse patient survival in Kaplan-Meier analysis (HR = 2.15, Plog-rank = 0.05). Conclusion: NNMT mRNA and protein are highly expressed not only in primary tissue of ccRCC, but also in metastases derived from different organs, offering it as a potential drug-target for metastatic ccRCC. Although NNMT expression is a good prognostic indicator for patient survival, its molecular function in ccRCC tumorigenesis and metastasis formation has to be further evaluated. Supported by the Robert Bosch Foundation, Stuttgart, Germany and the ICEPHA Grant Tuebingen-Stuttgart, Germany Citation Format: Anna Reustle, Steffen Rausch, Stefan Winter, Florian Büttner, Stephan Kruck, Joerg Hennenlotter, Marcus Scharpf, Falko Fend, Jens Bedke, Arnulf Stenzl, Matthias Schwab, Elke Schaeffeler. Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1693.

Research about NNMT gene in cancer

Nicotinamide N-methyltransferase (NNMT) is a S-adenosyl-L-methionine (SAM) dependent cytoplasm enzyme,which plays a vital role in the biotransformation and detoxification of many drugs and xenobiotic compounds.Recent studies have revealed abnormal expression of NNMT in many tumors,which may contribute to tumorigenesis and tumor development and radiotherapy or chemotherapy resistance. Key words: Nicotinamide N-methyltransferase; Nicotinamide; Neoplasms

Expression of nicotinamide N-methyltransferase in hepatocellular carcinoma is associated with poor prognosis

BackgroundHepatocellular carcinoma (HCC) is the most common tumor in the adult liver, with high relapse and mortality rates despite diverse treatment modalities. In this study, nicotinamide N-methyltransferase (NNMT), a key enzyme in drug metabolism, was investigated as a potential prognostic factor.MethodsFrozen tumors and non-cancerous surrounding tissues from 120 patients with primary HCC were studied. Expressions of NNMT and internal control genes were measured by real-time reverse-transcription PCR (RT-PCR). The relationship of NNMT mRNA level with clinicopathologic parameters and clinical outcome was evaluated.ResultsNNMT mRNA level is markedly reduced in HCCs compared to non-cancerous surrounding tissues (P < 0.0001), and NNMT expression in tumors was significantly correlated with tumor stage (P = 0.010). Moreover, stratification of patients based on tumor NNMT mRNA levels revealed that the patients who expressed higher NNMT mRNA levels tended to have a shorter overall survival (OS) time (P = 0.053) and a significantly shorter disease-free survival (DFS) time (P = 0.016). Both NNMT expression (P = 0.0096) and tumor stage (P = 0.0017) were found to be significant prognostic factors for DFS in a multivariate analysis.ConclusionThe results of this study indicated that NNMT gene expression is associated with tumor stage and DFS time in HCC cases. Because of the broad substrate specificity of NNMT, which could alter the efficacy and adverse effects of chemotherapy, NNMT merits further investigation regarding its role as a prognostic factor with a larger cohort of HCC patients.

Nicotinamide N‐methyl transferase (NNMT) gene polymorphisms and risk for spina bifida

Moderate hyperhomocysteinemia is a known risk factor for NTDs in a variety of experimental model systems and is believed to be important in humans as well. The enzyme nicotinamide N-methyl transferase (NNMT) was identified in a genome-wide linkage scan as being an important regulator of homocysteine homeostasis in a Spanish population, making it an interesting candidate gene for NTDs. We evaluated 11 SNPs (single nucleotide polymorphism) of the NNMT gene in our study population. In this study, 252 cases (infants with spina bifida) and 335 controls (nonmalformed infants), born during the period 1983-1986 in selected counties in California, were genotyped for variants of the NNMT gene. Allelic, genotype, and haplotype associations with spina bifida risk were evaluated and analyzed. None of the SNPs studied alone showed allelic or genotypic associations with spina bifida. However, the TCAG haplotype for block 3 (rs2852447, ra2852425, rs4646337, and rs11569688) showed a decreased risk for spina bifida among non-Hispanic Whites (OR 0.4; 95%CI: 0.1-1.0). No association was found between infant NNMT gene variants and risk for spina bifida in our study population. However, small sample sizes for most variant groups and for phase-unknown haplotype data limited the power of the study.

The relation between nicotinamide N-methyltransferase gene polymorphism and plasma homocysteine concentration in healthy Japanese men

0049-3848/$ see front matter © 2007 Elsevier Ltd. All rights reserv doi:10.1016/j.thromres.2007.02.012 polymorphisms are reported to associate with tHcy concentration, including the most notable methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Recently, a quantitative trait linkage (QTL) analysis on tHcy concentrationwas conducted in a Spanish family study of venous thrombosis [3]. The chromosome region 11q23 was shown to have a positive linkage signal, and in this region the nicotinamide Nmethyltransferase (NNMT) gene was located. Subsequent analysis for 10 single nucleotide polymorphisms (SNPs) in the NNMT gene identified one SNP (rs694539 in GenBank, dBSNP database) that was associated with the tHcy concentration. Since the SNP resides in the first intron, the functional significance of it is still unknown. NNMTenzyme activity was shown to have a significant variability among individual, and this difference was attributed not to the structure of the enzyme but to the expression levels of mRNA and protein [4,5]. Thus, this noncoding SNP might be related to the regulation of transcription, which correlates to the phenotype.

Identification of Nicotinamide N-Methyltransferase as a Novel Tumor Marker for Renal Clear Cell Carcinoma

To explore the involvement of enzymes of drug metabolism in renal cell carcinoma we analyzed the gene expression profiles of tumor and nontumor tissues from the same patient by DNA macroarray. The enzyme nicotinamide N-methyltransferase was selected for further evaluation. Nicotinamide N-methyltransferase mRNA expression was investigated in paired tissue samples from cancerous and noncancerous parts of the kidneys of 30 patients with clear cell renal cell carcinoma who underwent tumor nephrectomy. Measurements were performed by semiquantitative reverse transcriptase-polymerase chain reaction and quantitative real-time polymerase chain reaction. Paired tissue samples were also obtained from 1 patient with chromophobe renal cell carcinoma and from another with oncocytoma to compare the specificity of changes in nicotinamide N-methyltransferase expression among tumors that are related to different renal epithelial cell types. Western blot analysis and catalytic activity assay were also performed to study nicotinamide N-methyltransferase expression. Expression correlated with tumor characteristics. A marked increased expression in tumor tissue was found for nicotinamide N-methyltransferase, which is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. Differential gene expression measurements in tumor vs normal tissue revealed up-regulation in all clear cell renal cell carcinomas at between 3 and 294-fold (mean 41). In contrast, in chromophobe renal cell carcinoma and oncocytoma nicotinamide N-methyltransferase expression did not increase. In addition, nicotinamide N-methyltransferase expression significantly correlated inversely with tumor size. Our results indicate that a marked nicotinamide N-methyltransferase increase is a peculiar feature of clear cell renal cell carcinoma. Additional studies may establish the role of nicotinamide N-methyltransferase in tumor formation and progression.

Histone Deacetylase Inhibitor Depsipeptide Represses Nicotinamide N-Methyltransferase and Hepatocyte Nuclear Factor-1βGene Expression in Human Papillary Thyroid Cancer Cells

Nicotinamide N-methyltransferase (NNMT) catalyzes N-methylation of nicotinamide and other structural analogues. NNMT gene expression is enhanced in many papillary thyroid cancer cells and activated by hepatocyte nuclear factor (HNF)-1beta. In this work, we studied the effects of depsipeptide, a histone deacetylase inhibitor, on NNMT gene expression in BHP 18-21 papillary thyroid cancer cells. Depsipeptide reduced NNMT mRNA level in a dose-dependent and time-dependent manner as determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In contrast, expression of the sodium iodide symporter (NIS), a gene with differentiated function, waas enhanced in the treated cells. NNMT protein level determined by Western blot analysis and NNMT catalytic activity was also reduced significantly in the depsipeptide-treated cells. To study the mechanism of NNMT gene repression by depsipeptide, effects of depsipeptide on NNMT promoter activity were determined by luciferase reporter gene assay. NNMT promoter activity was significantly reduced in the HNF-1beta-positive BHP 18-21 cells but not in the HNF-1beta-negative BHP 14-9 papillary cancer cells. A mutant reporter construct with mutations in a HNF-1 site in the NNMT basal promoter region did not respond to depsipeptide in both HNF-1beta protein levels, and abolished activity of DNA binding to the HNF-1 site in the NNMT promoter region. Protein synthesis inhibitor cycloheximide and proteasome inhibitor MG-132 enhanced HNF-1beta stability in the depsipeptide-treated cells. In summary, depsipeptide represses NNMT and HNF-1beta gene expression in some papillary thyroid cancer cells. the repression of NNMT by depsipeptide is at the transcription level through downregulation of transcription activator HNF-1beta.

A Genomewide Exploration Suggests a New Candidate Gene at Chromosome 11q23 as the Major Determinant of Plasma Homocysteine Levels: Results from the GAIT Project

Homocysteine (Hcy) plasma level is an independent risk marker for venous thrombosis, myocardial infarction, stroke, congestive heart failure, osteoporotic fractures, and Alzheimer disease. Hcy levels are determined by the interaction of genetic and environmental factors. The genetic basis is still poorly understood, since only the MTHFR 677 C-->T polymorphism has been consistently associated with plasma Hcy levels. We conducted a genomewide linkage scan for genes affecting variation in plasma Hcy levels in 398 subjects from 21 extended Spanish families. A variance-components linkage method was used to analyze the data. The strongest linkage signal (LOD score of 3.01; genomewide P = .035) was found on chromosome 11q23, near marker D11S908, where a candidate gene involved in the metabolism of Hcy (the nicotinamide N-methyltransferase gene [NNMT]) is mapped. Haplotype analyses of 10 single-nucleotide polymorphisms within this gene found one haplotype associated with plasma Hcy levels (P = .0003). Our results, to our knowledge, represent the first genomic scan for quantitative variation in Hcy plasma levels. They strongly suggest that the NNMT gene could be a major genetic determinant of plasma Hcy levels in Spanish families. Since this gene encodes an enzyme involved in Hcy synthesis, this finding would be consistent with known biochemical pathways. These data could be relevant in determining the relationships between Hcy level, cardiovascular disease, osteoporosis, and Alzheimer disease.