Genetic aspects of vitamin D-deficient rickets: genetic markers of blood]

Polymorphism of the AB0 blood groups, haptoglobin Hp, vitamin-D-binding protein (Gc), transferrin (Tf), alpha 1-antitrypsin (alpha 1-AT) and serum alkaline phosphatase (Pp) was studied in a group of children suffering from rickets (VDDR) and in a adequate control group of healthy individuals of the same sex-age composition. Considerable differences were revealed between the VDDR patients and healthy individuals in frequencies of the PIM1 and PIM2 factors on the alpha 1-AT system, r and p of the AB0 system as well as the Hp. Increase in a portion of one of the homozygotes for the Hp and for the alpha 1-AT system took place at the expense of other homozygote proportion (the latter being decreased). Heterozygotes frequencies remained intact in both compared groups. Atypical combination of phenotypes and gene frequencies was observed in a group of patients in the alpha 1-AT and AB0 systems as compared with usual distribution in European population. Higher frequencies of rare alleles of the loci under study were observed in the VDDR patients, which is partially reflected in increase in heterozygosity level in total within a cogort of patients analysed. Combination of the Hp 1-1 (Hp)--A(AB0)--M2M2 (alpha 1-AT) factors should be considered as unfavourable in rickets prognosis.     

Serum protein groups in renal cell carcinoma

Hp, C3, Gc and Tf serum groups were determined in 66 patients with renal cell carcinoma. Previously reported associations between renal cell carcinoma and the C3F and Gc2 genes were not confirmed. Among female patients a significant excess of GcIF was observed whereas male patients showed a significant excess of TfC3.     

Serum protein polymorphisms in four populations of Afghanistan.

Gene frequencies of the serum proteins third component of complement (C3) transferrin (Tf), haptoglobin (Hp), group specific component (Gc), serum cholinesterase (E1), alpha1-antitrypsin (Pi), beta2-glycoprotein I (Bg), and ceruloplasmin (Cp) in the Tajiks, Pushtoons, Hazaras, and Usbeks in Afghanistan were reported. Rare variants were observed in the C3, Tf, and Pi systems.     

Distribution of ABO, MN, Rh blood groups and Hp, Tr, Gc and C3 serum factors in the Buryat population

Polymorphism of blood groups ABO, MN, Rh and serum proteins Hp, Tf, Gc, C3 was studied in Buryat populations of Zabaikalie, Pribaikalie, Olkhon island. No indication of significant heterogeneity was observed. Gene frequencies varied in different systems within the ranges: ABO (p-0.142-0.183; q-0.205-0.324; r-0.567-0.630); MN (m-0.531-0.624), Rh(d) (0-0.214), Hp (Hp 1-0.268-0.339), C3 (C3F-0.023-0.090), Tf (TfC-0.971-1.0), Gc (Gc1-0.728-0.840). Genetic distances between main Buryat groups were estimated.     

Serum protein and red cell enzyme polymorphisms in affective disorders

Frequencies of serum groups (Hp and Gc) and red cell enzyme types (PGM1, 6-PGD and ES D) were studied in 195 patients with affective disorders. The patients were classified into four groups: (1) bipolar (manic-depressive) psychosis; (2) unipolar, recurrent, depressive psychosis; (3) non-psychotic reactive depression, and (4) unclassifiable. The Hp2 gene was increased in reactive and unclassifiable patients, the PGM1 1 gene was increased in bipolar patients and the ES D1 gene in reactive patients. No associations were found between affective disorders and the Gc and 6-PGD systems.     

Serum protein polymorphisms in a Liberian population

Serum protein variations were studied in a Liberian population living in Buchanan town. Of the alpha 1-antitrypsin genes only M1 and M3 were polymorphic. The frequencies of the haptoglobin and Gc genes were in accordance with earlier known estimates in African populations. There was, however, a relatively low frequency of Hp 0 which may be related to the low malarial parasite prevalence in this group. The transferrin C2 gene was found in a significantly lower frequency among Liberians compared to European and Asiatic populations. A new transferrin variant was observed by isoelectric focusing. This variant could not be identified with conventional starch or polyacrylamide electrophoresis.     

Group-specific component, alpha1-antitrypsin and esterase D in Canadian Eskimos.

Three genetic markers - group-specific component (Gc), alpha1-antitrypsin, and esterase D - were examined in a population of Eskimos from Igloolik in the eastern Canadian Arctic. Gc and esterase D were found to be polymorphic. In addition to the common Gc types, an anodal variant called Gc Igloolik was found, probably identical to previously reported Gc Eskimo. Gene frequencies were Gc1: 0.6524, Gc2: 0.3373, GcIgl: 0.0104, for 338 Eskimos. Genetic types of alpha1-antitrypsin (Pi types) were mostly M, with two MS sibs who were half Caucasian, in 170 Eskimos. Frequencies of the esterase D allele in 336 Eskimos were EsD1: 0.7083, EsD2: 0.2917. The frequencies of Gc2 and EsD2 are both higher than are found in Caucasian populations.     

Dissection and cytological mapping of barley chromosome 2H in the genetic background of common wheat

We used gametocidal (Gc) chromosomes 2C and 3C(SAT) to dissect barley 2H added to common wheat. The Gc chromosome induces chromosomal breakage resulting in chromosomal aberrations in the progeny of the 2H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying structurally rearranged aberrant 2H chromosomes and characterized them by sequential C-banding and in situ hybridization. We established 66 dissection lines of common wheat carrying single aberrant 2H chromosomes. The aberrant 2H chromosomes were of either deletion or translocation or complicated structural change. Their breakpoints were distributed in the short arm (2HS), centromere (2HC) and the long arm (2HL) at a rough 2HS/2HC/2HL ratio of 2:1:2. We conducted PCR analysis of the 66 dissection lines using 115 EST markers specific to chromosome 2H. Based on the PCR result, we constructed a physical or cytological map of chromosome 2H that were divided into 34 regions separated by the breakpoints of the aberrant 2H chromosomes. Forty-seven markers were present in 2HS and 68 in 2HL. We compared the 2H cytological map with a previously reported 2H genetic map using 44 markers that were used in common to construct both maps. The order of markers in the distal region was the same on both maps but that in the proximal region was somewhat contradictory between the two maps. We found that the markers distributed rather evenly in the genetic map were actually concentrated in the distal regions of both arms as revealed by the cytological map. We also recognized an EST-marker or gene-rich region in the 2HL interstitial region slightly to the telomere.     

Induction of haptoglobin by all-trans retinoic acid in THP-1 human monocytic cell line

Haptoglobin (Hp) is one of the acute-phase proteins and is mainly synthesized in the liver. During our study on the differentiation of leukemia cells, we have found that Hp is synthesized in human monocytic cells by all-trans retinoic acid (ATRA). The synthesis of Hp by ATRA is induced in a dose- and time-dependent manner. Hp cDNA cloned from ATRA-treated THP-1 cells corresponds to the Hp alpha 2(FS)-beta form. Whereas ATRA acted as a strong inducer in THP-1 cells, IL-1 beta, TNF-alpha, IL-6, and LPS had little effect on Hp gene expression in these cells. These findings suggest that THP-1 cells express the Hp gene through a signal pathway different from hepatocytes, and that ATRA is a potent Hp-inducer in these cells.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.     

Evolution of haptoglobin: comparison of complementary DNA encoding Hp alpha 1S and Hp alpha 2FS.

Haptoglobin is a transport glycoprotein which removes free hemoglobin from the circulation of vertebrates. In human populations haptoglobin is polymorphic due to three alleles, Hp alpha 1F, Hp alpha 1S and Hp alpha 2. The Hp alpha 2 allele is roughly twice the length of the Hp alpha 1 alleles and is the product of a partial gene duplication possibly resulting from an unequal crossover event in a heterozygous genotype Hp alpha 1F/Hp alpha 1S. In the study described here we compare the cDNA encoding Hp alpha 1S to that encoding Hp alpha 2FS . Both have a leader sequence followed by the genotypic alpha chain sequence, a beta sequence and an untranslated sequence in the 3' end. The cDNA encoding Hp alpha 2FS is composed of alpha 1F and alpha 1S domains differing by four nucleotide replacements. Hp alpha 1S cDNA contains the same replacement site mutations found in the alpha 1S domain of Hp alpha 2FS , indicating that this coding region has sustained few, if any, mutations since its incorporation into the Hp alpha 2FS gene.     

Multiple domains contribute to heparin/heparan sulfate binding by human HIP/L29

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is thought to be involved in the promotion of cell adhesion, the promotion of cell growth in the cancerous state, and the modulation of blood coagulation. These activities are consistent with the proposed function of HIP/L29 as a heparin/heparan sulfate (Hp/HS) binding growth factor that has a preference for anticoagulantly active Hp/HS. Previous studies showed that a peptide derived from the C terminus of human HIP/L29 (HIP peptide-1) can selectively bind anticoagulant Hp and support cell adhesion. However, a murine ortholog does not have an identical HIP peptide-1 sequence, yet still retains the ability to bind Hp, suggesting that there may be additional Hp/HS binding sites outside of the HIP peptide-1 domain. To test this hypothesis, a systematic study of the domains within human and murine HIP/L29 responsible for Hp/HS binding activity was undertaken. Using deletion mutants, proteolytic fragments, and protease protection of HIP/L29 by Hp, we demonstrate that multiple binding domains contribute to the overall Hp/HS binding activity of HIP/L29 proteins. Furthermore, a conformational change is induced in human HIP/L29 upon Hp binding as detected by circular dichroism spectroscopy. These studies demonstrate the multiplicity of Hp/HS binding sequences within human and murine HIP/L29.     

Genetic linkage analysis of the carpal tunnel syndrome

Two generations of a family with autosomal dominant carpal tunnel syndrome were studied for genetic linkage to 20 informative polymorphic blood markers. No linkage was demonstrated between the syndrome and the markers tested; exclusion of close linkage (lod score less than -2.0) was found for MNSs, ACP, GALT, GPT, GLO, Hp, Gc, and Pi.     

Post-translational heterogeneity of the human vitamin D-binding protein (group-specific component)

The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.     

Isolation and characterization of the O-glycan chain of the human vitamin-D binding protein

On a highly purified preparation, the structure of the carbohydrate chain of the human vitamin D-binding protein was investigated and two genetic forms of this protein were considered (Gc 2 and Gc 1 proteins). It was found that only the Gc 1 protein (Gc1a isoform) was glycosylated, the glycan moiety representing about 1% of the protein. The structure of this O-glycosidically linked glycan was determined to be: Neu Ac alpha (2 leads to 3) Gal beta (1 leads to 3) GaINAc alpha (1 leads to 0) Ser (or Thr). A tetrasaccharidic O-glycan with two N-acetylneuraminic residues was also characterized. The vitamin D-binding protein is a rare example of a serum protein O-glycosylated only on some genetic forms.     

Genetic markers and malaria. Observations in Gujarat, India

189 healthy controls and 175 patients suffering from malaria vivax have been investigated with regard to associations between this disease and 22 genetic polymorphisms of the blood (ABO, MN, Ss, Rh, Kell, P, Lutheran, Kidd, Duffy, Diego, Xg; ABH-Secretor; Hp, Gc, Gm, Km; aP, AK, PGM1, 6-PGD, EsD; Hb variants) Significant associations could be demonstrated only for P and Hp systems, though in accordance with other investigations it cannot be excluded that the ABO system plays also a role in this connection.     

Genetic structure of the Mongols as derived from the ABO, MN, Rh, EsD, GLO1, PGM1, AcP, 6-PGD, Hp, Gc, Tf, C'3 and ChE2 loci

According to integral characterization of gene frequencies of the investigated loci AB0, MN, Rh, GLO1, PGM1, EsD, AcP, 6-PGD, Hp, Tf, Gc, C'3 and ChE2, Mongolian population has high level of polymorphism, with the exception of haplotypes R" (cdE) and Ry(CdE) at the Rh locus and TfB0-1 at the Tf locus. The data on biochemical and immunological polymorphic gene markers analysed in the population of Mongolia show that the Mongolians have some distinctive features, in comparison with the mean-in-the-world characteristics: high frequencies of the B genes at the AB0 locus; D, E, R1 and R2 at the Rh locus; GLO11, PGDc, TfDChi, E2(C5+), PGM1(1+); low frequencies of the genes A(AB0), R0(Rh), AcPc, Hp1, Gc2, C'3F, PGM 1(2-); the rest of the genes at the above-mentioned loci and the genes of the locus MN have the mean-in-the-world frequencies.     

The haptoglobin-gene deletion responsible for anhaptoglobinemia.

We have found an allelic deletion of the haptoglobin (Hp) gene from an individual with anhaptoglobinemia. The Hp gene cluster consists of coding regions of the alpha chain and beta chain of the haptoglobin gene (Hp) and of the alpha chain and beta chain of the haptoglobin-related gene (Hpr), in tandem from the 5' side. Southern blot and PCR analyses have indicated that the individual with anhaptoglobinemia was homozygous for the gene deletion and that the gene deletion was included at least from the promoter region of Hp to Hpr alpha but not to Hpr beta (Hpdel). In addition, we found seven individuals with hypohaptoglobinemia in three families, and the genotypes of six of the seven individuals were found to be Hp2/Hpdel. The phenotypes and genotypes in one of these three families showed the father to be hypohaptoglobinemic (Hp2) and Hp2/Hpdel, the mother to be Hp2-1 and Hp1/Hp2, one of the two children to be hypohaptoglobinemic (Hp2) and Hp2/Hpdel, and the other child to be Hp1 and Hp1/Hpdel, showing an anomalous inheritance of Hp phenotypes in the child with Hp1. The Hp2/Hpdel individuals had an extremely low level of Hp (mean+/-SD = 0.049+/-0. 043 mg/ml; n=6), compared with the level (1.64+/-1.07 mg/ml) obtained from 52 healthy volunteers having phenotype Hp2, whereas the serum Hp level of an individual with Hp1/Hpdel was 0.50 mg/ml, which was approximately half the level of Hp in control sera from the Hp1 phenotype (1.26+/-0.33 mg/ml; n=9), showing a gene-dosage effect. The other allele (Hp2) of individuals with Hp2/Hpdel was found to have, in all exons, no mutation, by DNA sequencing. On the basis of the present study, the mechanism of anhaptoglobinemia and the mechanism of anomalous inheritance of Hp phenotypes were well explained. However, the mechanism of hypohaptoglobinemia remains unknown.     

Group systems of serum proteins and erythrocyte enzymes in malignant lymphoma.

Four group systems of serum proteins (Hp, Gc, Gm, Km) and five group systems of erythrocyte enzymes (AP, PGM1, GPT, AK, EsD) were determined in 63 patients with malignant lymphoma. Statistical analysis of the distribution of the above mentioned systems in patients and Polish population samples did not reveal any significant differences, which points to the lack of any correlation between the disease and the group systems under examination.     

Isolation and characterization of bovine haptoglobin from acute phase sera

A macromolecular hemoglobin-binding protein, which was not detectable in normal bovine sera but appeared during acute phase inflammation, was purified, characterized, and designated as bovine haptoglobin (Hp). The purified protein had a molecular mass of 1,000-2,000 kDa, and was composed of two kinds of peptides, a 20-kDa peptide (alpha chain) and a 35-kDa glycopeptide (beta chain) linked by disulfide bonds. Amino acid composition and N-terminal sequence analyses revealed that both peptides were homologous to each counterpart of human Hp. Studies using some reducing reagents proved that highly polymerized Hp in serum was composed of 2-20 polymerized forms of alpha 2 beta 2 tetramer. Hp could bind one molecule of hemoglobin/alpha 2 beta 2 unit. Hp with smaller sizes obtained from native Hp by partial reduction with cysteine showed almost the same Hb-binding capacity.