A new method for FXIIIA genetic variants determination using isoelectric focusing in 1 M urea

Summary A new method for separating genetic variants of the A subunit of human coagulation factor XIII using ultrathin layer polyacrylamide gel isoelectric focusing in 1M urea followed by immunoblotting is described. The pattern obtained by this method differs from that reported previously: Three sets of unrelated band patterns are observed and can be explained by the existence of two additional gene loci, designated FXIIIA2 and FXIIIA3, besides the previously reported FXIIIA locus, now renamed FXIIIA1. The FXIIIA2 locus is polymorphic and shows three commonly occurring phenotypes, FXIIIA2 1, FXIIIA2 2-I, and FXIIIA2 2. These are determined by two common alleles, FXIIIA2^*1 and FXIIIA2^*2, with respective frequencies of 0.7965 and 0.2035 in the Japanese population. The studied population conforms to a Hardy-Weinberg equilibrium, and family data confirmed autosomal codominant transmission. The FXIIIA3 locus is monomorphic.     

PGM1 system. Report on the international workshop, October 10-11, 1983, Munich, West Germany.

Because of the increase in the number of PGM1 polymorphisms and the existence of four distinct nomenclatures for expressing subtypes by isoelectric focusing, a nomenclature workshop was held in 1983 to compare variants and arrive at a single method for reporting PGM1 data. A total of 30 rare variants were identified and the recommended method for expressing the four common alleles is PGM1*1A, PGM1*1B, PGM1*2A, and PGM1*2B.     

FXIIIA subtypes in Indonesians, Bangladeshis, Tibetans, South African blacks and South African whites

The polymorphism of FXIIIA was investigated in 105 Indonesians, 141 Bangladeshis, 186 Tibetans, 101 South African Blacks and 100 South African Whites using isoelectric focusing followed by immunoblotting. These population data conformed to the Hardy-Weinberg equilibrium. A circum-pan-Pacific cline in the FXIII*1A (or FXIII*1B) allele frequency appeared to exist among the Mongoloids.     

Genetic polymorphism of human factor H (beta 1H globulin)

Polyacrylamide gel isoelectric focusing (PAGIEF) of EDTA plasma and neuraminidase-treated plasma samples at pH 3.5-9.5 containing 8.0 M urea followed by an electroblotting with enzyme immunoassay was applied for the detection of factor H (HF) phenotypes in 536 unrelated Japanese blood donors living in Tokyo. In the major cathodal components, phenotypes of HF were classified into three common and five rare patterns, and these were considered to be controlled by two common and two rare alleles. The data suggest that the HF*Q0 allele also exists in the Japanese population. Family studies confirm the hypothesis that the HF polymorphism is controlled by autosomal codominant Mendelian inheritance.     

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.     

Allele frequencies of human complement factor I in a sample from Iwate, northern Japan, with the description of geographical cline.

Serum samples from 270 healthy blood donors of Iwate prefecture, northern Japan, were examined for polymorphism of factor I (IF) by using polyacrylamide gel isoelectric focusing followed by semidry horizontal electroblotting with enzyme immunoassay. In 270 individuals four different patterns were observed, and these were controlled by two common alleles, IF*A and IF*B, and one rare allele, IF*A2. Allele frequencies were estimated to be 0.1019, 0.8963 and 0.0018 for IF*A, IF*B and IF*A2, respectively. The data of IF allele frequencies thus far reported in Japan excluding Okinawa Island were compared, and a statistically significant (p less than 0.01) geographical cline was detected for IF*A and IF*B alleles.     

Biochemical and genetic studies on GP43, a 43-kD glycoprotein detected immunologically in human urine and serum

A new and previously undescribed glycoprotein with a molecular weight of 43,000 has been isolated from human urine. This protein, designated GP43; copurified with ribonuclease, which has the same molecular weight, but ribonuclease activity was removed by passage through an affinity column of agarose-5'-(4-aminophenyl phosphoryl) uridine 2'(3') phosphate. GP43 contains about 5.9% neutral sugar, 2.3% hexosamine, and 1.6% sialic acid. A rabbit antibody to the purified GP43 reacted with human urine and serum as well as with the purified GP43. The genetic polymorphism of GP43 was then studied in desialylated human serum samples by urea-polyacrylamide gel isoelectric focusing, followed by immunoblotting with the specific antibody for GP43. Three common phenotypes, designated GP43 1, 1-2, and 2, were easily recognized using this technique and represented homozygosity or heterozygosity for two autosomal codominant alleles, GP43*1 and GP43/2. The frequencies of the GP43*1 and GP43*2 alleles in a Japanese population were 0.7683 and 0.2317, respectively.     

Genetic studies of low abundance human plasma proteins. III. Polymorphism of the C1R subcomponent of the first complement component.

Genetic polymorphism of the C1R subcomponent of human complement component C1 has been detected in normal plasma samples using the high resolving power of isoelectric focusing in 6 M urea followed by immunoblotting. There are two common alleles at the C1R structural locus that show autosomal codominant inheritance. The C1R*1 and C1R*2 allele frequencies in U.S. white and U.S. black blood donors are: .934, .066, and .899, .101, respectively.     

Factor I (C3b inactivator) polymorphism among five populations in Eurasia

Factor I (C3b inactivator) polymorphism in the Japanese (in Western and Southern Japan), Taiwanese, Nepalese and French was studied using isoelectric focusing on polyacrylamide gels. The exposure of passively blotted nitrocellulose membranes to glutaraldehyde vapor facilitated the subsequent immunodetection of a low concentration of factor I and permitted the reliable identification of the three phenotypes determined by two codominant alleles FI*A and FI*B. The data indicated a west-to-east genocline, ranging from France to Western Japan, in which FI*A changed from 0.006 to 0.120.     

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A¹ (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.     

An extended survey of the genetic polymorphism at the human coagulation factor XIII: a subunit structural locus

The distribution of the three previously reported alleles, with normal products at the factor XIII A subunit structural locus, FXIIIA*1, FXIIIA*2 and FXIIIA*4 has been studied in populations from the region extending from the Indonesian archipelago through Papua New Guinea, Australia and New Zealand to the Pacific Islands of Micronesia, Melanesia and Polynesia. In addition a population from the Caspian Littoral of Iran and a population of South American Indians were studied. The FXIIIA*1 and FXIIIA*2 alleles were polymorphic in all populations studied. The distribution of the FXIIIA*4 allele suggests that it may be a Melanesian marker.     

Two new Bf S subtypes revealed by isoelectric focusing and immunofixation

Summary The genetic types of the properdin factor B were analyzed by isoelectric focusing on polyacrylamide gels and subsequent immunofixation. Sera from 516 unrelated, healthy individuals from Southern Germany were examined. Two new subtypes of the Bf*S allele were observed. They were provisionally named Bf*Sb1 and Bf*Sb2 (b = basic), since the position of their bands is located slightly towards the cathode. Whereas Bf Sb2 has, thus far, been found only in a single individual, Bf Sb1 was found in five unrelated persons and in a mother and her child indicating a simple codominant mode of inheritance. The combined frequency of the Bf*Sb alleles was calculated to be 0.0067.     

Genetic polymorphism of human plasminogen.

Using isoelectric focusing (IEF) in polyacrylamide gel of neuraminidase-treated serum or plasma samples and immunofixation or caseinolytic overlay after urokinase activation of gels, a common genetic polymorphism in human plasminogen has been delineated. Two alleles PLGN*A and PLGN*B, were observed with gene frequencies in whites of .69 and .30; in Orientals of .96 and .03; and in blacks of .80 and .18. Several rare alleles were also found. The distribution of phenotypes fits the Hardy-Weinberg equilibrium. Inheritance is autosomal codominant and fits the expectations of Mendelian inheritance. There is fetal synthesis, but no transplacental passage of plasminogen in either direction.     

Isoelectric focusing studies of human red cell PGM1 in Japanese, with special reference to the characterization of PGM17

The distribution of phosphoglucomutase (PGM1) subtypes in human red cells was determined by isoelectric focusing in 218 Japanese samples. Nine common phenotypes were observed corresponding to the following frequencies of the four alleles at the PGM1 locus: PGM11+ 0.6560, PGM11- 0.1170, PGM12+ 0.1674 and PGM12- 0.0505. In addition, a characterization of the PGM17 allele was performed. Our results obtained in the present study revealed the possibility that the PGM17 allele may be differentiated in the two alleles of PGM17+ and PGM17- through an investigation of isoelectric focusing.     

Alpha-2-HS-glycoprotein polymorphism detected in human urine by isoelectric focusing and immunoblotting

Polymorphism of alpha 2-HS-glycoprotein (AHSG) was revealed in human urine by isoelectric focusing and immunoblotting on polyacrylamide gels. More than 200 urine samples were examined in this manner and correct AHSG typing of the urine samples was achieved, in comparison with the results of direct grouping for plasma. Three phenotypes, AHSG 1, 2-1 and 2, were observed and found to be determined by two common alleles, AHSG*1 and AHSG*2. The frequencies of AHSG*1 and AHSG*2 calculated in a Japanese population were 0.7637 and 0.2363, respectively.     

Genetic polymorphism of human factor I (C3b inactivator)

Summary Genetic polymorphism of human factor I (C3b inactivator) has been described using polyacrylamide gel isoelectric focusing electrophoresis of neuraminidase-treated EDTA plasma samples followed by electrophoretic blotting technique. In 435 individuals three different common patterns were observed, and these were controlled by two common alleles at a single locus. The results of typing family material confirmed autosomal codominant Mendelian inheritance. Two common alleles were designated FI^*B and FI^*A, and gene frequencies were estimated to be 0.8931 and 0.1069 for FI^*B and FI^*A, respectively. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium. Linkage studies failed to show close linkage between factor I and the major histocompatibility complex.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

Genetic polymorphism of the seventh component of complement in a Japanese population

Summary Genetic polymorphism of C7 in a Japanese population has been described, using polyacrylamide gel isoelectric focusing electrophoresis followed by an electrophoretic blotting technique. Phenotypes of C7 were classified into six common patterns, and observed phenotypes were produced by autosomal codominant at a single locus with three alleles. Three common alleles, designated C7^*B, C7^*M and C7^*A, were found, and gene frequencies calculated from 494 individuals showed C7^*B=0.858, C7^*M=0.096 and C7^*A=0.046, respectively. It is noteworthy that both C7^*M and C7^*A have polymorphic frequencies in the Japanese population. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium. Results indicate that the electrophoretic blotting technique, which has high specificity and sensitivity, is applicable in the study of heterogeneity of protein antigens.     

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.     

Genetic polymorphism of human C4-binding protein

Two different forms of human C4-bp, C4-bp A and C4-bp B, have been identified by isoelectric focusing (IEF) of neuraminidase-treated EDTA-plasma samples. Family studies demonstrate Mendelian segregation of these forms, indicating that they are under gentic control. This conclusion is supported by IEF analysis of the two variants purified by affinity chromatography. Under completely denaturing conditions, C4-bp B was found to be composed of two subunits that focused at different pH, whereas C4-bp A contains only the more basic one. These results suggest that a single autosomal locus with at least two codominant alleles coding for the subunits controls the IEF variation of C4-bp in humans. The allele designated C4BP*1 codes for a subunit that, after neuraminidase treatment, focuses at pH = 6.65. The allele C4BP*2 codes for a different subunit that focuses at pH = 6.60. The C4-bp A phenotype corresponds to the genotype C4BP*1,C4BP*1 and the phenotype C4-bp B to the genotype C4BP*1,C4BP*2. The phenotype corresponding to the C4BP*2,C4BP*2 homozygous genotype has not been encountered thus far. Initial linkage data indicate that the C4BP locus is not closely linked to either the HLA or to the C3 loci.