Orosomucoid (ORM) typing by isoelectric focusing: an analysis of ORM haplotypes

A new separator isoelectric focusing method for typing of orosomucoid (ORM) was developed. This method provided a superior resolution of ORM patterns: two close bands of ORM1*5.2 products were clearly separated. A total of 364 subjects from Okinawa (Japan) were classified into 21 ORM phenotypes determined by 6 ORM1 and 7 ORM2 alleles including a polymorphic silent allele, ORM2*QO, and 2 new rare variants, ORM2*18 and ORM2*19. These phenotypes were also explained by 12 ORM haplotypes, half of which were polymorphic.     

Orosomucoid (ORM) typing by isoelectric focusing: evidence for two structural loci ORM1 and ORM2

Summary Orosomucoid (ORM) phenotyping was performed by isoelectric focusing and immunoprinting. The band patterns of desialyzed ORM indicated that the ORM system is controlled by two structural loci ORM1 and ORM2. In a total of 253 samples from two Caucasoid populations, five phenotypes determined by three polymorphic alleles, ORM1 ^*1, ORM1 ^*2, and ORM1 ^*3 were identified. The ORM1 ^*3 was characteristic of the Caucasoids. The ORM2 locus was monomorphic.     

Two new orosomucoid (ORM2) variants in Japanese

Orosomucoid (ORM) of plasma from 287 Japanese was typed by polyacrylamide gel isoelectric focusing followed by immunoprinting with specific antiserum to ORM. Two new variants were observed and they were designated ORM2 16 and ORM2 17.     

Orosomucoid (ORM) typing by isoelectric focusing: evidence for an additional duplicated ORM1 locus haplotype and close linkage of two ORM loci.

Human serum orosomucoid (ORM) exhibits a high variability. Several alleles including a duplicated ORM1 gene (ORM1*2.1) have been identified at two functional ORM loci, ORM1 and ORM2. In this study a modified isoelectric focusing, in which glycerin was omitted from gels, was used to differentiate a new variant ORM1 5 from ORM2 3. The ORM1 5 band was always observed together with the ORM1 2 band. The simultaneous expression could be explained in terms of an additional duplicated ORM1 locus haplotype, ORM1*5.2, whose average frequency was .016 in two Japanese populations. The ORM2*6 found at a polymorphic frequency (.023) was demonstrated to be in association with ORM1*2, indicating the close proximity between ORM1 and ORM2 loci.     

Orosomucoid (ORM1 and ORM2) types in the Spanish Basque Country, Galicia and northern Portugal

The genetic variation of orosomucoid (ORM1 and ORM2) in three south-western European populations (Galicia, Spanish Basque Country and northern Portugal) was investigated using hybrid isoelectric focusing. Three common ORM1 alleles were observed in these populations, the frequencies of ORM1 *S observed in Galicia and northern Portugal being the highest found among populations of European origin. Rare variants were observed for both the ORM1 and ORM2 loci.     

Three new orosomucoid (ORM) variants revealed by isoelectric focusing and print immunofixation

Summary Phenotypes of orosomucoid (ORM) in human sera have been analysed by isoelectric focusing and print immunofixation. After neuraminidase treatment the band patterns indicated that the polymorphism of the structural locus ORM1 is controlled by three autosomal codominant alleles. According to the previous nomenclature they were called ORM1^*F1, ORM1^*F2, and ORM1^*S. In a study of 272 unrelated individuals from southern Germany, five of the six expected common ORM1 subtypes were observed. Furthermore, we found three ORM variant phenotypes which have not been reported previously. These variants were characterized by additional bands in a cathodal position. One variant had additional double bands and presumably represents a rare ORM1 variant named ORM1S1. Two variants had additional single bands. They were assigned tentatively to the ORM2 gene locus. While the common gene product of ORM2 may be called ORM2A, the two variants are named ORM2B1 and ORM2B2, respectively. ORM2B1 has, thus far, been found only in a single individual; the variants ORM1S1 and ORM2B2 were found in a father-child pair and a mother-child pair, respectively. The frequency for variants tentatively assigned to the ORM2 locus is very low and was calculated to be 0.0037.     

Genetic Polymorphism of Cat (Felis catus) Plasma Orosomucoid

Genetic polymorphism of orosomucoid (ORM) was observed in 22 breeds of cats (Felis catus) using isoelectric focusing (pH 4.0–6.5) of desialylated plasmas followed by immunoblotting with rabbit antiserum to human ORM. From a total of 943 plasma samples examined, 15 phenotypes were identified and family studies demonstrated an inheritance of five codominant alleles, ORM^A, ORM^B, ORM^C, ORM^D, and ORM^E, at a single locus.     

Genetic polymorphisms of alpha-2-HS-glycoprotein, group-specific component and orosomucoid in the Han population, Chengdu, China.

The Han population in Chengdu, China, was investigated for genetic polymorphisms of alpha-2-HS-glycoprotein (A2HS), group-specific component (GC) and orosomucoid (ORM) using isoelectric focusing followed by immunofixation. The allele frequencies were: A2HS*1 = 0.6958,A2HS*2 = 0.3042, GC*1F = 0.4021, GC*1S = 0.3182, GC*2 = 0.2745, GC*1A = 0.0052, ORM1*F1 = 0.7028, ORM1*S = 0.2762, ORM1*F2 = 0.0210, ORM2*A = 0.9965, ORM2*Var = 0.0035.     

Orosomucoid typing of apes (family Pongidae) by isoelectric focusing: Among primates do only humans have two functional orosomucoid loci?

It has been demonstrated that human orosomucoid (ORM) is controlled by more than one functional loci, while Macaca ORM is controlled by one locus. To examine the time when the ORM gene was duplicated in the evolution of primates, plasma samples from 118 apes (family Pongidae) belonging to 4 genera and 12 species were investigated for ORM polymorphism using isoelectric focusing followed by immunoprinting. The band patterns of ORM in the subfamily Ponginae showed quantitatively different products as in humans. A pedigree study of common chimpanzees supported the two-locus model for ORM. Gibbons (subfamily Hylobatinae) displayed highly variable band patterns, but the number of loci was not determined unequivocally. Thus, this study shows that duplication of the ORM gene in primates occurred either before or after the divergence of Hylobatinae and Ponginae, consistent with a previous prediction from the molecular evolutionary rate of ORM.     

Distribution of ORM1, C6, C7 and APO C-II allele frequencies in populations from mainland Italy and Sardinia.

The genetic variation of the human plasma proteins ORM1, C6, C7 and APO C-II was investigated by isoelectric focusing followed by immunoblotting in populations from mainland Italy and Sardinia. The frequencies of ORM1*1 were 0.621 and 0.564, while those of C6*A were 0.657 and 0.706 on mainland Italy and in Sardinia, respectively. In the Roman sample, 8 heterozygotes with C6 variant alleles were encountered, while none were observed in Sardinians. For C7 and APO C-II a number of heterozygotes with the rare alleles C7*2 and APO C-II*2 were found, but their frequency did not reach polymorphic levels in either population. The two populations showed a significant difference in the gene frequencies distribution for ORM1.     

The rearrangement of the human alpha(1)-acid glycoprotein/orosomucoid gene: evidence for tandemly triplicated genes consisting of two AGP1 and one AGP2

The human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is controlled by the two tandemly arranged genes, AGP1 and AGP2. The further duplication of the AGP1 gene has been suggested by a few duplicated ORM1 locus haplotypes including ORM1*F1. S and ORM1*B9. S, detected by isoelectric focusing. To clarify the triplication of the AGP gene, 39 DNA samples from Japanese subjects were studied by the long-range PCR of intergenic regions. The analysis of PCR products showed that the tandemly triplicated genes, AGP1A-AGP1B-AGP2, occurred on about 20% of chromosomes. These composites were divided into ORM1A*F1-ORM1B*S-ORM2*M and ORM1A*B9-ORM1B*S-ORM2*M by allelic variations. Furthermore, the former was classified into a few haplotypes by three synonymous sequence variations, which might have arisen through gene conversion-like events. The recombination breakpoints existed between the 5' flanking region and intron 2 of the AGP1B gene. Thus, it is likely that the rearrangement of the AGP gene has often occurred.     

Method for the typing of the C3 polymorphism in stored sera by means of isoelectric focusing

A method is described by which the three common phenotypes of C3 can be typed by desialylation and isoelectric focusing in serum samples stored at -20 degrees C for several years.     

A/G Gln20Arg (exon 1) and G/A Val156Met (exon 5) polymorphisms of the human orosomucoid 1 gene in Mexico

The human orosomucoid 1 gene (ORM1) codes an alpha-1-acid glycoprotein that has been classified as an acute-phase reactive protein, and a major drug-binding serum component, as well as an immunomodulatory protein with genetic polymorphisms. Evaluation of ORM variation through isoelectric focusing and immunobloting has revealed a world-wide distribution of the ORM1 F and ORM1 S alleles. We evaluated and examined the genetic characteristics of two Mexican populations that have different anthropological and cultural antecedents, examining two ORM1 genotypes (exon 1 - A/G (Gln20Arg) and exon 5 G/A (Val156Met)) in 145 individuals, using nested polymerase chain reaction, sequencing, and restricted fragment length polymorphism. Mexican Mestizos had higher frequencies of the exon 1 A allele (P = 0.020) and AA genotype (P = 0.018) and lower frequency of the G allele (P = 0.020) when compared to Teenek Amerindians. When we examined exon 5 G/A (Val156Met) polymorphisms, we found significantly higher frequencies of the G allele (P = 0.0007) and the GG genotype (P = 0.0003) in the Mexican Mestizo population. The Teenek population had a significantly higher frequency of the A allele than has been reported for Chinese and African (P < 0.05) populations, and the G/A genotype was more frequently found in this Mexican population than in Chinese, African and European populations (P < 0.05).     

Apolipoprotein A-IV polymorphism in the Finnish population: gene frequencies and description of a rare allele

The apolipoprotein A-IV (apoA-IV) allele frequencies were determined in 387 adult Finns by immunoblotting after isoelectric focusing of serum. The gene frequencies were: A-IV1 = 0.942 and A-IV2 = 0.058. The phenotypes of 147 mother-child pairs studied were in accordance with the two allelic modes of inheritance. In 2 subjects, a rare apoA-IV variant was found.     

Orosomucoid (ORM) typing by isoelectric focusing: evidence for gene duplication of ORM1 and genetic polymorphism of ORM2

Summary It has been demonstrated that the genetic polymorphism of human serum orosomucoid (ORM) is controlled by polymorphic ORM1 and monomorphic ORM2 loci. In this study a Japanese family was encountered in which several members had puzzling electrophoretic patterns consisting of four bands. The ORM patterns were due to the products of a duplicated ORM1 locus haplotype (ORM1 ^* 2·1) or the products of new variant alleles at the ORM2 locus. The ORM1 ^* 2·1 haplotype is very common in the Japanese population, occurring at an allele frequency of 0.16. The increased occurrence of ORM1 2-1 and the heterogeneity in band intensity among ORM1 2-1 phenotypes could be explained in terms of a duplicated gene ORM1 ^* 2·1. The ORM2 locus proved to be polymorphic, with six alleles in the Japanese population. Dedicated to Professor Dr. K. Nishigami on the occasion of his 60th birthday     

Orosomucoid (ORM) typing by print lectinofixation: a new technique for isoelectric focusing. Two common alleles in Japan

Summary The genetic types of orosomucoid (ORM) were analyzed by isoelectric focusing (IEF) on polyacrylamide gels and subsequent print lectinofixation with a lectin from the beetle, allo A. In this paper, the newly devised print lectinofixation for ORM typing is described. This technique is faster, easier to perform, and has been found to be a useful tool in population genetics and forensic medicine. The results of typing for two alleles, ORM ^*1 and ORM ^*2 are described for a population of Northern Japan (n=500). We use the designation “lectinofixation” to denote the method using lectin in place of monospecific antibody in the immunofixation     

Changes in expression and microheterogeneity of the genetic variants of human α1-acid glycoprotein in malignant mesothelioma

Human α₁-acid glycoprotein (AAG), an acute-phase plasma protein, is heterogeneous in the native state and polymorphic in the desialylated state. The AAG heterogeneity is mainly explained by a variable glycan chain composition in its five glycosylation sites. The AAG polymorphism is due to the presence of genetic variants. Three main variants are observed for AAG, ORM1 F1, ORM1 S and ORM2 A, which have a separate genetic origin. In this paper, we have used different isoelectric focusing (IEF) methods and chromatography on immobilized metal affinity adsorbent to study the relative occurrence of the genetic variants of AAG in relation to changes in microheterogeneity, in plasma and pleural effusions of patients with malignant mesothelioma (MM). The results were compared to those obtained with the variants in plasma of healthy individuals. Significant changes in variant distribution were observed in the MM samples, that corresponded to a rise in the proportion of the ORM1 variants and a fall in that of the ORM2 variant. However, the concentration in MM plasma increased for both variants. The AAG in MM plasma and effusion fluids was found to be more heterogeneous on IEF than AAG of healthy plasma. The evidence of stronger concentrations of both the high and low pI forms of AAG in the MM samples suggested two kinds of changes in charge heterogeneity. These two changes were shown to be attributed to different variants — i.e. the high pI forms to ORM1 F1 and S and the low pI forms to ORM2 A, after fractionation of AAG by chromatography on immobilized copper(II) ions. These results indicate specific changes in both the expression and glycosylation for each AAG variant, according to its separate genetic origin, in MM.     

Changes in relative mobility of pancreatic amylase variants in isoelectric focusing

Using isoelectric focusing with one ampholytic solution, double- and single-banded amylase phenotypes were found in a sample of rhesus monkeys,Macaca mulatta. When applying different ampholytic solutions, these variants were shown to change their position relative to each other. Single-banded phenotypes showed either a position corresponding to one of the bands of the double-banded phenotype or to an intermediate one. Family studies, however, suggested that the differences between the observed patterns were not caused by genetic differences. This discloses a problem with respect to the interpretation of electrophoretic data, i.e. bands with different positions produced by isoelectric focusing may not necessarily represent genetic differences.     

Genetic studies of low-abundance human plasma proteins. V. Evidence for a second orosomucoid structural locus (ORM2) expressed in plasma.

Orosomucoid (ORM) or alpha-1-acid glycoprotein is an acute-phase protein of human plasma whose function is suggested to be the competitive inhibition of cellular recognition by infective agents. Genetically determined variation in ORM has been reported, with two major alleles segregating in all populations studied to date. Isoelectric focusing-immunoblotting studies of ORM revealed the presence of isoprotein species that did not segregate with the predominant alleles at the ORM locus and suggested the expression of a second structural gene locus for orosomucoid (ORM2). Genetically independent variation consistent with expression of the ORM2 locus was observed in plasma samples from American blacks but was not observed in U.S. whites or sampled populations of North- and South-American Indians, Eskimos, Aleuts, or New Guinea Highlanders. The population allele frequencies for this locus were .958, .025, .006, and .011 for alleles ORM*1, ORM2*2, ORM2*3, and ORM2*4, respectively. Family studies confirm the autosomal codominant inheritance of the observed phenotypes.     

Identification of sunfish species by muscle protein isoelectric focusing

Muscle protein phenotypes of nine recognized Lepomis species were developed by isoelectric focusing. Four classes were described based on putative parvalbumin patterns. All nine species could be distinguished by their protein phenotypes. Natural hybrids and their parentage were also identified, and the utility of this technique in studying natural sunfish hybridization is discussed.