Serology and gene sequence analysis of rare subgroup A3 blood group

To investigate the serological phenotypic characteristics and possible mechanism of subgroup A₃, a blood donor's ABO phenotypes were detected by the conventional microcolumn gel method and classic tube method. N-acetylgalactosaminyl transferase activity was detected by the non-radioactive phosphate coupling method. ABO subtype genotyping was determined by PCR-SSP and exons 1-7 of ABO gene were analyzed by Sanger sequencing. The donor's blood type was subgroup A₃ as evaluated by serological test. There was no N-acetylgalactosaminyl transferase activity in the red blood cells and weak N-acetylgalactosaminyl transferase activity in the plasma. The ABO blood group genotyping result was ABO*AO1, and the gene sequencing result was confirmed as A221/O01. Sequencing results showed two mutations, 467C>T and 607G>A in exon 7 in ABO*A allele. In conclusion, it is suggested that the ABO blood group of the donor be subgroup A₃, which may be induced by mutations 467C>T and 607G>A, and led to a decrease in N-acetylgalactosaminyl transferase activity and resulted in weakened A antigen.     

中国人罕见的cisAB变异型分子机制分析

为研究中国人群ABO血型系统中罕见的cisAB变异型的分子遗传背景, 用血型血清学方法鉴定12例ABO血型疑难样本和116例随机无血缘关系样本, 应用聚合酶链反应和DNA序列分析等方法对样本ABO基因酶活性编码区外显子6、7和侧翼内含子进行突变筛选和检测, 并对不同基因型的扩增片段进行单倍体序列分析。血清学检测发现12个样本均为(A强B弱)或(A弱B强), PCR产物直接序列分析表明这些样本均为cisAB变异型, 并存在4种基因型。通过单倍体序列分析, 从中发现2种cisAB等位基因, 其中cisAB01不仅存在外显子7的467C>T和803G>C变异, 还在第6内含子存在未经报道的163T>C和179C>T变异; 另一种等位基因是在B101基础上保留了A101的803G位点, 编码一条176G、235S、266M和268G组合的多肽链。经检索, 未发现该组合的ABO等位基因, 该等位基因已被国际血型抗原基因突变数据库命名为cisAB05等位基因。文章系统研究了中国人群cisAB变异型分子机制, 发现了一种新的cisAB05等位基因, 同时根据内含子序列推测cisAB01等位基因可能是通过A101和B101等位基因间的同源交换而形成的。     

Serological and genetic analysis of a rare CisAB01/O01 blood group

The paper aims to analyze a rare blood sample in Ganzhou City Hospital with CisAB subtype and explore a feasible pattern for blood typing of rare blood type patients, so as to ensure clinical transfusion safety. The routine serological methods were used for ABO forward and reverse blood typing and the fluorescence real-time PCR technique was used for sample genotyping. A human ABO blood group 6-7 exon sequencing kit was used for sequence analysis. The nucleic acid sequence of the sample was compared with reference sequences. The forward typing results demonstrated that the sample was ABw, RhD positive. The sample exhibited 4+ agglutination with anti-H and anti-AB antibodies. Reverse typing by microcolumn gel method showed an AB result, but the serum sample demonstrated weak agglutination with B cell under room temperature, 4 °C and 37 °C in saline when tested with tube method respectively. The serological results matched with the A₂B₃ serotype. The fluorescent real-time PCR genotyping results displayed A/O01. The sequence analysis demonstrated deletion of guanine in 261-position 467C>T (heterozygote) and 803G>T (heterozygote) mutation respectively. The mutation caused the A glycosyltransferase peptide chain to change from proline to leucine (P156L) at 156 and from glutamate to alanine (G268A) at 268. The result demonstrated that the sample''s genotype was CisAB01/O01. The mutation of glycosyltransferase coding gene leads to an abnormal serological reaction pattern. Only by combining the results of genetic analysis can we get the true sample blood type and better ensure the safety of clinical blood transfusion.     

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A¹ (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.     

Serological and gene sequencing analysis of a case of para-Bombay phenotype Amh

The paper aims to study the serological and genetic characteristics of a case of para-Bombay Am^h. The serological method was applied to identify the proband's ABO phenotype and PCR-SSP assay was used to analyze the genotype of the para-Bombay blood. DNA sequencing of the PCR products of the first exon of FUT1 gene was used to analyze the genotype and nucleic acid sequence mutation. The serological results showed that the ABO phenotype of the proband was O-type. However, while after absorption-elution test, the ABO phenotype showed weak A-type. The serological test also showed that the irregular antibody anti-H was positive. PCR-SSP assay showed that the proband was h4 para-Bombay type and sequence analysis showed a point mutation c.35C > T of FUT1 gene. The study suggests that genetic analysis is necessary for blood typing in those who have elusive immunological typing results.     

Serological and gene sequencing analysis of a case of para-Bombay phenotype Amh

The paper aims to study the serological and genetic characteristics of a case of para-Bombay Am^h . The serological method was applied to identify the proband''s ABO phenotype and PCR-SSP assay was used to analyze the genotype of the para-Bombay blood. DNA sequencing of the PCR products of the first exon of FUT1 gene was used to analyze the genotype and nucleic acid sequence mutation. The serological results showed that the ABO phenotype of the proband was O-type. However, while after absorption-elution test, the ABO phenotype showed weak A-type. The serological test also showed that the irregular antibody anti-H was positive. PCR-SSP assay showed that the proband was h4 para-Bombay type and sequence analysis showed a point mutation c.35C>T of FUT1 gene. The study suggests that genetic analysis is necessary for blood typing in those who have elusive immunological typing results.     

Study of NAT2 genetic polymorphism in West African subjects: example of an healthy non-smoker Senegalese population

The NAT2 genetic polymorphism determines the individual acetylator status and, consequently, the capacity to metabolize, or not, drugs and xenobiotics which are substrates of NAT2. As the nature and frequency of the NAT2 polymorphisms vary remarkably between populations of different ethnic origins, genotyping strategies used to predict the acetylation phenotype need to be adapted for each particular population regarding their genetic backgrounds at this locus. As few data on the genetic polymorphism of NAT2 are available in the Senegalese population, we performed an extensive identification of NAT2 variants in 105 healthy non-smoker Senegalese subjects by direct PCR sequencing of the coding region. Eleven previously described SNPs were identified in this Senegalese population. Upon allele analysis, the four most frequent alleles were of the NAT2*5- (35.7?%), NAT2*6- (21.0?%), NAT2*12- (16.7?%) and NAT2*14- (10.0?%) type, the remaining alleles, including the wild-type NAT2*4, having each a frequency lower than 10?%. According to the observed genotypes, 51 and 50 subjects were predicted to be of the rapid (48.6?%) and slow (47.6?%) acetylator phenotype, respectively, while four individuals (3.8?%) were considered of unknown phenotype as they carry at least one allele with a yet unknown functional effect. These baseline data would be of particular interest to set up an efficient genotyping strategy to predict the acetylation status of Senegalese patients with tuberculosis and, thus, to optimize their isoniazid treatment.     

Polymorphisms of Flanking Region of the ASB15 Gene and Their Associations with Performance Traits in Chicken

Research on the identity of genes and their relationship with traits of economic importance in chickens could assist in the selection of poultry. In this study, an F₂ resource population of Gushi chickens crossed with Anka broilers was used to detect single-nucleotide polymorphisms (SNPs) in the flanking region of the ASB15 gene by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). One SNP of ?1271 C>T in 5′ flanking region of the chicken ASB15 gene and two SNPs of the 10618 A>G and 10716 G>A in 3′ flanking region were identified. Furthermore, the 10618 A>G and 10716 G>A in 3′ flanking region were in complete linkage. Association analysis results showed that ?1271 C>T was not associated with performance traits, while the 10618 A>G and 10716 G>A were significantly associated with BW2, 4, 6, 8, 10, 12, SL12, CD8, CW4, 8, 12, BSL4, 8, 12, and SEW, EW, WW, BMW, LW, CW, SFT. Our results suggest that the ASB15 gene profoundly affects chicken performance traits.     

B para-Bombay phenotype in China caused by homozygous mutation for site 328 G>A of FUT1 gene: a case report

The aim of this paper is to accurately identify a case of B para-Bombay and to analyze the genetic mutation. ABO and Lewis blood groups were identified by standard serological methods, and trace antigens on RBCs were detected by adsorption-elution test, while blood group substances in the saliva were detected by agglutination inhibition test. The ABO gene exons 6-7, FUT1 gene exon 4 and FUT2 gene exon 2 were directly sequenced. Serological results showed that there were B antigens on RBCs without H antigens, anti-A and anti-HI antibodies in serum, and B and H blood group substances in the saliva. The Lewis phenotype was Le (a-b+). According to gene sequencing analysis, ABO, FUT1 and FUT2 genotypes were B101/O02, h^328G/Ah^328G/A and Se^357C/TSe^357C/T, respectively. This rare phenotype can be mislabeled as "O" if any of the detailed investigations are not performed. Therefore, in order to ensure the safety of blood transfusion, genetic and serological tests are necessary for the correct identification of difficult blood groups.     

B para-Bombay phenotype in China caused by homozygous mutation for site 328 G > A of FUT1 gene: a case report

The aim of this paper is to accurately identify a case of B para-Bombay and to analyze the genetic mutation. ABO and Lewis blood groups were identified by standard serological methods, and trace antigens on RBCs were detected by adsorption-elution test, while blood group substances in the saliva were detected by agglutination inhibition test. The ABO gene exons 6-7, FUT1 gene exon 4 and FUT2 gene exon 2 were directly sequenced. Serological results showed that there were B antigens on RBCs without H antigens, anti-A and anti-HI antibodies in serum, and B and H blood group substances in the saliva. The Lewis phenotype was Le (a-b+). According to gene sequencing analysis, ABO, FUT1 and FUT2 genotypes were B101/O02, h^328G/Ah3^28G/A and Se^357C/TSe^357C/T, respectively. This rare phenotype can be mislabeled as "O" if any of the detailed investigations are not performed. Therefore, in order to ensure the safety of blood transfusion, genetic and serological tests are necessary for the correct identification of difficult blood groups.     

Interaction between ABO and haptoglobin systems in a Bengalee population.

Blood samples from 2,232 individuals of a Bengalee Caste Hindu population were investigated in an attempt to confirm the association between the ABO and haptoglobin (HP) systems previously found in populations of European origin. Indians differ from Europeans in having lower HP*1 and higher ABO*B frequencies. In spite of this, as in previous studies, a weak HP/ABO association was found with a significantly lower HP*1 allele frequency in blood group O versus other ABO groups.     

ABO genotyping in leukemia patients reveals new ABO variant alleles

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*O01 was the most common allele found, followed by ABO*O22 and by ABO*A103. We identified 22 new ABO*variants in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.     

Molecular analysis to FUT-1, 2, 3 and ABO genotyping may instead of serological typing to diagnose para-Bombay in Chinese

The aim of this study was to evaluate the consistency between serotyping and molecular analysis in Chinese with para-Bombay. The molecular analysis of gene fragments in FUT-1, FUT-2, FUT-3 and ABO genotyping and serotyping were used including a saliva test to examine the A, B, H substance and an absorption elution test to examine the A, B, H; and further routine tests including ABO, H and Lewis phenotype. From eleven samples with anti-H negative, 10 samples were confirmed with para-Bombay by sequencing to FUT-1, from which six samples were 547-548delAG, three samples were 880TT deletion, one sample was 35C>T and one sample was 649G>T heterozygous (h7, China) as carrier. The sequencing to FUT-2 confirmed 357C>T in 11 samples, meaning H, A and B substance was secreted in saliva except for one sample which occurred 385A>T (I129F) heterozygous, which is a weak secretor. The FUT-3 sequence result demonstrated four samples with heterozygous mutations to 59T>G (L20R) combined with 508G>A (G170S) and seven samples without mutations in FUT-3 gene fragment same as reference. The consistency between sequencing with FUT-1/FUT-2 and serotyping by anti-H reported an identical result, except for one sample, which interestingly showed the H/h7 carrier with serotyping negative to anti-H. The result of sequencing with FUT-2/FUT-3 and Lewis phenotyping also reported a complete consistency. The saliva test to A, B, H substance and absorption elution test examining the A, B, H antigens on the surface of red blood cells completely matched the ABO exon 6, 7 sequence results. The sequencing of FUT-1, FUT-2, FUT-3 and ABO exon 6, 7 may become a useful tool to confirm the para-Bombay blood type.     

Compound heterozygous POMT1 mutations in a Chinese family with autosomal recessive muscular dystrophy‐dystroglycanopathy C1

Muscular dystrophy‐dystroglycanopathy (MDDG) is a genetically and clinically heterogeneous group of muscular disorders, characterized by congenital muscular dystrophy or later‐onset limb‐girdle muscular dystrophy accompanied by brain and ocular abnormalities, resulting from aberrant alpha‐dystroglycan glycosylation. Exome sequencing and Sanger sequencing were performed on a six‐generation consanguineous Han Chinese family, members of which had autosomal recessive MDDG. Compound heterozygous mutations, c.1338+1G>A (p.H415Kfs*3) and c.1457G>C (p.W486S, rs746849558), in the protein O‐mannosyltransferase 1 gene (POMT1), were identified as the genetic cause. Patients that exhibited milder MDDG manifested as later‐onset progressive proximal pelvic, shoulder girdle and limb muscle weakness, joint contractures, mental retardation and elevated creatine kinase, without structural brain or ocular abnormalities, were further genetically diagnosed as MDDGC1. The POMT1 gene splice‐site mutation (c.1338+1G>A) which leads to exon 13 skipping and results in a truncated protein may contribute to a severe phenotype, while the allelic missense mutation (p.W486S) may reduce MDDG severity. These findings may expand phenotype and mutation spectrum of the POMT1 gene. Clinical diagnosis supplemented with molecular screening may result in more accurate diagnoses of, prognoses for, and improved genetic counselling for this disease.     

Histo-Blood Group Gene Polymorphisms as Potential Genetic Modifiers of Infection and Cystic Fibrosis Lung Disease Severity

Background

The pulmonary phenotype in cystic fibrosis (CF) is variable; thus, environmental and genetic factors likely contribute to clinical heterogeneity. We hypothesized that genetically determined ABO histo-blood group antigen (ABH) differences in glycosylation may lead to differences in microbial binding by airway mucus, and thus predispose to early lung infection and more severe lung disease in a subset of patients with CF.

Methods and Principal Findings

Clinical information and DNA was collected on >800 patients with the ΔF508/ΔF508 genotype. Patients in the most severe and mildest quartiles for lung phenotype were enrolled. Blood samples underwent lymphocyte transformation and DNA extraction using standard methods. PCR and sequencing were performed using standard techniques to identify the 9 SNPs required to determine ABO blood type, and to identify the four SNPs that account for 90–95% of Lewis status in Caucasians. Allele identification of the one nonsynonymous SNP in FUT2 that accounts for >95% of the incidence of nonsecretor phenotype in Caucasians was completed using an ABI Taqman assay. The overall prevalence of ABO types, and of FUT2 (secretor) and FUT 3 (Lewis) alleles was consistent with that found in the Caucasian population. There was no difference in distribution of ABH type in the severe versus mild patients, or the age of onset of Pseudomonas aeruginosa infection in the severe or mild groups. Multivariate analyses of other clinical phenotypes, including gender, asthma, and meconium ileus demonstrated no differences between groups based on ABH type.

Conclusions and Significance

Polymorphisms in the genes encoding ABO blood type, secretor or Lewis genotypes were not shown to associate with severity of CF lung disease, or age of onset of P. aeruginosa infection, nor was there any association with other clinical phenotypes in a group of 808 patients homozygous for the ΔF508 mutation.     

Human-type blood groups and Gm factors in gibbons (Hylobates)

Blood specimens from 69 gibbons (63Hylobates lar, 4Hylobates concolor, and 2Hylobates pileatus) were tested for human-type ABO, MN, and Rh blood groups. AmongH. lar, three phenotypes were noted in the ABO and MN blood groups respectively, but all fourH. concolor were grouped as AM. All group A gibbons were of subgroup A₁; subgroups A₂B and A₁₂B were observed at a low frequency in group AB gibbons. Le^b antigen was detected in about 30% of the red cell samples fromH. lar, but all the samples were negative for Le^a. All the gibbons tested had c(hr) antigen but no other Rh antigens (D, C, E, and e) in their red cells. Some selected blood samples fromH. lar were also tested for some other blood group antigens and for the Gm and Inv factors. The Jk^a antigen was detected in all the red cell samples tested, but the S, s, U, K, k, and Fy^a antigens were not. In the tests of plasma with anti-Gm (1),H. lar could be divided into two groups, i.e., Gm(1)^Gi and Gm(–1)^Gi; Gm(2), Gm(4), and Inv(1) were absent in the species.     

On the incidence of blood group O and Gm(−1) phenotypes in patients with malignant melanoma

Summary Fifteen polymorphic systems of the blood (ABO, MNSs, Rhesus, P, Kell, Duffy, Kidd, Hp, Gc, Gm, Inv, aP, PGM₁, EsD, and 6-PGD) were examined in 191 unrelated male and female patients suffering from malignant melanoma. These polymorphic systems were compared with the corresponding phenotype and gene frequencies of controls from the same geographical area (Rhineland-Palatinate). The only associations discovered were the ABO and Gm polymorphisms: The incidence of O and Gm(-1) phenotypes in patients is obviously higher than in controls. These observations agree with the findings in other population samples from Germany and Bulgaria.     

Molecular evidence for human alpha2-HS glycoprotein (AHSG) polymorphism

Alpha2-HS glycoprotein (AHSG) is a human plasma glycoprotein that exhibits genetic polymorphism on isoelectric focusing (IEF). To identify the origin of two common alleles, AHSG*1 and *2, we examined nucleotide exchanges in the gene. AHSG cDNA was obtained by RT-PCR from poly(A) RNA of seven liver tissue samples and subcloned into a plasmid vector. After sequencing, we found six single nucleotide differences in comparison with the originally reported sequence. In particular, the nucleotide substitutions of C to T at amino acid position 230 and C to G at position 238 were common among the samples exhibiting phenotype 2–1 or 2. Since these substitutions might give rise to a NlaIII site and a SacI site, respectively, for the potential AHSG*2, we analyzed these substitutions by PCR-RFLP using genomic DNA of 68 individuals. The result was consistent with the IEF analysis of the corresponding serum, indicating that AHSG*1 was characterized by ACG (Thr) at position 230 in exon 6 and ACC (Thr) at position 238 in exon 7, and that AHSG*2 was characterized by ATG (Met) at position 230 and AGC (Ser) at position 238. Received: 5 March 1996 / Revised: 25 June 1996     

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8⁺ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8⁺ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8⁺ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8⁺ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8⁺ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.     

Genetic variations estimated from PCR-RFLP analysis of two morphs of the freshwater goby <Emphasis Type="Italic">Rhinogobius</Emphasis> in the Lake Biwa water system

The freshwater goby Rhinogobius is the most abundant fish in the shore area of Lake Biwa, Japan. Recently, it has been reported that two morphs of Rhinogobius inhabit this lake. In this study, genetic variations in Rhinogobius sp. OR (Orange-type) and Rhinogobius sp. BW (Biwa-type) in the Lake Biwa water system have been investigated using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial DNA, including the variable D-loop region. Samples of Rhinogobius sp. OR were collected from the middle sites of three rivers, two outlets, and two lakeshore sites, whereas samples of Rhinogobius sp. BW were taken from two lakeshore sites. Rhinogobius sp. OR and Rhinogobius sp. BW did not share any haplotypes, suggesting that PCR-RFLP analysis is effective for distinguishing between these species of goby. The haplotype diversity of Rhinogobius sp. OR (0.214–0.543) was lower than that of Rhinogobius sp. BW (0.543–0.682). There were no significant differences in haplotype frequencies between Rhinogobius sp. BW groups from the two localities. In addition, haplotype frequencies in Rhinogobius sp. OR did not differ significantly among samples from the middle sites of rivers, the outlets, and the shores. These results indicate that in Rhinogobius sp. OR there is frequent gene flow among fish inhabiting different sites, and that this species of goby consists of a single population throughout the Lake Biwa water system.