Prevalence, serologic and genetic studies of high expressers of the blood group A antigen on platelets*.

OBJECTIVE/AIM: The aim of this study is to describe the distribution of the platelet blood group A antigenicity in Euro-Brazilians (EUBs) and Afro-Brazilians (AFBs). BACKGROUND: A small but significant proportion of individuals express high levels of A or B antigen on their platelets corresponding to the erythrocyte ABO group. The mechanism of increased antigen expression has not been elucidated. MATERIAL/METHODS: A cohort of 241 blood group A donors was analysed by flow cytometry. Although mean fluorescence intensity (MFI) is a typical continuous variable, platelets were screened and divided into two categories: low expressers (LEs) and high expressers (HEs). A three-generation family was investigated looking for an inheritance mechanism. RESULTS: The prevalence of the HE platelet phenotype among group A(1) donors was 2%. The mean of MFI on platelets of A(1) subgroup of EUBs differs from that of AFBs (P = 0·0115), whereas the frequency of the HE phenotype was similar between them (P = 0·5251). A significant difference was found between sexes (P = 0·0039). Whereas the serum glycosyltransferase from HE family members converted significantly more H antigen on group O erythrocytes into A antigens compared with that in LE serum, their ABO, FUT1 and FUT2 genes were consensus. The theoretically favourable, transcriptionally four-repeat ABO enhancer was not observed. CONCLUSION: The occurrence of HE in several members suggests familial aggregation. Indeed, in repeated measures, stability of the MFI values is suggesting an inherited condition. Factors outside the ABO locus might be responsible for the HE phenotype. Whether the real mechanism of inheritance is either of a polygenic or of a discrete Mendelian nature remains to be elucidated.

The mutation G298A®Ala100Thr on th coding sequence of the Duffy antigen/chemokine receptor gene in non-caucasian Brazilians

Ala100Thr has been suggested to be a Caucasian genetic marker on the FY*B allele. As the Brazilian population has arisen from miscegenation among Portuguese, Africans, and Indians, this mutation could possibly be found in Euro- and Afro-Brazilians, or in Brazilian Indians. Fifty-three related individuals and a random sample of 100 subjects from the Brazilian population were investigated using the polymerase chain reaction and four restriction fragment length polymorphisms. Confirming the working hypothesis, among the related individuals three Afro-Brazilians (two of them a mother and daughter) and a woman of Amerindian descent had the Ala100Thr mutation on the FY*B allele. Five non-related Euro-Brazilians also carried the mutation. All nine individuals presented the Fy(a-b+) phenotype. We conclude that the Ala100Thr mutation can occur in populations other than Caucasians and that this mutation does not affect Duffy expression on red blood cells. Gene frequencies for this allele in the non-related individuals were in agreement with those of other populations. The Duffy frequencies of two Amerindian tribes were also investigated.

A novel blood group B subgroup: serological and genetic studies.

A discrepancy in the ABO blood groups between a newborn child and her parents was identified. Serological and DNA investigative techniques were performed. A weak variant of B (B(w)) was detected on the erythrocytes of the child, her grandmother and great-uncle. Adsorption-elution studies showed that their erythrocytes adsorb and yield anti-B on elution. The B(w) antigenic strength of the A(1)B(w) cells of her mother and maternal aunt was reduced when compared to that of the A(2)B(w) from another family member. Only one of 15 different anti-B sera agglutinated the A(1)B(w) erythrocytes. Agglutinin anti-B that reacted strongly with normal B erythrocytes and did not agglutinate the B(w) cells, was found in the sera of the A(1)B(w) individuals. The B(w) serum glycosyltransferase could not convert O cells into B cells and no B substance was found in saliva. All family members with the B(w)/AB(w) phenotypes were heterozygous for a B allele and DNA sequencing revealed a novel missense mutation in exon 7 of the B allele (556A > G), resulting in M186V. This substitution changes a highly conserved region of the enzyme, proposed to be a disordered loop near the enzyme cleft, and is expected to diminish the enzyme's activity, leading to this B(w) phenotype.

New genetic data on Amerindians from the Paraguayan Chaco.

New data on 17 blood group and protein genetic systems obtained among the Ayoreo and Lengua Indians of Paraguay are presented. They include the first report on the red cell band-3 protein investigated among South American Indians. This information was integrated with previous results available for these two and four other groups. Five of the six populations reside in the Chaco area, while the sixth was included as an outgroup living elsewhere in Paraguay. Four of the five Chaco tribes exhibit good genetic homogeneity, but the Ayoreo are somewhat different. The results confirm the Chaco as a distinct biological (as well as cultural and economic) region, which should be considered in evaluations of genetic variability among South American Indians.

Genetic relationships between Amerindian populations of Argentina.

A total of 495 individuals from five different Argentinian tribes was examined for variation in 23 blood group and protein genetic systems, and the results were integrated with previous data on some of these systems. These tribes generally present RH * R1, PGM1 * 1, and ACP * A frequencies lower and RH * R2, ESD * 1, and GLO * 1 prevalences higher than those observed in other South American Indian groups. Earlier studies with mitochondrial DNA showed that haplogroup A was present in low frequencies in these tribes, but haplogroup B showed a high prevalence among the Mataco. Average heterozygosities are very similar in the five tribes, while estimates of non-Indian ancestry are generally low. Both the blood group and protein, as well as the mtDNA data sets, divide the five tribes into two groups, and the relationships obtained with the blood group and protein systems are exactly those expected on the basis of geography and language. However, the topology obtained with the mtDNA results was different, possibly due to sampling effects or diverse patterns of exchange between the groups related to sex.

Electrophoretic variation of hair proteins.

Hair follicle cells secrete a complex assortment of proteins that form the hair shaft, and can be classified into two major groups. The low-sulfur proteins are keratins that contribute to the backbone of intermediate filaments, and the high-sulfur proteins are associated with these filaments. In the present investigation we describe a comparative electrophoretic study of normal human hair proteins from 182 individuals, including some families. Hair proteins were extracted in urea buffer (pH 9.3), examined by 10% polyacrylamide gel electrophoresis (pH 8.8) in the presence of sodium dodecyl sulfate and stained with Coomassie brilliant blue. Eighteen bands appeared and were reproducible in most individuals, with apparent molecular mass ranging from 10.0 to approximately 100 kDa. Based on the most prominent bands, an electrophoretic profile defined as the "frequent profile" was observed. This profile was observed in 180 individuals and consisted of 6 prominent bands, 4 of them of apparent molecular mass in the 40-70-kDa range, which is characteristic of keratins (61.9 +/- 1.02, 58.5 +/- 1.21, 47.9 +/- 1.58, and 45.4 +/- 1.53 kDa), and 2 bands with lower molecular mass (18.9 +/- 0.75 and 13.7 +/- 0.91 kDa). In 2 samples from unrelated women, an additional band of 42.1 +/- 1.72 kDa appeared. The meaning of this variant is still under investigation.

Electrophoretic variation of hair proteins

Hair follicle cells secrete a complex assortment of proteins that form the hair shaft, and can be classified into two major groups. The lowsulfur proteins are keratins that contribute to the backbone of intermediate filaments, and the high-sulfur proteins are associated with these filaments. In the present investigation we describe a comparative electrophoretic study of normal human hair proteins from 182 individuals, including some families. Hair proteins were extracted in urea buffer (pH 9.3), examined by 1O per cent polyacrylamide gel electrophoresis (pH 8.8) in the presence of sodium dodecyl sulfate and stained with Coomassie brilliant blue. Eighteen bands appeared and were reproducible in most individuals, with apparent molecular mass ranging from 10.0 to approximately 100 kDa. Based on the most prominent bands, an electrophoretic profile defined as the "frequent profile" was observed. This profile was observed in 180 individuais and consisted of 6 prominent bands, 4 of them of apparent molecular mass in the 407O-kDa range, which is characteristic of keratins (61.9 ñ 1.02, 58.5 ñ 1.21, 47.9 ñ 1.58, and 45.4 ñ 1.53 kDa), and 2 bands with lower molecular mass (18.9 ñ 0.75 and 13.7 ñ 0.91 kDa). In 2 samples from unrelated women, an additional band of 42.1 ñ 1.72 kDa appeared. The meaning of this variant is still under investigation.

An improved method for the isolation of erythrocyte antigen band-3.

An improved method for isolation of human and Rhesus monkey band-3 separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. Purified band-3 was obtained from human hemoglobin-free ghosts (Hb-free ghosts) after SDS-PAGE by chemical elution+sonication (CE+S). The section of the gel corresponding to the antigen was cut out, mechanically disrupted and incubated in 1% NaHCO3 containing 1% SDS, for 2 h, with shaking, at room temperature, followed by overnight incubation at 4 degrees C. The preparation was subsequently sonicated and clarified by centrifugation. Supernatants were dialyzed against distilled water, their protein contents were measured, and the presence of purified band-3 was demonstrated by SDS-PAGE. A calibration curve was developed for assay of CE+S material using densitometric evaluation of the protein profile on SDS-PAGE. An amount of 37.5 mg of Hb-free ghosts gave 3.15 mg of purified band-3 after CE+S, corresponding to an 8.4% yield. Rabbits were immunized with 50 micrograms CE+S antigen. Sera were collected and assayed by Western blot analysis against its proteolytic fragments, which were obtained from packed red blood cells by treatment with protease type VI from Streptomyces griseus (1 h at 37 degrees C), followed by extensive washing and hypotonic lysis. Specific antibodies recognized band-3 and its proteolytic fragments 60 and 63 kDa in human ghosts obtained from different blood donors, confirming the genetic polymorphism. Analogous serum obtained against the Rhesus monkey band-3 proteolytic fragment 63 kDa recognized the human antigen and its respective fragments. These results indicate the existence of similarities between these two species of band-3, suggesting the potential use of this technique in taxonomic and phylogenetic studies. Purification by CE+S is an efficient and rapid method for isolation of band-3 and its fragments with satisfactory yields and maintenance of both their immunogenic and antigenic properties.

An improved method for the isolation or erythrocyte antigen band-3

An improved method for isolation of human and Rhesus monkey band-3 separated by sodium dodecyl sulfate poliacrylamide gel electrophoresis (SDS-PAGE) is described. Purified band-3 was obtained from human hemoglobin-free ghosts (Hb-free ghosts) after SDS-PAGE by chemical elution + sonication (CE + S). The section of the gel corresponding to the antigen was cut out, mechanically disrupted and incubated in 1 per cent NaHCO3, containing 1 per cent SDS, for 2h, with shaking, at room temperature, followed by overnight incubation at 4ºC. The preparation was subsequently sonicated and clarified by centrifugation. Supernatants were dialyzed against distilled water, their protein contents were measured, and the presence of purified band-3 was demonstrated by SDS-PAGE. A calibration curve was developed for assay of CE+S material using densitometric evaluation of the protein profile on SDS-PAGE. An amount of 37.5 mg of Hb-free ghost grave 3.15 mg of purified band-3 after CE+S, corresponding to an 8.4 per cent yield. Rabbits were immunized with 50µg CE+S antigen. Serawere collected and assayed by Western blot analysis against its proteolytic fragments, which were obtained from packed red blood cells by treatment with protease type VI from Streptomyces griseus (1h at 37ºC), followed by extensive washing and hypotonic lysis. Specific antibodies recognized band-3 and its proteolytic fragments 60 and 63 kDa in human ghosts obtained from different blood donors, confirming the genetic polymorphism. Analogous serum obtained against the Rhesus monkey band-3 proteolytic fragment 63 kDa recognized the human antigen and its respective fragments. These results indicate the existence of similarities between these two species of band-3, suggesting the potential use of this technique in taxonomic and phylogenetic studies. Purification by CE+S is an efficient and rapid method for isolation of band-3 and its fragments with satisfactory yield and maintenance of both their immunogenic and antigenic properties