No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Variation in the FCγR3B gene among distinct Brazilian populations
Abstract
The FCGR3B gene codes for the FcgammaR3b receptor, which occurs in three polymorphic forms representing the human neutrophil antigens (HNA)-1a, HNA-1b, and HNA-1c. The alleles that code for these antigens are FCGR3B*1, FCGR3B*2, and FCGR3B*3, respectively. New variants of these alleles have been recently described. In order to study the frequency of these alleles and the occurrence of variant forms, we sequenced part of the FCGR3B gene in 149 individuals belonging to four distinct Brazilian populations, i.e., 60 Amerindians, 30 Whites of European descent, 30 Afro-Brazilians, and 30 Japanese. The FCGR3B*1 allele showed high frequency among Amerindians (0.850), with the value detected representing the highest frequency described thus far for this allele in population studies. Its frequency was 0.660 in the Japanese population studied, a value equal to that observed in Afro-Brazilians (0.600) and higher than that observed in Whites (0.480). The FCGR3B*3 allele was only found among Afro-Brazilians, where it occurred at a frequency of 0.080, which was lower than the frequency observed among Afro-North Americans (0.207) and Ugandans (0.166). Two variant haplotypes were detected among Amerindians and Afro-Brazilians, occurring in six individuals (four Amerindians and two Afro-Brazilians). The variant haplotype FCGR3B*1 A227G, which occurred in homozygosis in two Amerindians and in heterozygosis in two Afro-Brazilians, is described for the first time in the present report. In general, these data reveal variability in the frequency of alleles of the FCGR3B gene compared to other populations of the same genetic background in other regions of the world.
... Frequency of FCGR3B*02 was comparable to Chinese and Thais but was higher than Japanese and lower than Caucasians, Germans, Brazilians, and Turkish [22, 26, 30, 33, 35-37, 39, 42]. Frequency of FCGR3B*03 (8.76%) was highest as compared to all other populations [22,[25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] except Zambians. This may be due to the ethnic differences among both population groups and/or sample size variation. ...
... The allele frequency of ITGAM*01 (95.5%) found in the present study is higher than that found among the Germans, Caucasians, and Turkish, but it is slightly lower than that seen among the other Asian groups such as Japanese, Thais, and Chinese ( Table 2). The genotype IT-GAM*01+*02− (HNA-4a homozygous) was found to be the most common genotype in all the previous studies [22,[25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. This suggests that antibodies to HNA-4a may also be involved in Indian patients with NAN and AIN as reported previously in other studies [40]. ...
... The frequency of HNA-5a i.e., ITGAL*01 (23.7%) found in Indians is significantly lower (p < 0.05) as compared to that seen among all other populations worldwide (range: 50-89.6%) ( Table 2) [22,[25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. These data confirm that the chances of alloimmunization and immune neutropenia due to anti-HNA-5a in the Indian population will occur more frequently as compared to any other population worldwide. ...
Background:
Antibodies to human neutrophil alloantigens (HNA) are involved in the pathophysiology of several clinical conditions including transfusion-related acute lung injury (TRALI), alloimmune and autoimmune neutropenia, and febrile nonhemolytic transfusion reactions leading to neutropenia. The cognate antigens are polymorphic structures expressed on several glycoproteins on the neutrophils, i.e., antigens HNA-1a, -1b, -1c, and -1d on Fc-γ-receptor IIIb; HNA-2 on CD177; HNA-3a and -3b on choline transporter-like protein 2; HNA-4a and -4b on CD11b/αM subunit of the αMβ2-integrin (CD11b/CD18, Mac-1, CR3); and HNA-5a and -5b on αL-subunit (CD11a) of the αLβ2 integrin (CD11a/CD18), leukocyte function associated molecule (LFA)-1. Currently, there is a lacuna of diagnostic methods for detection of HNA in India. This study aimed to determine the HNA frequencies in Indians, estimate the risk of alloimmunization, and prepare typed neutrophil panels, which can be used to detect HNA antibodies in neutropenia cases.
Material and methods:
EDTA blood samples were collected from random 1,054 blood donors. HNA-2 was phenotyped on fresh EDTA samples using FITC labelled monoclonal anti-CD177 by flowcytometry. HNA-1 (FCGR3B) genotyping was carried out by DNA sequencing and PCR-RFLP. Antigens of HNA-3 (SLC44A2) and HNA-5 (ITGAL) were genotyped by PCR-RFLP using TaqαI and Bsp1286I restriction enzymes, respectively, while HNA-4 (ITGAM) was genotyped by PCR-SSP.
Results:
Allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 were found to be 0.433, 0.444, and 0.087, respectively. FCGR3B*01+*02+*03- was the most common genotype (33.78%). Ten individuals showed deficiency of FCGR3B individuals, while 23 showed hyperexpression, i.e., FCGR3B*01+*02+*03+. FCGR3B*04and *05 occurred with a frequency of 0.002 and 0.024. HNA-2 was found to be a high frequency antigen occurring in 98.8% population. Four percent individuals showed atypical expression of CD177 on their neutrophils. Allele frequencies of SLC44A2*01 and SLC44A2*02were 0.812 and 0.188, respectively, and that of ITGAM*01, ITGAM*02, ITGAL*01, and ITGAL*02 were 0.9546, 0.0454, 0.2372, and 0.7628, respectively.
Conclusion:
This is the first study in India to report the frequencies of HNA among blood donors. Typed neutrophil panels identified in the present study will enable us to investigate suspected cases of immune neutropenia in future.
... The sequencing technique allowed the detection of one new variant of the FCGR3B*01 allele (FCGR3B*01A227G/ G277A). In addition, four already described variants, one of the FCGR3B*01 allele 29,31 and three of the FCGR3B*02 allele [20][21][22]31 were also Bonferroni correction for multiple comparisons method between alleles and genotypes. (Table 3). ...
... (Table 3). Ten single nucleotide polymorphisms (SNPs) at non-polymorphic positions were also observed in 139 DNA samples, 59 in patients and 80 in controls; four of these SNPs had already been described in healthy individuals, [20][21][22]29,31 while the other six are original findings. Table 4 depicts the frequency of the most common clinical manifestations in the present series of SLE patients from whom clinical data were available. ...
... that the technique used in these studies might not have allowed the correct distinction between FCGR3B*01 and *02 alleles due to the variants that were reported by studies using the sequencing technique. 9,[20][21][22]29,31,32 The sequencing approach has previously been used in genotyping studies of the FCGR3B gene conducted in healthy individuals, 9,[20][21][22] and this is the first study to sequence the third exon of the FCGR3B gene in SLE patients. Our results share some similarity with the study by Hatta et al. in a Japanese population, in which there was an association between the FCGR3B*01/02 heterozygote genotype and SLE. 3 In our study the FCGR3B*01 allele and the FCGR3B*01/01 and FCGR3B*01/02 genotypes were associated with susceptibility to SLE (P < 0.001, P ¼ 0.028 and P ¼ 0.012, respectively). ...
Objective:
The objective of this study was to evaluate the association between Fc gamma receptor IIIb polymorphism and susceptibility to systemic lupus erythematosus and clinical traits of the disease.
Methods:
Genomic DNA was obtained from 303 consecutive systemic lupus erythematosus patients and 300 healthy blood donors from the southeastern region of Brazil. The polymorphic region of the FCGR3B gene was sequenced and the alleles FCGR3B*01, FCGR3B*02 and FCGR3B*03 were analyzed.
Results:
The FCGR3B*01 allele was more frequent in systemic lupus erythematosus patients (43.1%) while the FCGR3B*02 allele prevailed among controls (63.7%) (P = 0.001). The FCGR3B*03 allele was found equally in both groups. The FCGR3B*01/*01 (20.7%) and FCGR3B*01/*02 (41.1%) genotypes were more frequent among systemic lupus erythematosus patients (P = 0.028 and P = 0.012, respectively) while the FCGR3B*02/*02 genotype was more frequent in controls (45.5%) (P < 0.001). One variant of the FCGR3B*01 allele previously described in Germany was found in only one control. A new variant of the FCGR3B*01 allele with two substitutions (A227G/G277A) was found in one control. Three variants of the FCGR3B*02 allele previously described in African-Americans, Brazilians, Chinese and Japanese were found in ten 10 patients and two controls. In addition, several single nucleotide polymorphisms at non-polymorphic positions were identified in both patients and controls.
Conclusion:
Susceptibility to systemic lupus erythematosus was associated with the FCGR3B*01 allele, as well as with the FCGR3B*01/*01 and FCGR3B*01/*02 genotypes. No association was found between FCGR3B genotypes and clinical manifestations, disease severity or the presence of autoantibodies.
... The ISBT Granulocyte Antigen Working Party in 1998 established a new nomenclature of granulocyte antigens consisting of five antigen systems, which has subsequently been used worldwide by researchers in the field of granulocyte serology and molecular biology [2]. However, in the meantime, the molecular basis of the HNA-3a antigen has been elucidated [3,4], and new antigens and new alleles of existing genes have been published [5][6][7][8][9][10], including proposed denotation of the new alleles, which have not been approved by the ISBT Granulocyte Immunobiology Working Party (GIWP) [11]. A revision of the HNA nomenclature is therefore required. ...
... Different nucleotides can be present at each of the known polymorphic positions of FCGR3B, due to gene recombination which leads to a high degree of FCGR3B polymorphism [5][6][7][8]22]. Some of the genomic variants were reported repeatedly, for example FCGR3B*02 244A>G (formerly FCGR3B*02 277A>G) [5,6,8,10] and FCGR3B*01 316G>A (formerly FCGR3B*01 349G>A) [5,24], but in most cases, the informative value of these observations was limited by the lack of phenotype and clinical data and by varying experimental approaches such as PCR-SSP methods, genomic DNA sequencing and sequencing of cloned DNA fragments. ...
... Different nucleotides can be present at each of the known polymorphic positions of FCGR3B, due to gene recombination which leads to a high degree of FCGR3B polymorphism [5][6][7][8]22]. Some of the genomic variants were reported repeatedly, for example FCGR3B*02 244A>G (formerly FCGR3B*02 277A>G) [5,6,8,10] and FCGR3B*01 316G>A (formerly FCGR3B*01 349G>A) [5,24], but in most cases, the informative value of these observations was limited by the lack of phenotype and clinical data and by varying experimental approaches such as PCR-SSP methods, genomic DNA sequencing and sequencing of cloned DNA fragments. Matsuo and colleagues were the first to present phenotype data of some of the genomic variants [25]. ...
In 1998, the ISBT Granulocyte Antigen Working Party established a new nomenclature for the human neutrophil antigens (HNA) which included five antigen systems and a total of seven antigens. In the meantime, new antigens and new alleles have been described and these now make it necessary for the ISBT HNA nomenclature subcommittee to present a proposal for an HNA nomenclature update. HNA-1 includes four antigens, HNA-1a, HNA-1b, HNA-1c and HNA-1d, which can result from five different Fcγ receptor IIIb (FcγRIIIb)-encoding alleles, FCGR3B*01, *02, *03, *04 and *05. Due to copy number variation, individuals may exhibit between zero and four alleles leading to either the HNA-1 null type or combinations of the known HNA-1 antigens. Current data indicate that HNA-2 is an isoantigen without allelic variation, encoded by the CD177 gene. Individuals without any HNA-2 expression on their neutrophils are addressed as HNA-2 null. The two antithetical antigens of the choline-transporter-like protein 2 (CTL2), HNA-3a and HNA-3b, are encoded by the three different alleles SLC44A2*01, *02 and *03, which discriminate in one or two nucleotide substitutions. HNA-4a, which is encoded by the ITGAM*01 allele, is an antigen of the αMβ2-integrin. The αLβ2-integrin contains HNA-5a as the only antigen which is encoded by the ITGAL*01 allele. Antigens and alleles other than those listed have to be approved by the ISBT Granulocyte Immunobiology Working Party (GIWP) before they can be assigned to the official nomenclature.
... The Fc-γ-IIIb receptor is encoded by the FCGR3B gene and occurs in 3 polymorphic forms representing the human neutrophil antigens, that is, HNA-1a, HNA-1b, and HNA-1c, which are encoded by the FCGR3B*1, FCGR3B*2, and FCGR3B*3 alleles, respectively [13]. In addition to their clinical significance, the FCGR3B allele frequencies could be used as an additional marker in population studies [14][15][16]. ...
... It was found that the FCGR3B*1 allele was the most common allele in 800 Thai blood donors. This finding confirms that the FCGR3B*1 allele is more frequent than the FCGR3B*2 allele in Asian populations, Afro-Brazilians, and Native Americans [4,6,7,9,13,14]. On the other hand, the FCGR3B*1 and FCGR3B*2 allele frequencies in the northern Thai population were significantly different from those in the central Thai and Korean populations [20]. ...
Human neutrophil antigens (HNAs) are involved in autoimmune and alloimmune neutropenia and transfusion-related acute lung injury. The HNA-1 system is important in immunogenetics, and allele frequencies have been described in different populations. This study investigated the frequency of FCGR3B alleles encoding HNA-1a, HNA-1b, and HNA-1c among Thai blood donors and compared these frequencies with those previously reported for other populations.
Eight hundred DNA samples obtained from unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok, and the Blood Bank, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, were included. Samples were simultaneously typed for each FCGR3B allele using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique.
The frequencies of FCGR3B*1, FCGR3B*2, and FCGR3B*3 alleles in central Thai blood donors were 0.548, 0.452, and 0.004, respectively; only FCGR3B*1 and FCGR3B*2 alleles were found in northern Thai blood donors (0.68 and 0.32, respectively). Compared with other Asian populations, central Thais had higher frequencies of the FCGR3B*2 allele (P<0.001), while the frequencies of the FCGR3B*1 and FCGR3B*2 alleles in northern Thais were similar to those previously reported in Taiwanese and Japanese populations. In contrast, the frequencies of the FCGR3B*1 and FCGR3B*2 alleles in the northern Thai population were statistically different from those observed in central Thai, Korean, German, and Turkish populations.
FCGR3B allele frequencies were significantly different between central and northern Thai blood donors. Our in-house PCR-SSP method is a simple, cost-effective, and convenient method for FCGR3B allele detection.
... We were interested in the characterization of the Argentinean general population because of known differences in the HNA-1 alleles frequencies between the migratory component from Europe, particularly from Italy (1) and Spain (2), and the indigenous component (3,4). ...
... In the Toba Amerindian group, the HNA-1a and HNA-1b gene frequencies (0.77 and 0.23, respectively) were similar to those reported in Japanese (0.622 and 0.378) and others geographically distant Amerindians populations (3,4,11). ...
Human neutrophil antigens (HNA) are polymorphic structures located in the neutrophil membrane. The neutrophil-specific antigens HNA-1a (NA1), 1b (NA2) and 1c (SH) are well-recognized allotypic forms of FcgammaRIIIb and the most frequent targets of neutrophil alloantibodies. The aim of this study was to determine the gene frequencies of the neutrophil-specific antigens belonging to the HNA-1 system in blood donors and Toba Amerindians from Rosario, Argentina. Two hundred and eighteen unrelated healthy Argentinean blood donors and Toba Amerindians from Rosario were typed for HNA-1a, HNA-1b and HNA-1c using polymerase chain reaction with sequence-specific primers. For the Argentinean blood donors, the HNA-1a and HNA-1b gene frequencies were 0.44 and 0.56 and for the Amerindians Toba were 0.77 and 0.23, respectively. The HNA-1c antigen is present in 4.7% (gene frequency=0.023) of the blood donors but in none of the Amerindian individuals. The present data showed that the HNA-1 allele frequencies in the major population and the Toba Amerindians from Rosario are similar to those described in European and others distant Amerindians populations, respectively.
... Extended testing in some populations such as, Africans and Brazilian Indians, has demonstrated that genetic variability is larger than previously anticipated. [12][13][14][15][16][17][18] Here, we aimed to define the epitopes of HNA-1a and HNA-1b by permutation of all four amino acids and subsequent serological characterization of the variant CD16b proteins. ...
Background:
Neutrophil specific Fcγ receptor IIIb (CD16b) is a low-affinity IgG receptor. Its polymorphic variants are associated with human neutrophil antigens (HNA). HNA-1a and HNA-1b differ in four amino acids. Immunization can lead to the production of alloantibodies. The exact contribution of four amino acid exchanges for the formation of HNA-1a, -1b epitopes is currently unknown.
Study design and methods:
Permutation of each polymorphic amino acid from wild-type CD16b cDNA constructs was performed and expressed on HEK293 cells. All 16 receptor variants were produced and tested against 19 well-characterized HNA antisera in an antigen capture assay.
Results:
Analyzing the reaction pattern revealed that anti-HNA-1a antibodies can bind whenever asparagine (N) is present in position 65, regardless of the three other positions (CD16b *N**). Anti-HNA-1b antibodies can bind when serine (S) is present in position 36 (CD16b S***), when N is present in position 82 (CD16b **N*), or both (CD16b S*N*). CD16b variants with N65 and S36 and/or N82 (such as CD16b SNN*) bind both, anti-HNA-1a and anti-HNA-1b alloantibodies. If these specific amino acids are missing (as in CD16b RSD*), no antibodies will bind.
Conclusion:
Whereas the primary structure of HNA-1a and HNA-1b usually differs in four amino acids, epitope composition is not "antithetical". N65 alone determines the presence of HNA-1a, and S36 and/or N82 determine the presence of HNA-1b. Amino acid 106 does not participate in epitope formation. Our findings are of specific relevance when a HNA-1 phenotype is predicted from a genotype.
Objectives
Our study aimed to establish a novel multiplex polymerase chain reaction (PCR) for rapid simultaneous detection of all relevant human neutrophil antigen (HNA)‐1, ‐3, ‐4 and ‐5 alleles.
Background
Granulocyte‐reactive antibodies are involved in several diseases, such as neonatal alloimmune neutropenia, autoimmune neutropenia and transfusion‐related acute lung injury. A panel of well‐defined test granulocytes is required for diagnostic antibody detection and prospective blood donor screening. Several genotyping methods for the detection of HNA alleles have been described, but most approaches require separate amplification of each HNA allele or at least a separate amplification of the HNA‐1 alleles.
Methods
The new method is based on simultaneous detection in one reaction tube, where each HNA‐1 allele is amplified by two allele‐specific primers, one primer of which is labelled with a fluorescent dye (HEX, FAM). Allelic polymorphisms for HNA‐3, ‐4 and ‐5 were amplified with one common unlabelled primer and two fluorescence‐labelled (HEX, FAM) allele‐specific primers. DNA fragments of HNA alleles are analysed on a Genetic Analyser 3130xl by amplicon size and fluorescent dye. A total of 110 blood donors with known genotypes were studied.
Results
In the 110 DNA samples studied, all HNA‐1, ‐3, ‐4 and ‐5 alleles could be detected precisely. All results matched perfectly with those from reference typing by PCR‐sequence‐specific primer. Amplification performed well even at low DNA concentrations (10 ng μL⁻¹).
Conclusion
Our method enables fast and easy genotyping of all relevant HNA‐alleles in one PCR reaction. Results are easy to analyse due to different amplicon sizes and fluorescent dyes. Furthermore, the method is suitable for high sample throughput.
Neutrophils are dynamic, motile cells that provide the first line of defence against many pathogens. Therefore, neutrophils are equipped with a number of receptors. Some of these receptors are known polymorphic carrying immunogenic structures. Immunization against these antigenic structures, known as human neu-trophil antigen (HNA), can lead to the production of antibodies. These antibodies are implicated in a number of clinical conditions including immune-mediated neutropenia, refractoriness to granulocyte transfusions and transfusion-related acute lung injury. Based on the development of molecular genotyping, intensive studies on the allelic distribution of HNAs have been conducted all over the world. Significant disparity on the allelic distribution was observed, indicating different clinical relevance and priority of HNAs in different ethnic groups. Currently , except in Caucasians, only limited studies on the occurrence of neutrophil alloantibodies have been reported among other populations. These studies may be hampered by the current approach of neutrophil antibody detection due to the fact that several methods still need to be used to obtain reliable results. In this short review, current state on the distribution of HNAs, occurrence of antibodies against HNA and their clinical relevance among different populations will be illustrated.
Background and objective:
Antibodies to human neutrophil antigens (HNAs) have been implicated in transfusion-related acute lung injury and allo- and autoimmune neutropenia. To date, five HNA systems are assigned, and during the last decades enormous efforts have been undertaken to identify the underlying genes and to characterize the antigens. This review of the literature will provide the current genetic, molecular and functional information on HNAs.
Recent findings:
New information on alleles and antigens has been added to nearly each of the five HNA systems. HNA-1d has been added as the antithetical epitope to HNA-1c that is located on the glycoprotein encoded by FCGR3B*02 but not by FCGR3B. FCGR3B*04 and *05 now are included as new alleles. A CD177*787A>T substitution was demonstrated as the main reason for the HNA-2-negative phenotype on neutrophils. The target glycoprotein of HNA-3 antibodies could be identified as choline transporter-like protein 2 (CTL2) encoded by SLC44A2. The conformation sensitive epitope discriminates between arginine and glutamine at position 152 resulting in HNA-3a and HNA-3b. An additional Leu151Phe substitution can impair HNA-3a antibody binding. Recently an alloantibody against HNA-4b which discriminates from HNA-4a by an Arg61His exchange of the glycoprotein encoded by the ITGAM gene was reported in neonatal alloimmune neutropenia. An update of the current HNA nomenclature based on the new findings was provided in 2016 by the ISBT Granulocyte Immunobiology Working Party nomenclature subcommittee.
Conclusions:
The molecular basis of each of the five HNA antigen systems has been decoded during the past decades. This enables reliable molecular typing strategies, antibody detection and specification as well as development of new assays based on recombinant antigens. However, research on HNA alleles, antigens, and antibodies is not finally terminated and also in the future will add new findings.
Objectives:
This study aimed to determine human neutrophil antigen (HNA) frequency, estimate possible HNA incompatibilities and predict the risk of HNA alloimmunisation in the Northeastern Thai, Burmese and Karen populations.
Background:
Alloantibodies against HNA are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions.
Methods:
A total of 400 unrelated healthy Thais, 261 Burmese and 249 Karen was included in this study. DNA samples were typed for HNA-1, -3, -4 and -5 systems using polymerase chain reactions with sequence-specific primers (PCR-SSP).
Results:
In this cohort, HNA-1a was more prevalent than HNA-1b. Accordingly, the possible risk of HNA-1a alloimmunisation against HNA-1a is lower than HNA-1b (0·0802-0·1351 vs 0·2293-0·2497). This is in contrast to the situation reported in Caucasian and African populations. The predicted risk of HNA-3 incompatibility in Thais, Burmese and Karen were 28·09%, 30·66% and 22·77%, respectively. The possible risks of HNA-3a alloimmunisation were 0·0493 in Thais, 0·0608 in Burmese and 0·0196 in Karen, respectively. No individuals were found to be homozygous for HNA-4bb. The probability of developing alloantibodies against HNA-4a was low in these populations and every population in Asia. In contrast, the overall frequency of HNA-5bb homozygous individuals was high in this study, peaking at 0·192.
Conclusions:
This is the first study that reported the allele frequencies of HNA-1, -3, -4, and -5 in a large sample of healthy unrelated individuals from ethnic Thais, Burmese and Karen. Our results indicated the high possible risk of HNA-1, -3 and -5 alloimmunisation in these populations.
Neutrophils are dynamic, motile cells that provide the first line of defence against many pathogens. Therefore, neutrophils are equipped with a number of receptors. Some of these receptors are known polymorphic carrying immunogenic structures. Immunization against these antigenic structures, known as human neutrophil antigen (HNA), can lead to the production of antibodies. These antibodies are implicated in a number of clinical conditions including immune-mediated neutropenia, refractoriness to granulocyte transfusions and transfusion-related acute lung injury. Based on the development of molecular genotyping, intensive studies on the allelic distribution of HNAs have been conducted all over the world. Significant disparity on the allelic distribution was observed, indicating different clinical relevance and priority of HNAs in different ethnic groups. Currently, except in Caucasians, only limited studies on the occurrence of neutrophil alloantibodies have been reported among other populations. These studies may be hampered by the current approach of neutrophil antibody detection due to the fact that several methods still need to be used to obtain reliable results. In this short review, current state on the distribution of HNAs, occurrence of antibodies against HNA and their clinical relevance among different populations will be illustrated.
Objectives. To examine the association between Fcγ receptor (FcγR) polymorphisms and the development of hypersensitivity reactions to adalimumab in patients with rheumatoid arthritis.
Methods. Sixty-five patients receiving adalimumab were enrolled in the study. Genetic polymorphisms for FcγR3B were genotyped in FCGR3B NA1/2 alleles by real allelic discrimination assay. Clinical information and the occurrence of a hypersensitivity reaction to adalimumab were collected from the patients’ charts.
Results. A hypersensitivity reaction was observed in 12% of the patients. Clinical information obtained from patients with a reaction and those without were the same. The FCGR3B NA1/NA1, NA1/NA2 and NA2/NA2 alleles were found in 75%, 13% and 13% of the patients with hypersensitivity reaction, respectively, and in 28%, 42%, and 30% of those without a hypersensitivity reaction, respectively (P = 0.04). Multivariate logistic regression analysis identified only the NA1/NA1 as an independent relevant factor for a hypersensitivity reaction to adalimumab (OR 7.7, p = 0.01).
Conclusions. The FCGR3B NA1/NA1 genotype is associated with hypersensitivity reactions to adalimumab.
Background
Neonatal immune neutropenia (NIN) is a rare, but potentially life-threatening, disorder caused by maternal alloantibodies recognizing paternal neutrophil antigens on fetal cells. Alloantibodies directed against the human neutrophil alloantigen system (HNA)-1 located on Fc receptor IIIb (FcRIIIb) are most frequently implicated in NIN. In this report, we describe two cases of NIN with alloantibodies against FcRIIIb, which did not match one of the known HNA-1a, -1b, or -1c specificities, but define a new antigen, HNA-1d. Study Design and Methods
Neutrophil-reactive antibodies were detected by agglutination, microscopic immunofluorescence, and monoclonal antibody (MoAb)-specific immobilization of neutrophil antigens (MAIGA) assay. For epitope mapping of FcRIIIb-reactive antibodies, recombinant chimeric variants of FcRIIIb were used in the MAIGA assay. Genotyping of FCGR3B was performed by allele-specific polymerase chain reaction. ResultsBoth mothers were typed FCGR3B*01+, *02-, *03+. Antibody screening revealed the presence of alloantibodies reactive with FcRIIIb encoded by FCGR3B*02, but not with FcRIIIb encoded by FCGR3B*03. MAIGA with recombinant, partly chimeric FcRIIIb variants demonstrated that the antigen recognized by maternal antibodies is characterized by two amino acids, Ala78 and Asp82. Among the FCGR3B alleles, the sequence Ala78---Asn82 is exclusively encoded by FCGR3B *02. ConclusionA previously unrecognized second antigen, HNA-1d, is present on FcRIIIb encoded by FCGR3B*02. This antigen is characterized by the sequence Ala78---Asn82. It appears that only individuals carrying the HNA-1c phenotype can form anti-HNA-1d alloantibodies. The HNA-1 system now consists of four antigens encoded by three alleles.
The importance of human neutrophil antigens (HNA) in immunogenetics and their involvement in hematologic diseases have accelerated the elucidation of their molecular basis and their allele frequencies distribution has been described in many populations over the world. In this study, our aim was to evaluate the frequency of FCGR3B alleles encoding HNA-1a, 1b and 1c among Tunisians of sub-Saharan origin and to compare them to Tunisian blood donors and to a group from sub-Saharan Africa.
We typed the DNA of 106 individuals (62 Tunisians of sub-Saharan origin, 33 Tunisian blood donors and 11 from sub-Saharan Africa) for the three FCGR3B alleles by polymerase chain reaction using sequence specific primer (PCR-SSP).
FCGR3B*1, FCGR3B*2 and FCGR3B*3 allele frequencies were respectively 0.347, 0.573 and 0.080 among Tunisians of sub-Saharan origin, 0.379, 0.591 and 0.030 among Tunisian blood donors and 0.318, 0.546 and 0.136 among the group from sub-Saharan Africa.
These allele frequencies were similar to those previously reported in other black and white populations. The frequencies found in the two Tunisian groups confirm the intermixing origin from Europe, sub-Africa and Asia of the Tunisian population. Our results provide a database for future studies of the HNA system and associated diseases in Tunisia.
The FCGR3B gene encoding the FcyRIIIb receptor for immunoglobulin G has three polymorphic forms known as HNA-1a, HNA-1b, and HNA-1c, encoded by the alleles FCGR3B*01, FCGR3B*02, and FCGR3B*03, respectively. It is not clear whether the inheritance of the FCGR3B*03 allele, which encodes the HNA-1c, is linked or not to the other two alleles. The objective of this study was to identify the inheritance pattern of the FCGR3B*03 allele in Brazilians.
Blood samples from nine families with at least one FCGR3B*03(+) member, totalizing 47 individuals, were studied. The presence of the FCGR3B*01, FCGR3B*02, and FCGR3B*03 alleles was detected by the polymerase chain reaction with sequence-specific priming method, and all DNA samples were sequenced.
In three of the nine studied families, the FCGR3B*03 was passed down with the FCGR3B*02, while in one family the FCGR3B*03 was inherited in linkage with FCGR3B*01. The other families were not informative regarding FCGR3B*03 inheritance. Sequencing showed for the first time one single-nucleotide polymorphism at Position 264 resulting from a simple substitution C→T; three other different substitutions at Position 230, T→A, T→G; and the presence of three nucleotides at Position 230 (T, G, and A). The previously reported variants FCGR3B*01A227G and FCGR3BG330T were also found.
In this Brazilian FCGR3B*03(+) group we found that the inheritance of FCGR3B*03 took place by a linkage to FCGR3B*02 or to FCGR3B*01. Linkage of FCGR3B*03 to FCGR3B*02 was the most common. Additionally, we report SNPs that have not been described, suggesting that they might be more common than previously thought.
The genes encoding the Fcgamma receptors (FcgammaR) IIIa and IIIb (FCGR3A and FCGR3B) are clustered on chromosome 1 band q23-24 and exhibit allelic polymorphism. We investigated the molecular basis of additional new FCGR3 genomic variation.
A segment shared by FCGR3A and FCGR3B containing the polymorphic nucleotide positions 141, 147, 227, 266, and 277 in exon 3 was cloned and sequenced from genomic DNA of 30 donors and 3 bacterial artificial chromosome (BAC) clones. A mixture consisting of isolated FCGR3B*2- and FCGR3A- plasmids was cloned and sequenced as well. Additionally, nucleotide databases were screened for clones with variant FCGR3 sequences.
A total of 12 FCGR3 variants defined by the polymorphic positions were detected in whole blood genomic DNA from 23 of 24 FCGR3B*2 and/or FCGR3B*3 positive donors, the DNA from two of three BAC clones and in the DNA mixture of isolated FCGR3B*2- and FCGR3A- plasmids.
Nucleotide exchanges of the variants were non-random and resulted from two alternative nucleotides present in one of the polymorphic position of the basic FCGR3 forms. Polymerase chain reaction (PCR) artefacts cannot be excluded as origin of new variants, but there is strong evidence that at least two variants are the result of a somatic recombination.
The Fc receptors for human immunoglobulin G (FcgammaR) IIIb are encoded by genes clustered on the long arm of chromosome 1 (band q21 --> 24) and exhibit allelic polymorphisms. Several rare FCGR3B sequences were identified in both white and black donors. However, the origins of these genomic variants are unknown and their transcription has not yet been investigated. Blood from a donor with known FCGR3 variants was used to extract DNA from peripheral blood CD34+ cells, CD19+ B-cells, neutrophils and buccal cells, after which FCGR3 gene sequencing was performed. Additionally, RNA samples from 5 Caucasian individuals containing known variant FCGR3 genes were reverse-transcribed to cDNA and the FCGR3 genes were sequenced. Our results showed that the frequencies of variant clones were higher in B-cell preparations than in CD34+ hematopoietic progenitor cells from peripheral blood and neutrophils. Very high variant frequencies were found in buccal cell-derived clones. Variant cDNA sequences were identified in three of five individuals with known FCGR3 variants. We conclude that FCGR3 gene variants are differentially transcribed between cell types and tissues, increasing the likelihood of the presence of variant FcgammaRIII receptors on the cell surface. The significance of the high number of variant clones in buccal cells, however, is unclear.
Neutrophils express two distinct types of receptor for the Fc region of IgG, FcRII and FcRIII, in amounts of 10,000 to 20,000 FcRII (40 Kd) and 100,000 to 200,000 FcRIII (50 to 80 Kd) per neutrophil. We showed that the FcRIII exhibits genetically determined heterogeneity, detectable by differences in electrophoretic mobility with sodium dodecyl sulfate (SDS) as well as by reaction with antibodies against the biallelic neutrophil-specific antigen system NA. FcRIII was precipitated with an FcRIII-specific monoclonal antibody (MoAb) from the neutrophils of 35 donors. NA1NA1 donors expressed an FcRIII with a molecular weight (mol wt) of 50 to 65 Kd, NA1NA2 donors expressed an FcRIII with a mol wt of 50 to 80 Kd, and NA2NA2 donors expressed an FcRIII with a mol wt of 65 to 80 Kd. Statistical analysis showed that the electrophoretic heterogeneity corresponds with the NA polymorphism (k = 1). Sequential immunoprecipitation with a MoAb against NA1 and a MoAb against anti- FcRIII, followed by SDS-polyacrylamide gel electrophoresis (PAGE), showed that NA1-FcRIII is distinct from NA2-FcRIII. Moreover, immunoprecipitation with a MoAb against NA1 yielded a protein of 50 to 65 Kd, and immunoprecipitation with human anti-NA2 sera or an MoAb against NA2 yielded a protein of 65 to 80 Kd. Preincubation of NA1NA2 neutrophils with F(ab')2 fragments of an MoAb against anti-NA1 reduced binding of IgG dimers to these cells with about 50%, whereas it completely prevented binding of the dimers to NA1NA1 neutrophils. Inhibition experiments with the MoAb against NA2 yielded the same results for NA1NA2 cells, whereas binding of IgG dimers to NA2NA2 cells was completely prevented. Thus, the products of both NA alleles bind IgG. Immunoprecipitation from the medium of neutrophils either stimulated with formyl- methionyl-leucyl-phenylalanine (FMLP) or treated with glycosyl-phosphatidyl-inositol-specific phospholipase C (GPI- PLC) showed that both the NA1-FcRIII and the NA2-FcRIII are released from the cell surface, indicating that both forms of FcRIII have some structural features in common. Deglycosylation of FcRIII from homozygous donors yielded material that showed several bands on SDS- PAGE. GPI-PLC treatment of neutrophils indicated that all of this material is phosphatidyl-inositol linked.
Neutrophils express two distinct types of receptor for the Fc region of IgG, FcRII and FcRIII, in amounts of 10,000 to 20,000 FcRII (40 Kd) and 100,000 to 200,000 FcRIII (50 to 80 Kd) per neutrophil. We showed that the FcRIII exhibits genetically determined heterogeneity, detectable by differences in electrophoretic mobility with sodium dodecyl sulfate (SDS) as well as by reaction with antibodies against the biallelic neutrophil-specific antigen system NA. FcRIII was precipitated with an FcRIII-specific monoclonal antibody (MoAb) from the neutrophils of 35 donors. NA1NA1 donors expressed an FcRIII with a molecular weight (mol wt) of 50 to 65 Kd, NA1NA2 donors expressed an FcRIII with a mol wt of 50 to 80 Kd, and NA2NA2 donors expressed an FcRIII with a mol wt of 65 to 80 Kd. Statistical analysis showed that the electrophoretic heterogeneity corresponds with the NA polymorphism (k = 1). Sequential immunoprecipitation with a MoAb against NA1 and a MoAb against anti-FcRIII, followed by SDS-polyacrylamide gel electrophoresis (PAGE), showed that NA1-FcRIII is distinct from NA2-FcRIII. Moreover, immunoprecipitation with a MoAb against NA1 yielded a protein of 50 to 65 Kd, and immunoprecipitation with human anti-NA2 sera or an MoAb against NA2 yielded a protein of 65 to 80 Kd. Preincubation of NA1NA2 neutrophils with F(ab')2 fragments of an MoAb against anti-NA1 reduced binding of IgG dimers to these cells with about 50%, whereas it completely prevented binding of the dimers to NA1NA1 neutrophils. Inhibition experiments with the MoAb against NA2 yielded the same results for NA1NA2 cells, whereas binding of IgG dimers to NA2NA2 cells was completely prevented. Thus, the products of both NA alleles bind IgG. Immunoprecipitation from the medium of neutrophils either stimulated with formyl- methionyl-leucyl-phenylalanine (FMLP) or treated with glycosyl-phosphatidyl-inositol-specific phospholipase C (GPI- PLC) showed that both the NA1-FcRIII and the NA2-FcRIII are released from the cell surface, indicating that both forms of FcRIII have some structural features in common. Deglycosylation of FcRIII from homozygous donors yielded material that showed several bands on SDS-PAGE. GPI-PLC treatment of neutrophils indicated that all of this material is phosphatidyl-inositol linked.
Two polymorphic forms of Fc receptor III (FcR III) are expressed on human neutrophils. These differ with respect to their apparent molecular masses after digestion with N-glycanase, and with respect to their reactivity with MAb Gran 11 and alloantisera which recognize determinants (NA1 and NA2) of the biallelic neutrophil antigen (NA) system. To determine the molecular basis for this polymorphism we isolated RNA from neutrophils of NA1NA1 and NA2NA2 homozygotes and synthesized corresponding cDNAs. cDNAs encoding FcR III were then amplified using the polymerase chain reaction, cloned, and sequenced. The cDNA that encodes FcR III on NA1NA1 neutrophils differed from the cDNA that encodes FcR III on NA2NA2 neutrophils at five nucleotides, predicting four amino acid substitutions. As a result, NA1 FcR III has only four potential N-linked glycosylation sites as compared with six in NA2 FcR III. The amino acid substitutions and differences in the number of potential N-linked glycosylation sites probably account for the different forms of neutrophil FcR III observed after digestion with N-glycanase and for the antigenic heterogeneity of this receptor.
A low affinity receptor for IgG immune complexes, Fc gamma RIII(CD16), is expressed on human NK cells as an integral membrane glycoprotein anchored through a transmembrane peptide; on polymorphonuclear neutrophils (PMN) the receptor is anchored through a phosphatidylinositol (PI) linkage. The protein on NK cells has a molecular mass 6-10 kD larger than that on PMN, and, unlike the latter, is resistant to PI-specific phospholipase C (PI-PLC). Fc gamma RIII(CD16) transcripts isolated from PMN and NK cells of single donors revealed multiple single nucleotide differences, one of which converts an in frame UGA termination codon to a CGA codon. The resulting open reading frame encodes a longer cytoplasmic domain for Fc gamma RIII(CD16) in NK cells, contributing to its transmembrane anchor. Two nearly identical, linked genes that encode these transcripts have been cloned for Fc gamma RIII(CD16), one of which (III-1) is allelic for NA-1 and NA-2. The allelic sites have been mapped to two single nucleotides in the extracellular domain. These genes are transcribed in a cell type-specific fashion to generate the alternatively anchored forms of this receptor.
Recently, a new alloantigen on IgG Fc receptor type IIIb (FcγRIIIb), SH, was described (Bux et al, Blood 89:1027, 1997). We identified three healthy individuals whose neutrophils reacted positively with the SH antiserum. The neutrophil antigen (NA) phenotype of all three donors was NA(1+,2+). Analysis of genomic DNA showed that the three donors were positive for the described SH-encoding mutation in the NA2-FcγRIIIB gene, 266C→A. However, NA(1,2) genotyping and nucleotide sequencing of an NA2-specific fragment amplified from the genomic DNA fragment showed that these individuals carried three FcγRIIIBgenes, namely, NA1-FcγRIIIB, NA2-FcγRIIIB, andSH-FcγRIIIB, encoding NA1-FcγRIIIb, NA2-FcγRIIIb, and SH-FcγRIIIb, respectively. Southern blot analysis confirmed these findings. Furthermore, all three transcripts were isolated from neutrophil mRNA. To investigate whether the presence of threeFcγRIIIB genes resulted in a higher membrane expression of FcγRIIIb, we measured the reactivity of neutrophils from NA(1+,2+)SH(+) individuals with a panel of CD16 monoclonal antibodies (MoAbs) in comparison with neutrophils from NA(1+,2+)SH(−) controls. Reactivity of four different anti–pan-FcγRIII MoAbs and NA2-specific MoAb GRM1 was higher with SH(+) neutrophils compared with controls, whereas that of NA1-specific MoAbs was similar, which is in concordance with the results from the genomic analysis. We observed that reactivity with NA2-specific CD16 MoAb PEN1 was sixfold higher in SH(+) individuals compared with controls. Apparently, the 60Ala→Asp substitution in SH-FcγRIIIb influences the epitope recognized by PEN1. In conclusion, we identified three NA(1+,2+)SH(+) individuals carrying three FcγRIIIB genes and observed a clear gene-dosage effect on the level of expression of neutrophil FcγRIIIb.
The granulocyte antigens NA1 and NA2 are often targets of granulocyte antibodies causing immune neutropenia. Currently, NA typing relies on the properties of the typing sera or antibodies and the techniques used. Therefore, the technique of polymerase chain reaction with sequence-specific primers (PCR-SSP) was adapted for DNA-based NA typing and was used for determining the NA gene frequencies in the German population.
The genomic DNA of 160 unrelated healthy individuals was typed for NA1 and NA2 by PCR-SSP. In 60 granulocyte samples, the NA phenotype was additionally determined by the antigen capture assay and the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results.
Results of the antigen capture assay and PCR-SSP correlated precisely, whereas nine individuals were typed heterozygous only by GIFT. The gene frequencies were 0.35 for NA1 and 0.65 for NA2.
The NA2 gene is more frequent in the German population than the NA1 gene, as determined by genotyping using PCR-SSP. In contrast to GIFT, which showed an error rate for NA typing of 15 percent, PCR-SSP and the antigen-capture assay are more reliable methods of NA typing of granulocytes.
Receptors for immunoglobulin G immune complexes (Fc gamma RII and Fc gamma RIII) are expressed on most hematopoietic cells and show much structural and functional diversity. In order to determine the genetic basis for this diversity, a family of genes encoding the human and mouse receptors was isolated and characterized. Humans have five distinct genes for low-affinity Fc gamma Rs, in contrast to two in the mouse. With the use of yeast artificial chromosomes, the genes encoding the human receptors were oriented and linked, which established the structure of this complex locus. Comparison of the human and mouse genes generated a model for the evolutionary amplification of this locus.
The allele frequencies of six VNTRs (D1S80, D4S43, ApoB 3' VNTR, von Willebrand factor VNTR-I, DXS52 and DYS19) in 123 Amerindians from five tribes (Arara, Wayana-Apalai, Wayampi, Yanomama and Kayapo) were compared with three other Brazilian populations: Whites, Blacks, and individuals of Japanese extraction. The data clearly distinguished the four populations, and measurements of diversity show a decreasing average heterozygosity from Blacks to Whites, Japanese, and Indians. Seven novel alleles were observed: amongst them, two small DYS19 alleles and a large D4S43 allele occurred only in Indians, and may be useful genetic markers for this population. Other prominent features of the Amerindians were: (1) high frequency of ApoB allele 46; (2) absence of a shorter variant of D4S43 allele 1; (3) high frequency, limited to one tribe, of allele 12 of the von Willebrand VNTR. The study also demonstrated a heterogeneity of the Indian tribes, due to both different allele frequencies and the presence or absence of specific alleles. GST was 0.106 for the five Indian populations, and 0.065 for Whites, Blacks and Japanese. HS and HT demonstrated that 11% of the total diversity among Amerindians is caused by interpopulational variability, as compared with 7% for the other three racial groups. In contrast, diversity within each tribe is usually low, as demonstrated by a low average number of alleles per locus. These findings indicate that the study of a small number of tribes may not be representative of the variability of Amerindians, even if a large number of individuals are studied. To capture the whole range of genetic variability of Amerindians, it is necessary to study a large number of populations. The limited genetic diversity thus far observed for Amerindians seems to reflect both a genuine decrease of diversity and a bias caused by the study of limited numbers of tribes.
The granulocyte antigens NA1 and NA2 are the two recognized allelic forms of Fc gamma receptor IIIB. These antigens are clinically relevant, because they are the most frequent targets of neutrophil antibodies in alloimmune neonatal neutropenia, transfusion-related acute lung injury, and chronic benign autoimmune neutropenia of infancy.
A genotyping assay for NA1 and NA2 using polymerase chain reaction with sequence-specific forward and reverse oligonucleotide primers has been developed and validated. Genomic DNA was isolated from the peripheral blood of 478 unrelated individuals of five ethnic groups and used as template for NA genotyping.
A validation study of 22 serologically typed samples (2 NA1/NA1, 10 NA1/NA2, and 10 NA2/NA2) was performed. A concordance rate of 100 percent (22/22 samples) was observed between the genotyping assay and serologic typing. In the genotyping study conducted, the NA1 and NA2 gene frequencies observed were 0.31 and 0.69 for African Americans, 0.30 and 0.70 for Asian Indians, 0.37 and 0.63 for whites, 0.53 and 0.47 for Hispanics, and 0.55 and 0.45 for Native Americans, respectively.
Polymerase chain reaction with sequence-specific primers provides a simple and rapid alternative to neutrophil antigen typing by serologic tests. The NA1 and NA2 gene frequencies observed in Asian Indians and African American populations are similar to those observed in white populations, while those observed in Native American and Hispanic populations are more similar to those previously reported for Asian populations.
Polymorphic structures of the neutrophil Fc gamma receptor IIIb (Fc gamma RIIIb) result in alloantibody formation that causes alloimmune neonatal neutropenia and transfusion reactions. Alloantigens located on Fc gamma RIIIb include the antigens NA1 and NA2. In four cases of alloimmune neonatal neutropenia, granulocyte-specific alloantibodies directed against a thus far unknown antigen were detected by granulocyte agglutination and immunofluorescence tests in the maternal sera. By the use of the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay, the new antigen, termed SH, was located on the Fc gamma RIIIb. Nucleotide sequence analysis of the Fc gamma RIIIb coding region from a SH(+) individual showed a single-base C-->A mutation at position 266, which results in an Ala78Asp amino acid substitution. A family study confirmed that this nucleotide difference is inherited, and corresponds to the SH phenotype. Serologic typing of 309 randomly selected individuals showed an antigen frequency of 5% in the white population. The same frequency was found by genotyping, for which a technique based on polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP) was developed. Typing of all SH(+) individuals for NA1 and NA2, and PCR-restriction fragment length polymorphism analysis of the NA-specific PCR products from five SH(+) individuals using the SH-specific endonuclease SfaN 1 showed that SH antigen is very probably the result of an additional mutational event in the NA2 form of the Fc gamma RIIIB gene. Immunochemical studies also demonstrated that the SH determinants reside on the 65- to 80-kD NA2 isoform of the Fc gamma RIIIb. Our findings show the existence of an additional polymorphism of the Fc gamma RIIIb, which can result in alloantibody formation causing alloimmune neonatal neutropenia.
Human IgG receptors are very heterogeneous and we currently distinguish three Fc gamma receptor classes specifying at least 12 receptor isoforms. On top of this complexity, Fc gamma R are further found to differ between different individuals. Polymorphisms have been identified for all three Fc gamma R classes. The best-studied ones represent allelic variation of Fc gamma RIIa (CD32) and Fc gamma RIIIb (CD16). The Fc gamma RIIa polymorphism is now considered to be a heritable risk factor for autoimmune and infectious diseases, and support for a relevant role of the IIIb polymorphism has also been obtained. A detailed analysis of the exact contribution of each of these Fc gamma R polymorphisms in relation to previously implicated risk factors should unravel the pathophysiological importance of Fc gamma R polymorphisms in the near future.
Platelet-specific alloantigens are important in neonatal alloimmune thrombocytopenia, posttransfusion purpura, refractoriness to platelet transfusions, and population genetics. Data are scarce on allele frequencies in ethnic groups other than whites and Asians.
Using allele-specific restriction enzyme analysis, we studied the distribution of HPA-1 and HPA-2 alleles in six Brazilian Amazon tribes of Amerindians, belonging to five different language stocks. We compared these with the values obtained for blacks and whites.
Only the HPA-1a allele was found among 132 Amerindian chromosomes, compared with a gene frequency of HPA-1b of 0.115 and 0.113, respectively, among blacks and whites. The frequency of HPA-2b among the Amerindians (0.042) is lower than that obtained for blacks and whites (0.148 and 0.100, respectively), and the lowest thus far observed in a population of Asian origin.
Differences in DNA polymorphisms in Amerindian populations have not only anthropological and genetic interest, but also practical applications when they involve coding regions that may change the functional or immunologic features of the protein.
Recently, a new alloantigen on IgG Fc receptor type IIIb (Fc gamma RIIIb), SH, was described (Bux et al, Blood 89:1027, 1997). We identified three healthy individuals whose neutrophils reacted positively with the SH antiserum. The neutrophil antigen (NA) phenotype of all three donors was NA(1+,2+). Analysis of genomic DNA showed that the three donors were positive for the described SH-encoding mutation in the NA2-Fc gamma RIIIB gene, 266C-->A. However, NA(1,2) genotyping and nucleotide sequencing of an NA2-specific fragment amplified from the genomic DNA fragment showed that these individuals carried three Fc gamma RIIIB genes, namely, NA1-Fc gamma RIIIB, NA2-Fc gamma RIIIB, and SH-Fc gamma RIIIB, encoding NA1-Fc gamma RIIIb, NA2-Fc gamma RIIIb, and SH-Fc gamma RIIIb, respectively. Southern blot analysis confirmed these findings. Furthermore, all three transcripts were isolated from neutrophil mRNA. To investigate whether the presence of three Fc gamma RIIIB genes resulted in a higher membrane expression of Fc gamma RIIIb, we measured the reactivity of neutrophils from NA(1+,2+)SH(+) individuals with a panel of CD16 monoclonal antibodies (MoAbs) in comparison with neutrophils from NA(1+,2+)SH(-) controls. Reactivity of four different anti-pan-Fc gamma RIII MoAbs and NA2-specific MoAb GRM1 was higher with SH(+) neutrophils compared with controls, whereas that of NA1-specific MoAbs was similar, which is in concordance with the results from the genomic analysis. We observed that reactivity with NA2-specific CD16 MoAb PEN1 was sixfold higher in SH(+) individuals compared with controls. Apparently, the 60Ala-->Asp substitution in SH-Fc gamma RIIIb influences the epitope recognized by PEN1. In conclusion, we identified three NA(1+,2+)SH(+) individuals carrying three Fc gamma RIIIB genes and observed a clear gene-dosage effect on the level of expression of neutrophil Fc gamma RIIIb.
IgG immune complexes are of central importance in the humoral immune system and strongly implicated in the pathogenesis of hematologic and rheumatic autoimmune disorders. Cross-linking of receptors for the Fc domain of IgG antibodies (FcgammaRs) triggers a wide variety of effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. In this way, FcgammaR provide an essential feedback between the humoral and cellular immune response. In the past, significant advances have been made in the molecular dissection of FcgammaR function using cellular transfection systems. Current approaches designed to target and change individual FcgammaR genes in mice have given further insight into their specific contributions to systemic processes, also indicating them to be important immunoregulatory receptors involved in various disease states of allergy, autoimmunity, and inflammation. Future work on targeting FcgammaR binding sites in combination with humanized FcgammaR mouse models will lead to novel therapeutic strategies in the treatment of IgG-mediated human disease in which FcgammaR activation plays an integral part.
The neutrophil-specific antigens NA1, NA2, and SH are well-recognized allotypic forms of FcgammaRIIIB. Individuals carrying all three FcgammaRIIIB genes were recently described.
A Danish population (n = 200) was typed for NA1, NA2, and SH by polymerase chain reaction with sequence-specific primers (PCR-SSP). Twelve individuals with three FcgammaRIIIB genes were further genotyped by PCR-based restriction fragment length polymorphism and by DNA sequencing. Family studies were performed on three individuals who carry three FcgammaRIIIB genes.
The gene frequencies for NA1, NA2, and SH were 0.365, 0.635, and 0.030, respectively. In eight individuals (4%), all three FcgammaRIIIB genes were identified. All 12 SH+ individuals were NA1+.
The NA1, NA2, and SH gene frequencies observed in Danes are similar to those in other white populations. The distribution of FcgammaFIIIB genotypes in the Danish population strongly indicates that the NA and the SH systems are located in two closely linked loci and that SH is closely linked to NA1. Finally, a new PCR-SSP was developed to distinguish NA2 from SH.
Granulocyte-specific antigens play an important role in provoking immune neutropenia and transfusion reactions. We developed a new DNA-typing method, PCR-preferential homoduplex formation assay (PHFA), to determine granulocyte-specific antigens on the neutrophil Fcgamma receptor IIIb (FcgammaRIIIb, CD16b), namely, the NA1, NA2, and SH antigens and their gene frequencies in the Japanese population.
Four hundred unrelated healthy Japanese blood donors were typed using PCR-PHFA. To confirm the accuracy of the results of FcgammaRIIIB genotyping using PCR-PHFA, PCR-sequence-specific primer (SSP) typing and PCR-restriction fragment length polymorphism (RFLP) typing were carried out in another 20 samples for comparison.
The results of PCR-PHFA typing agreed well with other methods. The frequencies of the FcgammaRIIIB alleles were 62.2, 37.8, 0 and 0% for NA1, NA2, SH, and 'NA-null', respectively.
The PCR-PHFA method can be semi-automated easily with computer-based assignment and is suitable for typing both small and large numbers of samples. In the Japanese population, the frequency of NA1 is about double that in Caucasians (32.5%), and the SH allele is rare.
Neutrophil-specific antigens NA1 and NA2 are located on Fcgamma receptor IIIb (FcgammaRIIIb). NA1 and NA2 forms of FcgammaRIIIb differ by four amino acids and the corresponding genes by five nucleotides. Variations in NA gene frequencies are encountered among ethnic groups. Altered forms of the genes are expected among individuals.
RFLPs associated with four recognition sites were used to determine NA genotypes of 232 individuals. When atypical NA genotypes were identified, FcgammaRIIIB and FcgammaRIIIA regions were sequenced.
NA1 FcgammaRIIIB frequency in 100 Japanese (0.66) was greater than that in 53 African Americans (blacks) (0.40; p<0.01) and 79 whites (0.32; p<0.001). Sequencing of atypical FcgammaRIIIB in 16 people confirmed that four blacks had G-->A substitutions at 227; 7 blacks had A-->G substitutions at 277; and 1 Japanese person had C-->G at 141 and G-->T at 227. A at 227 and G at 277 represent expected nts of NA1 FcgammaRIIIB. One black had an NA1 FcgammaRIIIB with a G-->A substitution at 349; A is normally found in NA2 FcgammaRIIIB at 349. Sequencing atypical FcgammaRIIIA in three persons revealed that two blacks had G-->A substitutions at 277 plus C-->A substitutions at 266 and 1 white had previously described T-->G at 230. Two blacks with atypical NA2 FcgammaRIIIB had T-->G FcgammaRIIIA at 230. One black was NA(null).
NA2 FcgammaRIIIB is more polymorphic in blacks than in whites or Japanese persons. Chimeric FcgammaRIIIB alleles are most similar to NA2 FcgammaRIIIB. One alternate allele of NA1 (NA1*02) and four alternate alleles of NA2 (NA2*02, NA2*03, NA2*04, and NA2*05) are described.
Platelet antigens are of importance in several clinical situations and in population genetics. Data are scarce on allele frequencies in ethnic groups other than whites, Asians and African Americans. The frequencies of the alleles of the systems HPA-3 and HPA-5 were determined using the allele-specific restriction enzyme for five South American Amerindian populations and compared with those obtained for Afro-Americans, Japanese and whites from Brazil. The frequency of the HPA-3a allele among the Amerindians as a group did not differ from the values obtained for the other populations. However, differences were observed among the Amerindians, varying from 0.27 to 0.75, the highest frequency thus far observed for a population of Asian origin. Only the HPA-5a allele was found among 130 Amerindian chromosomes. The determination of gene frequencies of the HPA systems in different populations allows inference of gene flows and genetic constitution of populations and the estimation of the risk of platelet-specific alloimmunization.
The granulocyte antigens HNA-1a, -1b, and -1c (formerly named NA1, NA2 and SH) which reside on the neutrophil FcgammaReceptor IIIb (FcgammaRIIIb) play a major role in immune neutropenias and pulmonary transfusion reactions. In an attempt to shed some light on the origin and history of these antigens we typed the DNA of Blacks from South Africa (n=99), and Ghana (n=27), of 56 African Americans, and of 138 Chinese from Taiwan for HNA-1a,-1b, and -1c antigens using polymerase chain reaction with sequence-specific primers (PCR-SSP). In African and American Blacks, the HNA-1b antigen was more frequent than HNA-1a (77 vs. 67% and 77 vs. 59%, respectively). In contrast, in Chinese HNA-1a was more frequent than HNA-1b (91 vs. 54%). We observed 3 individuals with FcgammaRIIIB deficiency among the 126 tested African Blacks indicating a higher frequency of FcgammaRIIIB deficiency in Blacks than the reported 0.1% in Europeans. In addition, the frequency of HNA-1c in African and American Blacks (38 and 23%, respectively) was higher than the reported 5% in Europeans. Among the 57 HNA-1c (+) Blacks, all were HNA-1b (+) but only 26 were HNA-1a (+) supporting the idea that the HNA-1c antigen is the result of an additional point mutation in the allele coding for HNA-1b. Recently, HNA-1a, -1b, and -1c (+) Europeans have been reported to have three distinct FcgammaRIIIB genes. Among 26 Blacks who had been typed HNA-1a,b,c (+) by PCR-SSP we identified only 7 having three FcgammaRIIIB genes by DNA sequencing. When we sequenced the DNA of 6 HNA-1a,b,c (+) Europeans we found 4 of the individuals had three FcyRIIIB genes. Therefore, we assume that in Africa the point mutation occurred first in the HNA-1b allele resulting in the HNA-1c allele and the FcgammaRIIIB gene duplication took place later.
The FcgammaRIIA gene is expressed in two polymorphic forms, R131 and H131, which differ by the replacement of histidine by arginine at position 131. The FCGR3B (FcgammaRIIIB) gene exists in two allelic isoforms, known as FCGR3B1 (FcgammaRIIIB-NA1) and FCGR3B2 (FcgammaRIIIB-NA2), which differ in nucleotides 141, 147, 227, 277, and 349. An additional polymorphism is the SH antigen that is associated with the FCGR3B3 (FcgammaRIIIB-SH) allele.
By use of a PCR with allele-specific primers, the allelic polymorphisms of FcgammaRIIA and FcgammaRIIIB were determined among 263 unrelated Brazilian subjects, including Amazon Indians (n = 92), blood donors (n = 85), and patients with sickle cell disease (SCD) (n = 86).
Amazon Indians had a significantly higher frequency of the R131 allele than did blood donors and SCD patients (0.91 vs. 0.55 vs. 0.55; p<0.001). NA1 and NA2 gene frequencies were found to be 0.67 and 0.21 for Amazon Indians, 0.58 and 0.42 for blood donors, and 0.61 and 0.39 for SCD patients, respectively. The FcgammaRIIIB-SH allele was absent from the Amazon Indians, but 9 (10.6%) blood donors and 10 (11.6%) SCD patients expressed this allele.
Overall, the data indicate that the distribution of the FcgammaRIIIB alleles is significantly different in Amazon Indians from the distribution in Brazilian blood donors or African Brazilian patients with SCD, but that it is similar to the distributions reported in Asian populations. Moreover, the distribution of the FcgammaRIIA and FcgammaRIIIB alleles among Brazilian blood donors and SCD patients is comparable to the distributions reported in whites from the United States and Europe.
Human neutrophil antigen-1c (HNA-1c) (SH) has been described as the third alloantigen of the Fc receptor type IIIb (FcgammaRIIIb) for IgG beside the known alloantigens HNA-1a (NA1) and HNA-1b (NA2). Controversy exists on the assignment of the HNA-1c coding gene to the FCGR3B locus and on a possible linkage between the HNA-1c and HNA-1a coding genes.
Two hundred sixty northern German blood donors and 43 individuals from Uganda were typed for FCGR3B*1 (NA1), FCGR3B*2 (NA2), and FCGR3B*3 (SH) by allele-specific PCR. In a subset of FCGR3B*3-positive probands, PCR-amplified FCGR3 fragments were subcloned and sequenced. Transmission of FCGR3B*3 was analyzed in family studies. A possible correlation with the FcgammaRIIIb alloantigen expression was investigated by flow cytometry.
In the northern German population, FCGR3B*3 was found exclusively in individuals carrying FCGR3B*1 independent of the existence of FCGR3B*2 at a frequency of 5 percent. In the individuals from Uganda, each possible combination of FCGR3B*1, FCGR3B*2, and FCGR3B*3 was detected. FCGR3B*3 frequency was 34.9 percent. Within both populations, some individuals carried each of the three genotypes. DNA sequencing revealed new FCGR3 variants caused by single nucleotide exchanges at the typical polymorphic positions. In one individual, six different FCGR3 variants were detected.
The coincidence of the three known FCGR3B alleles varies within the population of Germany and Uganda. Three simultaneous FCGR3B forms may be explained by two gene loci, but the basis of the high number of different variants in some individuals still remains unclear. Possible explanations may be a hypermutation mechanism or a number of FCGR3 higher than expected hitherto.
The granulocyte antigens HNA-1a, -1b and -1c reside on the FcgammaIIIb receptor, which is exclusively expressed on neutrophils. They are involved in autoimmune and alloimmune neutropenias as well as in severe transfusion reactions. Recent family studies show the HNA-1c antigen to be inheritably linked to HNA-1a, resulting in individuals carrying three genes. Using sequence-specific primers, the HNA antigens can be discerned in a polymerase chain reaction (PCR), but not the number of coding FCGR3B genes present in each individual. Therefore a real-time kinetic PCR method using the LightCycler technique was established to ascertain the amount of FCGR3B genes in each individual. For this purpose, the FCGR3B genes of four HNA-1a,-1b,-1c (+), one HNA-1b,1c (+) and one HNA-1b (+) individual were quantified. In addition, two families, in which the mother was FCGR3B deficient, were analyzed. Quantification showed two of the four HNA-1a,-1b and-1c (+) individuals to have three genes and the other two to have only two genes. The HNA-1b,-1c (+) individual had also only two genes. As expected, the HNA-1b donor was typed for two genes. No FCGR3B genes could be detected in the FCGR3B-deficient mothers of either family; their husbands however, carried two FCGR3B genes. Accordingly, quantitative PCR showed the offspring of both families to have only one FCGR3B gene. Quantitative PCR with the Light Cycler has been revealed to be a fast and reliable method for the determination of FCGR3B genes present. FCGR3B quantification confirms the idea of the HNA-1c antigen to be inherited not only linked to HNA-1a, but also to be passed down on its own.
Between the 15th and 19th centuries ad, the Atlantic slave trade resulted in the forced movement of approximately 13 million people from Africa, mainly to the Americas. Only approximately 11 million survived the passage, and many more died in the early years of captivity. We have studied 481 mitochondrial DNAs (mtDNAs) of recent African ancestry in the Americas and in Eurasia, in an attempt to trace them back to particular regions of Africa. Our results show that mtDNAs in America and Eurasia can, in many cases, be traced to broad geographical regions within Africa, largely in accordance with historical evidence, and raise the possibility that a greater resolution may be possible in the future. However, they also indicate that, at least for the moment, considerable caution is warranted when assessing claims to be able to trace the ancestry of particular lineages to a particular locality within modern-day Africa.
Allelic polymorphisms of human Fcg receptor IIa and Fcg receptor IIIb among distinct groups in Brasil Determination of granulocyte-specific antigens on neutrophil Fcg receptor IIIb by PCR-preferential homoduplex formation assay, and gene frequencies in the Japanese population
- St Kuwano
- Jo Bordin
- Chiba
Kuwano ST, Bordin JO, Chiba AK et al. Allelic polymorphisms of human Fcg receptor IIa and Fcg receptor IIIb among distinct groups in Brasil. Transfusion 2000: 40: 1388–92. 17. Fujiwara K, Watanabe Y, Mitsunaga S et al. Determination of granulocyte-specific antigens on neutrophil Fcg receptor IIIb by PCR-preferential homoduplex formation assay, and gene frequencies in the Japanese population. Vox Sanguinis 1999: 77: 218–22.