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Identification of 197 Genetic Variations in Six Human Methyltransferase Genes in the Japanese Population

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Identification of 197 Genetic Variations in Six Human Methyltransferase Genes in the Japanese Population J Hum Genet (2001) 46:529–537 © Jpn Soc Hum Genet and Springer-Verlag 2001 ORIGINAL ARTICLE Susumu Saito · Aritoshi Iida · Akihiro Sekine Yukie Miura · Tsutomu Sakamoto · Chie Ogawa Saori Kawauchi · Shoko Higuchi · Yusuke Nakamura Identification of 197 genetic variations in six human methyltransferase genes in the Japanese population Received: May 17, 2001 / Accepted: June 18, 2001 Abstract Methylation is an important event in the biotrans- Introduction formation pathway for many drugs and xenobiotic com- pounds. We screened DNA from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in six Methylation is an important feature of the biotransforma- methyltransferase (MT) genes (catechol-O-MT, COMT; tion pathway for many drugs and xenobiotic compounds guanidinoacetate N-MT, GAMT; histamine N-MT, HNMT; (Weinshilboum 1989). The reaction involves transfer of nicotinamide N-MT, NNMT; phosphatidylethanolamine the activated methyl group of S-adenosyl-l-methionine N-MT, PEMT; and phenylethanolamine N-MT, PNMT) by (AdoMet) to the substrates. direct sequencing of their entire genomic regions except Among the enzymes involved in such reactions, called for repetitive elements. This approach identified 190 SNPs methyltransferases, catechol-O-methyltransferase (COMT; and seven insertion/deletion polymorphisms among the six EC 2.1.1.6) catalyzes the transfer of a methyl group from S- genes. Of the 190 SNPs, 33 were identified in the COMT adenosylmethionine to catecholamines, a class of molecules gene, 6 in GAMT, 41 in HNMT, 8 in NNMT, 98 in PEMT, that includes the neurotransmitters dopamine, epinephrine, and 4 in PNMT. Nine were located in 5Ј flanking regions, and norepinephrine (Kopin 1985). O-methylation drives 156 in introns, 10 in exons, and 15 in 3Ј flanking regions. one of the major pathways for degrading catecholamine These variants may contribute to a more precise under- transmitters (Zhu and Conney 1998). In addition to its role standing of possible correlations between genotypes and in the metabolism of endogenous substances, COMT is im- disease-susceptibility phenotypes or risk for side effects portant for metabolizing catechol drugs such as L-DOPA from drugs. (Lautala et al. 2001). COMT is found in two forms in tis- sues, a cytoplasmic soluble form (S-COMT) and a mem- Key words Single-nucleotide polymorphism (SNP) · brane-bound form (MB-COMT) located in the rough Catechol-O-methyltransferase gene (COMT) · endoplasmic reticulum (Salminen et al. 1990; Bertocci et al. Guanidinoacetate N-methyltransferase gene (GAMT) · 1991; Lundström et al. 1991; Ulmanen et al. 1997). The Histamine N-methyltransferase gene (HNMT) · Nicotina- soluble enzyme appears to be the predominant form of mide N-methyltransferase gene (NNMT) · Phosphatidyle- COMT in most tissues. However, in humans the membrane- thanolamine N-methyltransferase gene (PEMT) · bound enzyme predominates in the brain and adrenal me- Phenylethanolamine N-methyltransferase gene (PNMT) dulla, and also in pheochromocytomas (Tenhunen et al. 1994; Eisenhofer et al. 1998). The MB-COMT transcript differs from the S-COMT transcript in that the former contains a longer 5Ј untranslated region (UTR) and its translation-start site lies 50 codons upstream from that of S-COMT (Tenhunen et al. 1994). Lotta et al. (1995) identified a G to A polymorphism S. Saito · A. Iida · A. Sekine · Y. Miura · T. Sakamoto · C. Ogawa · S. Kawauchi · S. Higuchi · Y. Nakamura in the COMT gene at codon 158, which leads to sub- Laboratory for Genotyping, SNP Research Center, Institute of stitution of methionine for valine. This polymorphism Physical and Chemical Research, Tokyo, Japan is associated with a three- to fourfold difference in enzy- Y. Nakamura (*) matic activity (Lachman et al. 1996); i.e., homozygosity Laboratory of Molecular Medicine, Human Genome Center, for the A allele, COMTMet, is associated with low enzyme Institute of Medical Science, The University of Tokyo, 4-6-1 activity and thermolability. Individuals who are homozy- Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Tel. ϩ81-3-5449-5372; Fax ϩ81-3-5449-5433 gous for the normal (valine) allele display relatively high e-mail: [email protected] activity and heat stability of COMT; heterozygotes usually 530 N. Matsuda et al.: EGF receptor and osteoblastic differentiation display intermediate levels of COMT activity and heat sion increases dramatically. When hepatocyte proliferation stability. was induced in adult rats by partial hepatectomy, PEMT2 Guanidinoacetate methyltransferase (GAMT; expression was inhibited (Vance et al. 1997). Those results EC2.1.1.2) catalyzes the last step of creatine biosynthesis in suggested that this enzyme might play a role in the regula- mammals. Creatine is an important metabolite for cellular tion of hepatocyte growth and cell division (Walkey et al. energy metabolism in a variety of tissues, e.g., skeletal, 1999). cardiac and smooth muscle, and brain (Walker 1979; Phenylethanolamine N-methyltransferase (PNMT; EC Wallimann and Hemmer 1994). GAMT deficiency causes 2.1.1.28) catalyzes the final step in the catecholamine bio- an inborn error in creatine biosynthesis (Stöckler et al. synthetic pathway, converting norepinephrine to epineph- 1994, 1996a, 1996b, 1997; Carducci et al. 2000; Ilas et al. rine (Axelrod and Weinshilboum 1972). It is primarily 2000). Deficiency of GAMT readily explains the accumula- expressed in the adrenal medulla and the retina (Baetge tion of guanidinoacetate and the depletion of the total et al. 1988). Its activity increases after stress in response creatine pool in affected individuals. to glucocorticoids and neuronal stimulation (Betito et al. Histamine N-methyltransferase (HNMT; EC 2.1.1.8) 1994). Since defects in epinephrine metabolism have been catalyzes a major metabolic pathway of histamine, a known implicated in the etiology of attention-deficit hyperactivity neurotransmitter (Schwartz et al. 1991). Histamine plays an disorder (ADHD), aggression, and anxiety (Klinteberg important role in allergic responses and is involved in regu- and Magnusson 1989; Pliszka et al. 1994; Girardi et al. 1995; lating secretion of gastric acid (Wasserman 1983; Loiselle Hanna et al. 1996), PNMT is considered to be a candidate and Wollin 1993). Biochemical genetic studies of HNMT in for a range of psychiatric disorders. human red blood cells demonstrated fivefold individual dif- To investigate the nature of apparent genotype/pheno- ferences in the level of this enzyme’s activity, mainly reflect- type correlations for methyltransferases more precisely, ing common genetic polymorphisms (Scott et al. 1988; Price we began by searching for additional single-nucleotide et al. 1993). Preuss et al. (1998) demonstrated a C314T polymorphisms (SNPs) in the six methyltransferase genes polymorphism in exon 4 in the HNMT gene that resulted described above, including their promoter regions and in- in a Thr105Ile amino acid substitution; they found that the trons but excluding repetitive elements. We report here a T314 allele was associated with decreased levels of both total of 197 genetic variations, of which 144 have not been HNMT enzymatic activity and immunoreactive protein. reported before. Therefore, the presence of a T314 allele would be expected to result in reduced histamine metabolism and increased bronchoconstriction. A number of investigators have exam- ined potential associations of HNMT genotypes with al- Materials and methods tered susceptibilities to various diseases such as asthma (Yan et al. 2000a) and schizophrenia (Yan et al. 2000b). Exon–intron boundaries of the COMT, GAMT, HNMT, Nicotinamide N-methyltransferase (NNMT; EC 2.1.1.1) NNMT, PEMT, and PNMT genes were defined by compar- catalyzes the N-methylation of nicotinamide and other py- ing genomic sequences with mRNA sequences. Accession ridines to form pyridinium ions (Rini et al. 1989). Several numbers of the genomic sequences obtained for this study diseases have been associated with abnormal nicotinamide are as follows: metabolism that results in elevated levels of N- methylnicotinamide; these include idiopathic Parkinson’s COMT (AC000080.2), GAMT (NT_000879.1), disease (Williams et al. 1993) and hepatic cirrhosis (Cuomo HNMT (AC019304.3), NNMT (AC019290.3), et al. 1994). NNMT enzymatic activity in human liver varies PEMT (AC020558.3), PNMT (AC040933.3) in a fivefold range, with a bimodal frequency distribution Accession numbers of the mRNA sequences are as follows: (Rini et al. 1989). Phenotypic differences in NNMT activity could reflect genetic polymorphisms; however, no sequence COMT (NM_000754.2, M65213.1), differences were seen when investigators compared cDNAs GAMT (NM_000156.3), HNMT (NM_006895.1), of individuals with high and low NNMT activity (Smith NNMT (U20970.1, U20971.1), PEMT (NM_007169.1, et al. 1998; Yan et al. 1999). AF113126.1, AF176807.1), PNMT (NM_002686.1) Phosphatidylethanolamine N-methyltransferase (PEMT; EC2.1.1.17) converts phosphatidylethanolamine to Amplification of samples. Total genomic DNAs were iso- phosphatidylcholine in mammalian liver (Vance et al. lated from peripheral leukocytes of 48 unrelated Japanese 1997). Immunolocalization experiments have detected two individuals by the standard phenol/chloroform extraction isoforms of PEMT, encoded by a single gene (Walkey et al. method. On the basis of sequence information from 1997). PEMT1, which constitutes the majority of PEMT GenBank, we designed polymerase chain reaction (PCR) activity, is located at the endoplasmic reticulum (Vance and primers to amplify
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