Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for the genetic polymorphism of TPMT has been established for European Caucasians, African-Americans, Southwest Asians and Chinese, but it remains to be elucidated in Japanese populations. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in Japanese samples (n=192) using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 0.8% of the samples (three heterozygotes). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the samples analyzed. This study provides the first analysis of TPMT mutant allele frequency in a sample of Japanese population and indicates that TPMT*3C is the most common allele in Japanese subjects.     

C7 polymorphism in Japanese: new allele C7*8

The polymorphism of C7 was investigated in neuraminidase-treated sera from 513 unrelated Japanese individuals using isoelectric focusing followed by an electroimmunoblotting technique. Besides the common phenotypes 5 rare variants including 2 types of new variants were detected. The family analysis suggested the hereditary occurrence of a new allele C7*8. The allele frequencies were: C7*1 = 0.8314, C7*2 = 0.0926, C7*3 = 0.0380, C7*4 = 0.0331, C7*6 = 0.0010, and C7*8 = 0.0039.     

Three Japanese families with members carrying C7 silent allele (C7*Q0). Possibility for an association between C7*Q0 and C6*B

Three Japanese families with members carrying C7 silent allele(s) (C7*Q0) are presented. C6 types in the family members were also examined, and it was found that C7*Q0 was transmitted from a parent to offsprings as a haplotype, C7*Q0-C6*B. In another study of C6 types in sera from 3 volunteer blood donors with homozygous C7 deficiencies, the C6 phenotypes were found to be C6 B (homozygote). It seems remarkable that C7*Q0 can be associated with C6*B.     

Polymorphisms of serum proteins in Japanese patients with vascular diseases. I. Factor XIIIB, plasminogen and complement types in primary varicose veins.

The polymorphisms of the B subunit of coagulation factor XIII (F13B), plasminogen (PLG), complement C6, C7, factor B (BF) and factor I (IF) were studied among 21 unrelated Japanese patients with primary varicose veins (PVV) by isoelectric focusing followed by immunoblotting. The allele frequencies for F13B*2 and IF*A in PVV patients were significantly higher (F13B*2, p = 0.0047; IF*A, p = 0.0006) than those in healthy controls (n = 60). Significant associations of F13B 2 allotype [p = 0.0220, relative risk (RR) = 13.9] and IF A allotype (p = 0.0006, RR = 10.0) with PVV were observed; however, no significant association of PLG, C6, C7 or BF allotype with the disease was found.     

Complement phenotypes in patients with psoriasis

Phenotype frequencies for the complement proteins C4A, C4B, Bf (factor B) and C3 were performed for 49 Caucasian patients with psoriasis. The C4*A6 allele was present in 26.6% of the patients as compared to 5.4% of healthy regional Caucasian controls, p less than 0.001, relative risk = 6.28. The C4*A6 allele is known to be in linkage disequilibrium with the HLA B17 allele and to produce a non-functional gene product when it occurs with the B17 allele. HLA B17 is known to be associated with psoriasis in many Caucasian populations. Additional findings in the present study were a significant reduction in the C4B*2 allele frequency, a non-significant increase in the Bf*F allele frequency and no difference for Bf or C3 phenotype frequencies in the patients with psoriasis as compared to the controls.     

Genetic polymorphism of coagulation factor XIII B subunit in the Japanese population: description of three new rare alleles

Polyacrylamide gel isoelectric focusing (PAGIEF) of neuraminidase-treated EDTA plasma samples followed by electroblotting with enzyme immunoassay was performed to further investigate coagulation factor XIII B subunit (FXIII B) polymorphism. In 435 Japanese subjects PAGIEF patterns of FXIII B were classified into five common and three rare allotypes. This suggested that the FXIII B*2 allele existed in the Japanese population in the same manner as in Caucasians. Three new rare allotypes were considered to be controlled by three rare alleles which were designated FXIII B*13, FXIII B*14, and FXIII B*15, respectively. The gene frequencies calculated from 435 Japanese subjects were FXIII B*1 = 0.2977, FXIII B*2 = 0.0184, FXIII B*3 = 0.6805, FXIII B*13 = 0.0011, FXIII B*14 = 0.0011, and FXIII B*15 = 0.0011, respectively.     

I (C3b/C4b inactivator) typing by agarose gel isoelectric focusing and immunoblotting technique

Genetic polymorphism of I (C3b/C4b inactivator) was studied by the method of agarose gel isoelectric focusing followed by an immunoblotting technique. Serum or plasma samples were pretreated with neuraminidase. The method is rapid, and gives the simple and reliable patterns of I. The allele frequencies calculated from healthy Japanese individuals living in the western part of Japan were: IF* A = 0.126 and IF*B = 0.874.     

Polymorphism of plasminogen in healthy individuals and patients with cerebral infarction

Phenotyping of plasma plasminogen (PLG) was carried out by the method of agarose gel isoelectric focusing followed by immunofixation or immunoblotting. The allele frequencies calculated from healthy Japanese individuals (n = 795) were as follows: PLG*1 = 0.9440, PLG*2 = 0.0189, PLG*A = 0.0076, PLG*A2 = 0.0006, PLG*B = 0.0138, PLG*B2 = 0.0013, and PLG*C = 0.0138. The PLG phenotype distribution in a group of patients with cerebral infarction (n = 125) was also studied. The allele frequencies were PLG*1 = 0.960, PLG*2 = 0.016, PLG*A = 0.012, and PLG*B = 0.012. No statistically significant association was found between PLG types and cerebral infarction.     

Polymorphism of complement component I in Mongoloid populations: a new genetic variant IF A2

The genetic polymorphism of the complement component I (IF) was investigated in 282 Chinese, 239 Koreans and 198 Japanese. The 3 common IF phenotypes (A, AB and B) and a new rare IF phenotype (BA2) were observed. The obtained allele frequencies are as follows: IF*A = 0.0993 and IF*B = 0.9007 in Chinese; IF*A = 0.0921 and IF*B = 0.9079 in Koreans; IF*A = 0.0985, IF*B = 0.8990 and IF*A2 = 0.0025 in Japanese. These 3 Mongoloid populations showed a much higher degree of IF polymorphism than Caucasian populations.     

Analysis of active and inactive complement C4 complotypes associated with subtypes of HLA-B17 in different racial groups.

HLA-A, B, and C antigens and the HLA-linked markers BF, C2, and C4 were determined in 1,799 unrelated Caucasians, 140 North American blacks, 140 Chinese, and 66 Japanese. One allele of the C4A locus (Rodgers), C4A*6, was found to code for a functionally inactive product in HLA-B17 (Bw57)-positive individuals, but for a functionally active product in HLA-B37- or HLA-B27-positive individuals. Further studies revealed that the functionally inactive C4A*6 gene product was found only in one subtype of HLA-B17, namely, 17.1 (Bw57, long). In addition, it was found that the frequency of the other subtype of HLA-B17, namely, 17.2(Bw58, short) varies greatly in different populations and that HLA-Bw58 is associated with either C4A*3,B*1 or C4A*3,B*Q0.     

C1R subcomponent polymorphism in Japanese: description of a new allele

The polymorphism of C1R was investigated in 570 unrelated Japanese individuals using isoelectric focusing and immunoblotting. A total of 11 different C1R phenotypes including a new pattern designated C1R 11-1 were observed. The allele frequencies were C1R*1 = 0.4561, C1R*2 = 0.3377, C1R*5 = 0.1956, C1R*8 = 0.0088 and C1R*R (C1R*9 and C1R*11) = 0.0018. The population data fitted the Hardy-Weinberg equilibrium. The C1R polymorphism in Japanese was shown to be controlled by 3 common alleles, C1R*1, C1R*2 and C1R*5, as compared to Caucasians where only the former 2 are present commonly. This complement system can be a useful genetic marker for anthropological studies.     

CYP2A6 polymorphism reveals differences in Japan and the existence of a specific variant in Ovambo and Turk populations

CYP2A6 is a polymorphic enzyme, and CYP2A6 genotype has been shown to be associated with smoking habits and lung cancer. We investigated CYP2A6 polymorphism in Japanese from four different geographic areas of Japan and in the Ovambo and Turk populations. Using two polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLPs), we identified the functionally important variants of CYP2A6: *1A, *1B, *1F, *1G, *4A, and *4D. In the Japanese population the highest frequencies of the CYP2A6*1A allele were observed in subjects from the Fukuoka (Kyushu Island) and Ehime (Shikoku Island) prefectures, whereas subjects in Shimane and Tottori (both located on the Japan Sea side of Honshu Island) showed the highest frequencies of the CYP2A6*1B allele. In the Tottori and Shimane groups no subject was homozygous for the CYP2A6*4A allele, a whole gene deletion type that is prevalent among Asians. In the Ovambo and Turk populations the CYP2A6*1A allele was predominant. Furthermore, two alleles undetected in the Japanese were observed in these latter two ethnic groups: CYP2A6*1G was found solely in the Ovambos, and CYP2A6*1F was found solely in the Turks. The present study is the first to show interprefecture differences in CYP2A6 polymorphism in Japanese who live in relatively close but distinct geographic areas; this is also the first study to evaluate CYP2A6 variations among these Japanese and the Ovambo and Turk populations. The distribution results of these alleles could help to define the true significance of CYP2A6 polymorphism as a genetic susceptibility marker in worldwide populations.     

Transferrin subtypes and variants in Germany; Further evidence for a Tf null allele

Summary Isoelectric focusing (IEF) with carrier ampholytes was used for the determination of transferrin C subtypes and transferrin B and D variants in a sample of 1125 unrelated individuals from Southern Germany. The observed TfC allele frequencies were Tf*C1=0.7872, Tf*C2=0.1365, and Tf*C3=0.0675. The rare C subtype C6 was observed twice. A new C subtype, called C10, was observed and identified by IEF with immobilized pH gradients. The rare C subtypes C4 and C8 were also studied by this method. TfB and TfD variants were found with a heterozygous frequency of 1.53%. One new TfD was found which is located between D1 and D2 and therefore named D1-2. Evidence for a Tf null allele was obtained in a child and the putative father; they were considered to be heterozygous for an allele Tf0. The theoretical exclusion rate for paternity examinations was calculated for the Tf system and found to be 17.95%.     

Distribution of transferrin subtypes in Tarragona (east Spain).

The distribution of transferrin (TF) subtypes was determined by isoelectric focusing of sera from 284 unrelated individuals from Tarragona (south of Catalonia). The allele frequencies observed, TF*C1 = 0.805, TF*C2 = 0.162, TF*C3 = 0.026 and TF*B = 0.007 were similar to those reported for other Spanish populations.     

To the research of the gene pool of the Gagauz population of Moldavia

Population genetic data on Gagauzes from Moldavia are reported here for the first time. AB0 and Rhesus blood groups, serum protein group (HP, TF, GC) and the red cell enzyme polymorphism PGM1 were determined in 190 Gagauzes. In addition to this the ability to taste PTC was tested. The following allele frequencies were found: AB0*0 = 0.5241, AB0*A = 0.3279, AB0*B = 0.1480; RH*D = 0.6083, RH*d = 0.3917; HP*1 = 0.3544, HP*2 = 0.6456; TF*C1 = 0.7472, TF*C2 = 0.1770, TF*C3 = 0.0730, TF*B = 0.0028; GC*1F = 0.1025, GC*1S = 0.5932, GC*2 = 0.3043; PGM*1+ = 0.5932; PGM*1- = 0.1000, PGM*2+ = 0.2607, PGM*2- = 0.1107. The frequency of the PTC*T allele was found to be 0.5298. These frequencies and genetic distance analyses show that the gene pool of the Gagauzes is similar to that of neighbouring southeastern European populations.     

Multiplex pyrosequencing method to determine CYP2C9*3, VKORC1*2, and CYP4F2*3 polymorphisms simultaneously: its application to a Korean population and comparisons with other ethnic groups

Warfarin is an anticoagulant that is difficult to administer because of the wide variation in dose requirements to achieve a therapeutic effect. CYP2C9, VKROC1, and CYP4F2 play important roles in warfarin metabolism, and their genetic polymorphisms are related to the variability in dose determination. In this study we describe a new multiplex pyrosequencing method to identify CYP2C9*3 (rs1057910), VKORC1*2 (rs9923231), and CYP4F2*3 (rs2108661) simultaneously. A multiplex pyrosequencing method to simultaneously detect CYP2C9*3, VKORC1*2, and CYP4F2*3 alleles was designed. We assessed the allele frequencies of the polymorphisms in 250 Korean subjects using the multiplex pyrosequencing method. The results showed 100 % concordance between single and multiplex pyrosequencing methods, and the polymorphisms identified by pyrosequencing were also validated with the direct sequencing method. The allele frequencies of these polymorphisms in this population were as follows: 0.040 for CYP2C9*3, 0.918 for VKORC1*2, and 0.416 for CYP4F2*3. Although the allele frequencies of the CYP2C9*3 and VKROC1*2 were comparable to those in Japanese and Chinese populations, their frequencies in this Korean population differed from those in other ethnic groups; the CYP4F2*3 frequency was the highest among other ethnic populations including Chinese and Japanese populations. The pyrosequencing methods developed were rapid and reliable for detecting CYP2C9*3, VKORC1*2, and CYP4F2*3. Large ethnic differences in the frequency of these genetic polymorphisms were noted among ethnic groups. CYP4F2*3 exhibited its highest allele frequency among other ethnic populations compared to that in a Korean population.     

Placental glucose dehydrogenase polymorphism in Japanese

The polymorphism of glucose dehydrogenase (GDH) was investigated in 516 Japanese placentae. The allele frequencies were GDH*1 = 0.510, GDH*2 = 0.488 and GDH*3 = 0.002. GDH*3 appears to increase from Japan via Southeast Asia and India to Europe.     

Genotoxic effects of alcohol in human peripheral lymphocytes modulated by ADH1B and ALDH2 gene polymorphisms

Ethanol is almost totally broken down by oxidative metabolism in vivo. Ethanol per se is considered to be neither carcinogenic, mutagenic nor genotoxic. However, during the metabolic conversion of ethanol to acetaldehyde and acetate, the organism is exposed to both ethanol and acetaldehyde and therefore ethanol is suspected to be co-carcinogenic. The genetic polymorphisms of alcohol dehydrogenase-2 (ADH1B) and acetaldehyde dehydrogenase-2 (ALDH2) influence the metabolism of alcohol. The ADH1B*1/*1 genotype encodes the low-activity form of ADH1B, and ALDH2*1/*2 and ALDH2*2/*2 genotype encode inactive ALDH2. The aim of this study was to test the hypothesis that polymorphisms of the ADH1B and ALDH2 genes are significantly associated with genotoxicity induced by alcohol drinking, measured using the cytokinesis-block micronucleus (CBMN) assay, an established biomarker of genome instability, in peripheral blood lymphocytes of 286 healthy Japanese men. There was a significant trend for the mean micronuclei (MN) frequency in habitual or moderate drinkers without a smoking habit to increase as the numbers of the *1 allele in ADH1B increased (P=0.039 or P=0.029) and the *2 allele in ALDH2 increased (P=0.019 or P=0.037). A logistic regression analysis showed that the number of subjects with MN frequency levels more than median value of MN (3.0) was significantly higher in the subjects with the ADH1B*1 allele as adjusted estimates (OR 2.08, 95% C.I. 1.24-3.48), when the OR for the subjects with the ADH1B*2/*2 genotype was defined as 1.00. The number of subjects with MN frequency levels more than median value of MN was also significantly higher in the subjects with the ALDH2*2 allele as adjusted estimates (OR 1.79, 95% C.I. 1.04-3.11), when the OR for the subjects with the ALDH2*1/*1 genotype was defined as 1.00. The results of this study have identified important novel associations between ADH1B/ALDH2 polymorphisms and genotoxicity in alcohol drinkers.     

Protein subtype polymorphisms (Gc and Tf) in a Catalan population from Gerona (northeastern Spain)

The Tf and Gc polymorphic subtype variants have been examined by means of isoelectric focusing in a population sample from two subpyrenean regions in the province of Gerona (Northeast Spain). The estimated allele frequencies were Tf*C1 = 0.774, Tf*C2 = 0.167, TF*C3 = 0.055, TF*B = 0.004; Gc*1F = 0.129, Gc*1S = 0.555 and Gc*2 = 0.316. These values are in general similar to those so far reported in other Spanish populations. The comparisons between our data and those published in Spain, indicate that the present sample is closer to Barcelona than to the other groups compared.     

CYP2D6 polymorphism and the presence of anti-LKM-1 in patients with chronic hepatitis C

Anti-LKM-1 autoantibodies are directed mostly at cytochrome P450 2D6 (CYP2D6) autoantigen, whose activity ranges from "complete deficiency" to "extensive metabolism" due to genetic polymorphism. We aimed to find any relevance of CYP2D6 alleles to the presence/absence of anti-LKM-1 in Japanese patients with chronic hepatitis C. The frequency of an extensive metabolizer-type allele (CYP2D6*1) in anti-LKM-1-positive patients was higher than that in anti-LKM-1-negative patients (0.800 vs 0.431; P = 0.0035), while the CYP2D6*10 allele with moderately reduced activity was less frequent in the former than the latter (0.050 vs 0.389; P = 0.0069). Moreover, the rate of homozygosity for CYP2D6*1 showed a striking difference between the two groups (70% vs 19%; P = 0.0021). These findings suggest that a genetic predisposition to produce the enzyme CYP2D6 of extensive metabolizer-type is associated with the induction of anti-LKM-1 in chronic hepatitis C patients.