Molecular basis of inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) polymorphism

A genetic polymorphism of the inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) was analyzed at the nucleic acid level. Three common alleles, ITIH1*1, ITIH1*2 and ITIH1*3, were characterized by mutations at codons 551 and 561 in exon 14. ITIH1*1 was characterized by GAG (Glu) at codon 551 and CAG (Gln) at codon 561, ITIH1*2, by GTG (Val) and CGG (Arg), and ITIH1*3, by GAG (Glu) and CGG (Arg).     

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.     

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 (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 (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.     

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).     

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 1) subtyping and formal genetics

Summary Orosomucoid (ORM) phenotyping has been performed in 141 families with 407 children from southwest Germany. Eight families were observed in which duplicated ORM1 genes, F1F2, F1F3, segregated. The family data gave no information about the presence of tandemly duplicated ORM1 F1F4 and ORM1 F1F5 genes. To date, the segregation of the phenotypes of the children agrees with the extended formal model: two ORM1 loci with two common (^*F1, ^*S) and several rare (^*F1F2, ^*F1F3, ^*F4, ^*F5) alleles. The parental allele frequencies were calculated by gene counting as ORM1 ^*F1 = 0.5781, ^*S = 0.3901, ^*F1F2 = 0.0195, ^*F4 = 0.0053, ^*F1F3 = 00.0035, ^*F5 = 0.0035.     

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.     

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.     

Identification and characterization of polymorphisms at the HAS alpha1-acid glycoprotein (ORM*) gene locus in Caucasians

Human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a group of healthy Caucasians. Polymorphisms were identified using BamHI, EcoRI, BglII, PvuII, HindIII, TaqI and MspI. Nonrandom associations were found between the BamHI, EcoRI and BglII RFLPs. The RFLPs detected with PvuII, TaqI and MspI were all located in exon 6 of both AGP genes. The duplication of an AGP gene was observed in 11% of the individuals studied and was in linkage disequilibrium with the TaqI RFLP. The identification and characterization of these polymorphisms should prove useful for other population and forensic studies.     

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.     

Molecular analysis of the CYP2F1 gene: identification of a frequent non-functional allelic variant

The CYP2F1 is a human cytochrome P450 that is selectively expressed in lung tissue and involved in the metabolism of various pneumotoxicants with potential carcinogenic effects. In the present study, we report the first systematic investigation of the genetic polymorphism of this enzyme. We analyzed the nucleotidic sequence of the CYP2F1 gene in DNA samples from 90 French Caucasians consisting in 44 patients with lung cancer and 46 control individuals, using single-strand conformation polymorphism analysis of PCR products (PCR-SSCP). We identified 24 novel mutations distributed in the promoter region of the gene, as well as in the coding regions and their flanking intronic sequences. In addition to the wild-type CYP2F1*1 allele, seven allelic variant, CYP2F1*2A, *2B, *3, *4, *5A, *5B and *6, were characterized. The most frequent allelic variant, CYP2F1*2A (25.6%), harbors a combination of 9 mutations, including 2 missense mutations (Asp218Asn and Gln266His) and a 1-bp insertion (c.14_15insC) that creates a premature stop codon in exon 2, probably leading to the synthesis of a severely truncated protein with no catalytic activity. The identification of around 7% of homozygotes for the frameshift mutation in our Caucasian population suggests the existence of an interindividual variation of the CYP2F1 activity and, consequently, the possibility of interindividual differences in the toxic response to some pneumotoxicants and in the susceptibility to certain chemically induced diseases. However, our preliminary results did not show any evidence that the CYP2F1 genetic polymorphism has implications in the pathogenesis of lung cancer.     

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.     

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.     

猪POU1F1基因部分序列变异和同源性分析

对长白、杜洛克、约克夏、姜曲海、梅山和香猪等六个猪种的POU1F1基因第四、第五和第六外显子分别进行PCR扩增,并对含有第四、第六外显子的PCR产物和含有第五外显子的克隆产物进行测序。结果表明：六个猪种中,POU1F1基因的第四外显子存在碱基突变,为T→C。对该序列进行Nla Ⅲ 酶消化,产生两种不同的基因型(GG和HH);而第五和第六外显子则高度保守,未发现任何突变。将人POU1F1基因第四外显子、POU同源区核苷酸编码序列和氨基酸序列,分别与猪、小鼠、牛的POU1F1基因相应的核苷酸序列和氨基酸序列进行同源性比较,结果发现：人与猪、小鼠、牛的POU1F1基因第四外显子的核苷酸同源性分别高达93.9%、86.7%、92.1%,而由第四外显子编码的部分POU特异区的氨基酸序列则完全一致;人与猪、小鼠、牛POU同源区的核苷酸同源性分别为91.4%、85.1%、87.9%,氨基酸同源性分别为96.6%、94.8%、90.2%。这说明在哺乳动物中,其POU1F1蛋白的POU同源区和由第四外显子编码的POU特异区部分是高度保守的;猪可作为实验动物,建立人类相关疾病模型,为医学研究提供参考依据。     

Extended HLA-DPB1 polymorphism: an RNA approach for HLA-DPB1 typing

Most of the 119 human leukocyte antigen (HLA)-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We investigated how DPB1 polymorphism is represented in the entire coding region. An RNA sequencing-based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5′ untranslated region (UTR) through the 3′-UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5′ and 3′-UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages.     

Full-length sequence analysis of the HLA-DRB1 locus suggests a recent origin of alleles

The HLA region harbors some of the most polymorphic loci in the human genome. Among them is the class II locus HLA-DRB1, with more than 400 known alleles. The age of the polymorphism and the rate at which new alleles are generated at HLA loci has caused much controversy over the years. Previous studies have mostly been restricted to the 270 base pairs that constitute the second exon and represent the most variable part of the gene. Here, we investigate the evolutionary history of the HLA-DRB1 locus on the basis of an analysis of 15 genomic full-length alleles (10-15 kb). In addition, the variation in 49 complete coding sequences and 322 exon 2 sequences were analyzed. When excluding exon 2 from the analysis, the diversity at the synonymous sites was found to be similar to the intron diversity. The overall diversity in noncoding region was also similar to the genome average. The DRB1*03 lineage has been found in human, chimpanzee, bonobo, gorilla, and orangutan. An ancestral "proto HLA-DRB1*03 lineage" appeared to have diverged in the last 5 million years into the human-specific lineages *08, *11, *13, and *14. With exception to exon 2, both the coding- and the noncoding diversity suggests a recent origin (<1 million years ago) for most of the alleles at the HLA-DRB1 locus. Sites encoding for amino acids involved in antigen binding [antigen recognizing sites (ARS)] appear to have a more ancient origin. Taken together, the recent origin of most alleles, the high diversity between allelic lineages, and the ancient origin of sequence motifs in exon 2, is consistent with a relatively rapid generation of novel alleles by gene conversion like events.