Population genetic studies of the Philippine Negritos. I. A pilot survey of red cell enzyme and serum protein groups

Electrophoretic surveys of red cell enzyme and serum protein systems representing 21 genetic loci were carried out on 129 blood samples of the Negritos of Pampanga, Central Luzon, the Philippines. Nine (out of 16) red cell enzyme loci and four (out of five) serum protein loci showed polymorphic variation. Low frequencies of ACP 1A, GPTs1, ESD2, and Hp1, and a markedly high frequency of PGM12 were contrasted to those in non-Negrito Filipinos. Variant ESD phenotypes with a slowly migrating isozyme occurred in high frequency. The new allele designated as ESD3Negrito (ESD3N) had a frequency of .10 +/- .019. In AK, a variant phenotype indistinguishable from AK 2-1 was observed in 14% of the sample. In the Gc system, a fast migrating variant was discovered in high frequency which was distinct from Gc Ab and Gc J. The variant allele, denoted GcNegrito (GcN), had a frequency of .21 +/- .025. A relatively high degree of allelic diversity in the Negrito sample was also suggested by the average heterozygosity for 21 loci screened (.165), which is compared to that of the Japanese population (.140).     

Group-specific component (Gc) 'subtypes' of Gc1 by isoelectric focusing in US blacks and whites.

Isoelectric focusing was applied to the Gc polymorphism. In agreement with Constans et al., we found two common 'subtypes' of Gc1 that could not be identified by conventional electrophoretic procedures. They are labeled Gc1F and Gc1S. Gc1F has a slightly lower isoelectric point than Gc1S. In groups of US blacks the allele frequencies were for Gc1F; 0.732 and for Gc1S; 0.147. In whites these figures were 0.149 and 0.572. We also found GcAb in blacks with a frequency of 0.015. The concentrations in serum of Gc protein as measured by radial immunodiffusion did not differ according to phenotype.     

Subtypes of serum group specific component (Gc) in normal conditions and in pathology

In the framework of the ecogenetic research programme, the data are presented on the genetic polymorphism of the vitamin D-binding protein (Gc) in various USSR populations. Blood serum samples were studied, taken from the Russians of the town Yegorievsk, Moscow Region (p = 321) and 113 Russian patients with tuberculosis using the method of isoelectrofocusing. The information was obtained of the Gc frequencies in two population units of Buryats of Aginsky and Ost-Ordynsky Autonomous Districts of Chita and Irkutsk Regions, including the Olkhon island (on the lake Baikal), in totality, 593 individuals and 13 local groups. The position of the studied Russian and Buryat groups within the gene frequency co-ordinate space is well in line with the estimated area of their localization, with regard to the world distribution. Among the Buryat populations studied, there is distinct heterogeneity for which the factor Gc1F plays a leading role within the Gc system/responsible for 92% of all possible genetic variability. Gc factor frequencies in Buryats range within the following limits: 1F.-0.3864-0.6023, 1S-0.1895-0.4535, 2-0.1364-0.2581. For the Russians of Yegorievsk and the patients with tuberculosis of Moscow and Moscow Region following allele frequencies are established: 1-F0.1169, 1S-0.5476, 2-0.1364 and 1F-0.1106, 1S-0.5531, 2-0.3363, respectively, which indicates that no association exists between Gc variants and tuberculosis. The correlation of the Gc allele frequency distribution with the ratio of insulin-independent diabetes (type 2) world-wide indicates that expression of high frequency of diseases is accompanied with comparatively rare characteristic combination of frequencies of three Gc alleles.     

Serum Gc, Hp and alpha 2HS phenotypes in human T-lymphotropic leukemia virus type I infection

The serum Gc, Hp and alpha 2HS phenotypes were examined in 64 subjects known to have the human T-lymphotropic leukemia virus type I (HTLV-I) infection and in 60 uninfected subjects. There were no significant differences in the distributions of Gc, Hp and alpha 2HS phenotype and allele frequencies between any grouping of HTLV-I-infected subjects and the controls. No association between the Gc, Hp and alpha 2HS genotypes and susceptibility to adult T-cell leukemia was found.     

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.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum

Horizontal polyacrylamide gel electrophoresis, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, Gc F and Gc S , and three Pa alleles, Pa D, Pa F and Pa S , were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. Gc F and Pa F , ranged from 0.72–0.93 and from 0.58–0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the Gc S fraction and thus were found unsuitable for Gc typing.     

Gene conversion involving the pilin structural gene correlates with pilus+ in equilibrium with pilus- changes in Neisseria gonorrhoeae

Gonococci (Gc) exhibit pilus+----pilus- "phase transitions" at high frequency, but only some of the pilus- Gc can revert to pilus+ phenotype. We examined reversible phase transitions between pilus+ Gc and a particular pilus- variant (P-rp+ phenotype) whose pilin mRNA carries a unique block of nucleotides encoding an "assembly missense" pilin polypeptide. The results show that Gc pilus+ in equilibrium with P-rp+ transitions can result from intragenic recombination in which there is nonreciprocal exchange of partially homologous DNA sequences from a partial pilin gene (in silent, storage form) into the expression locus' complete pilin gene. Hence Gc pilus phase variation, like pilus antigenic variation, can occur by gene conversion of the pilin structural gene expression locus.     

Population distribution of the human vitamin D binding protein: anthropological considerations

The polymorphism of the serum vitamin D binding protein (DBP) in humans is based on the existence of three common alleles, Gc1F, Gc1S, and Gc2, and 84 rare alleles. The geographical distribution of Gc1F, Gc1S, and Gc2 alleles shows north to south clines, together with a balanced equilibrium between the Gc1F or Gc1S allele frequency and the Gc2 frequency. The distribution of the FST values shows high variability within a geographical area. For European and North Asiatic groups, the FST values are the lowest observed, and the reason may be a long process of homogenization. Aboriginal populations from Australia and New Guinea and groups from both North Africa and South America show the greatest heterogeneity of their allele frequencies. Systematic factors such as genetic drift and selection may account for this distribution. In contrast with the three main DBP alleles, the distribution of the rare alleles corresponds to patterns of human migrations that occurred during prehistoric and historic periods. Thus, the rare mutants are of particular relevance to anthropological and genetical investigations.     

HIV antigen and antibody detection: variable responses to infection in the Edinburgh haemophiliac cohort

Sequential serum samples from 18 haemophiliac patients exposed simultaneously to human immunodeficiency virus type 1 (HIV 1) in early 1984 were tested retrospectively for serological markers of infection. Assay for total antibodies to HIV established that the time to seroconversion might be as long as 110 days after exposure to contaminated factor VIII; serum samples were also tested by Western blotting, by enzyme linked immunosorbent assay (ELISA) for specific antibodies to envelope and core proteins, and for p24 antigen by two assay systems during the two years after infection. The studies showed that five of the 12 patients for whom serum samples obtained between exposure and seroconversion were available had transient p24 antigenaemia. Although amounts of total antibody to HIV and of antibodies to envelope proteins rose continuously during the two years of the study, amounts of antibody to the core protein were variable and tended to decline in patients who became symptomatic. Two patients had persistent p24 antigenaemia that began four months after seroconversion; these patients remained asymptomatic. One patient who developed the acquired immune deficiency syndrome (AIDS) had transient antigenaemia at the time of seroconversion but failed to show any antigen for the rest of the study; progression to AIDS was accompanied by an increase in antibodies to envelope proteins.Much of the variability in the course of infection with HIV must represent the differences in the susceptibility of the patients to infection.     

Signs of cellular immunosuppression correlate with HLA-DR phenotypes in healthy HIV-negative homosexuals: preliminary findings

Twelve healthy, anal-receptive, homosexual Caucasian males who were seronegative for HIV antibody were typed for HLA-DR antigens. Flow cytometry was used to immunophenotype peripheral blood lymphocytes bearing the CD4, CD8, LEU7, and combined CD8 and LEU7 antigens. These individuals had reported a large number of sexual partners within a five-year period preceding this study. Each individual was assigned a score based on the Hardy-Weinberg frequency of their HLA-DR phenotype in the Caucasian population. The larger the value of this score, the more common the HLA-DR phenotype, the smaller the score, the rarer the phenotype in the population at large. A significant inverse correlation was observed between this score and the proportion of lymphocytes with CD8 and LEU7 antigens. Lymphocytes bearing these two antigens have in vitro suppressor activity and are elevated in patients with human immunodeficiency virus (HIV) infection. The inverse association between CD8+/LEU7+ cells and frequency of HLA-DR phenotypes is consistent with the hypothesis that individuals with rarer phenotypes whose partners are drawn from the population at large are more likely to be challenged during anal insemination, which results in immunosuppression (alloantigenic challenge hypothesis). On the other hand, it is possible that an association exists between certain HLA-DR phenotypes and immune status. Although these observations were made in a very small sample, we believe that the strength of this association provides justification for further investigation into the possibility that alloantigenic challenge may increase the risk for infection, if exposed to HIV, and augment the immunosuppressive action of HIV once significant infection has occurred.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum.

Horizontal polyacrylamide gel electrophoreses, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, GcF and GcS, and three Pa alleles, Pa D, Pa F and Pa S, were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. GcF and PaF, ranged from 0.72-0.93 and from 0.58-0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the GcS fraction and thus were found unsuitable for Gc typing.     

The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae

The pilin antigenic variation (Av) system of Neisseria gonorrhoeae (Gc) mediates unidirectional DNA recombination from silent gene copies into the pilin expression locus. A DNA sequencing assay was developed to accurately measure pilin Av in a population of Gc strain FA1090 arising from a defined pilin progenitor under non-selective culture conditions. This assay employs a piliated parental Gc variant with a recA allele whose promoter is replaced by lac-regulatory elements, allowing for controlled induction of pilin Av. From this assay, the frequency of pilin Av was measured as 0.13 recombination events per cell, with a corresponding rate of pilin Av of 4x10(-3) events per cell per generation. Most pilin variants retained the parental piliation phenotype, providing the first comprehensive analysis of piliated variants arising from a piliated progenitor. Sequence analysis of pilin variants revealed that a subset of possible recombination events predominated, which differed between piliated and non-piliated progeny. Pilin Av exhibits the highest reported frequency of any pathogenic gene conversion system and can account for the extensive pilin variation detected during human infection.     

Quantitative studies of the interaction of cholecalciferol ((vitamin D3) and its metabolites with different genetic variants of the serum binding protein for these sterols.

Cholecalciferol (vitamin D3) and its 25-hydroxy metabolite are transported in plasma bound to a specific protein, the binding protein for cholecalciferol and its metabolites (DBP). DBP is identical with the group-specific component (Gc) proteins, which are known to display genetic polymorphism. Studies were conducted to explore whether or not major differences in the transport of cholecalciferol and its biological metabolites might exist among persons with different Gc phenotypes. Detailed quantitative studies were first carried out on the interaction of 25(OH)D3 with DBP in 21 different samples of serum, representing eight different Gc phenotypes. The studies used a filter disc assay method that provided highly reproducible quantitative results with cholecalciferol-related sterols. The Gc phenotypes studied included the three common types (Gc 1-1, 2-1, and 2-2) and several uncommon genetic variants (Gc Ab-Ab, Ab-1, Ab-2, Chip-1, and Chip-2). The binding affinities for 25(OH)D3 observed with these different sera were all fairly similar to each other. More extensive studies were then conducted to compare the binding of four cholecalciferol-related sterols to each of three genetic variants of DBP, by using sera from homozygous persons with the Gc 1-1, Gc 2-2 and Gc Ab-Ab phenotypes. The ligands tested included cholecalciferol, 25(OH)D3, 1,25(OH)2D3, and 24(R) 25(OH)2D3. The affinities of the three genetic types of DBP/Gc protein were found to be similar for each of the four cholecalciferol-related sterols. The apparent association constants for 25(OH)D3 and 24,25(OH)2D3 were similar (approx. 1--2 x 10(8) M-1); lesser affinities were observed for 1,25(OH)2D3 (kA approx. 1 x 10(7) M-1) and for cholecalciferol (kA approx. 3--4 x 10(5) M-1). Thus the common genetic variants of DBP/Gc protein, and the uncommon genetic variants studied here, all appear to have similar binding properties for cholecalciferol and its several metabolites.     

Role of N-linked glycans on bunyamwera virus glycoproteins in intracellular trafficking, protein folding, and virus infectivity

The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN, family Bunyaviridae) contain three potential sites for the attachment of N-linked glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the glycoproteins with endo-beta-N-acetylglucosaminidase H (endo H) or peptide:N-glycosidase F revealed that Gn and Gc differ significantly in their glycan status and that late in infection Gc glycans remain endo H sensitive. The roles of the N-glycans in intracellular trafficking of the glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the glycan on Gn, by changing N60 to a Q residue, resulted in the protein misfolding and failure of both Gn and Gc proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a cDNA containing the N60Q mutation. In contrast, mutant Gc proteins lacking glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of virus infection.     

Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili

Type IV pili are a major virulence factor of the obligate human pathogen Neisseria gonorrhoeae (the gonococcus; Gc). Pili facilitate bacterial adherence to epithelial cells, but their participation in later steps of epithelial infection, particularly intracellular replication and exit, is poorly understood. Using polarized T84 cells as a model for mature mucosal epithelia, pilus dynamics in piliated, Opa-expressing Gc were examined over time. T84 infection was characterized by a several-hour delay in the growth of cell-associated bacteria and by non-directional exit of Gc, the first time these phenomena have been reported. During infection, non-piliated progeny arose stochastically from piliated progenitors. Piliated and non-piliated Gc replicated and exited from T84 cell monolayers equally well, demonstrating that piliation did not influence Gc survival during epithelial infection. The frequency with which pilin variants arose from a defined piliated progenitor during T84 cell infection was found to be sufficiently high to account for the extensive pilin variation reported during human infection. However, the repertoire of variants appearing in association with T84 cells was similar to what was seen in the absence of cells, demonstrating that polarized epithelial cells can support Gc replication without selecting for a subset of pilin variants or piliation states.     

Antigen detection in primary HIV infection

Serial blood samples were obtained from 21 homosexuals who had developed symptomatic primary infection with human immunodeficiency virus (HIV) after a median incubation time of 14 days. During the first two weeks after the onset of illness HIV antigen (p24) was detected in the blood by enzyme linked immunosorbent assay (ELISA). During the second and third weeks after the onset of illness p24 antibody was detected by Western blot assay and antigen concentrations rapidly decreased to undetectable values. Dissociation of antigen-antibody complexes showed complexed antigen during the phase of declining concentrations of free antigen. Neither free nor complexed antigen was detected in any serum samples for several months thereafter, which suggested that failure to detect HIV antigen reflected low or absent synthesis of viral protein rather than masking of antigen by antibodies. Reappearance of HIV antigen with a fall in p24 antibody concentration was observed in a few patients six months or more after the onset of disease.The combined use of antigen and antibody assays made it possible to obtain evidence of infection with HIV in all of the 95 serum samples tested, illustrating the usefulness of these assays for diagnosing infection with HIV in its early stages.     

Genetic studies in the Markham Valley, northeastern Papua New Guinea: gamma globulin (Gm and Inv), group specific component (Gc) and ceruloplasmin (Cp) typing.

Genetic studies in the Markham Valley, northeastern Papua New Guinea; Gamma globulin (Gm and Inv), group specific component (Gc) and ceruloplasmin (Cp) typing. M. S. Schanfield, Eugene Giles and H. Gershowitz, Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48104. Immunoglobulin allotyping was carried out on 680 serum samples from inhabitants of the Markham Valley, Papua New Guinea (seven villages speaking the same Melanesian [PAP] speaking village). Family and population data verified the presence of Gm-ag, G-ab and Gm-afb among the MN speakers and Gm-ag, Gm-axg, Gm-ab and Gm-afb among the PAP speakers. The frequency of Gm-ag was between 0.048 and 0.235, while the frequency of Gm-ab was between 0.427 and 0.627 and the frequency of Gm-afb ranged between 0.261 and 0.424 among the seven MN villages; the single PAP village had frequencies of 0.568, 0.160, 0.213 and 0.059 for Gm-ag, Gm-axg, Gm-ab and Gm-afb respectively. The frequency of Inv1 ranged between 0.034 and 0.095 in the MN villages and 0.014 in the PAP village. The rare occurrence of Gm(x) without Gm(g) was explained by the presence of a Gm-axfb haplotype, while in two PAP families the presence of Gm(x) without Gm(g) was explained by the abnormally weak expression of Gm(g) in a Gm-axg haplotype. A total of 654 sera were typed for Gc, with the seven MN villages ranging between 0.350 and 0.650 for Gc-1, 0.312 and 0.575 for Gc-2 and between 0.017 and 0.112 for Gc-Ab; the single PAP village had a value of 0.627 for Gc-1, 0.165 for Gc-2 and 0.208 for Gc-Ab. A total of 693 sera were tested for ceruloplasmin type. All showed the common Cp(b) phenotype.     

Altered conformation of Gc (vitamin D-binding protein) upon complexing with cellular actin

Complexing of serum Gc (Vitamin D-binding protein) with cellular actin can occur in the extracellular space as a result of cell turnover, and particularly cell necrosis. The clearance of such complexes is significantly more rapid than that of Gc alone, and several tissues are involved in their uptake, but the mechanisms involved are unknown. We present evidence here that interaction with actin results in alteration of certain physicochemical properties of Gc. Fluorescence of the hydrophobic probe 2-p-toluidinylnaphthylene-6-sulfonate was abolished by complex formation with actin. In addition, isoelectric focusing of complexes between Gc, and actin from different tissues, revealed that complexes were generally more acidic than either protein individually. These findings indicate that complexing of Gc with actin results in altered conformation.     

Polymorphism of alpha-1-antitrypsin in hematological malignancies

Alpha-1-antitrypsin (AAT) or serine protease inhibitor A1 (SERPINA1) is an important serine protease inhibitor in humans. The main physiological role of AAT is to inhibit neutrophil elastase (NE) released from triggered neutrophils, with an additional lesser role in the defense against damage inflicted by other serine proteases, such as cathepsin G and proteinase 3. Although there is a reported association between AAT polymorphism and different types of cancer, this association with hematological malignancies (HM) is, as yet, unknown. We identified AAT phenotypes by isoelectric focusing (in the pH 4.2-4.9 range) in 151 serum samples from patients with HM (Hodgkins lymphomas, non-Hodgkins lymphomas and malignant monoclonal gammopathies). Healthy blood-donors constituted the control group (n = 272). The evaluated population of patients as well as the control group, were at Hardy-Weinberg equilibrium for the AAT gene (χ(2) = 4.42, d.f.11, p = 0.96 and χ(2) = 4.71, d.f.11, p = 0.97, respectively). There was no difference in the frequency of deficient AAT alleles (Pi Z and Pi S) between patients and control. However, we found a significantly higher frequency of PiM1M1 homozygote and PiM1 allele in HM patients than in control (for phenotype: f = 0.5166 and 0.4118 respectively, p = 0.037; for allele: f = 0.7020 and 0.6360 respectively, p = 0.05). In addition, PiM homozygotes in HM-patients were more numerous than in controls (59% and 48%, respectively, p = 0.044). PiM1 alleles and PiM1 homozygotes are both associated with hematological malignancies, although this is considered a functionally normal AAT variant.