Genetic heterogeneity at the glycosyltransferase loci underlying the GLOB blood group system and collection

The aim of this study was to further explore the molecular genetic bases of the clinically important but rare blood group phenotypes p, P(1) (k) and P(2) (k) by analysis of the 4-alpha-galactosyltransferase (P(k)) and 3-beta-N-acetylgalactosaminyltransferase (P) genes responsible for synthesis of the related P(k) (Gb(3)) and P (Gb(4)) antigens respectively. Lack of these glycolipid moieties is associated with severe transfusion reactions and recurrent spontaneous abortions but also offers immunity against certain infectious agents. Blood samples from 20 p and 11 P(1) (k) or P(2) (k) individuals of different geographic and ethnic origin were investigated. DNA sequencing by capillary electrophoresis was performed following amplification of the coding regions in the P(k) or P genes. In the P(k) gene, nine novel and five previously described mutations were detected. One of the newly found mutations introduced an immediate stop, five shifted the reading frame introducing premature stop codons and three were missense mutations causing amino acid substitutions in conserved regions of the transferase. Four new and two previously described mutations in the P gene were found. Three of the novel alleles reported here carried nonsense mutations whilst the fourth allele had a missense mutation. The finding of 13 novel mutations in 14 alleles emphasizes further the genetic heterogeneity at the glycosyltransferase loci underlying the GLOB blood group system and collection.     

Distribution of Rhesus blood group antigens and weak D alleles in the population of Albania

Background

Determination of Rhesus (Rh) status is of critical importance in the field of both transfusion and obstetric medicine. As the distribution of Rh phenotypes was unknown in the Albanian population, we investigated the donor population in Albania to estimate the prevalence of each phenotype, as well as to identify and characterise the variants at the molecular level.

Materials and methods

A total of 38,836 blood donors were phenotyped for Rh D, C, c, E and e antigens by routine serological methods, and samples with reduced D antigen expression underwent molecular characterisation by a Tm-shift genotyping method and direct sequencing.

Results

Among all donors 89.00% and 10.86% were D-positive and D-negative, respectively, while 0.14% (n=55) of the donors were found to be weak D-positive. Overall 45/55 samples (81.8%) were resolved by Tm-shift screening, showing that approximately 67% of the variant D alleles were weak D type 1, while weak D type 3 (9.1%) and weak D type 2 (3.6%) were less common. A novel c.932A>G (p.Y311C) variant was also found in the heterozygous state by direct sequencing.

Discussion

This extensive study reveals the distribution of Rh phenotypes in the Albanian population, the low prevalence of individuals with a weak D phenotype, and the specific pattern of distribution of the three most common variant alleles in this Caucasian population.     

Distribution of Diego blood group alleles and identification of four novel mutations on exon 19 of SLC4A1 gene in the Chinese Han population by polymerase chain reaction sequence–based typing

Background The Diego blood group system plays an important role in transfusion medicine. Genotyping of DI1 and DI2 alleles is helpful for the investigation into haemolytic disease of the newborn (HDN) and for the development of rare blood group databases. Here, we set up a polymerase chain reaction sequence–based typing (PCR‐SBT) method for genotyping of Diego blood group alleles. Study Design and Methods Specific primers for exon 19 of the solute carrier family 4, anion exchanger, member1 (SLC4A1) gene were designed, and our PCR‐SBT method was established and optimized for Diego genotyping. A total of 1053 samples from the Chinese Han population and the family members of a rare proband with DI1/DI1 genotype were investigated by the PCR‐SBT method. An allele‐specific primer PCR (PCR‐ASP) was used to verify the reliability of the PCR‐SBT method. Results The frequencies of DI1 and DI2 alleles in the Chinese Han population were 0·0247 and 0·9753, respectively. Six new single nucleotide polymorphisms (SNPs) were found in the sequenced regions of the SLC4A1 gene, and four novel SNPs located in the exon 19, in which one SNP could cause an amino acid alteration of Ala858Ser on erythrocyte anion exchanger protein 1. The genotypes for Diego blood group were identical among 41 selected samples with PCR‐ASP and PCR‐SBT. Conclusion The PCR‐SBT method can be used in Diego genotyping as a substitute of serological technique when the antisera is lacking and was suitable for screening large numbers of donors in rare blood group databases.     

Genomic typing of the Kidd blood group locus by a single-tube allele-specific primer PCR technique

The Kidd (JK) blood group system is clinically important in transfusion medicine. Alloantibodies to antigens in this system may be produced following blood transfusion or during pregnancy and can result in serious haemolytic transfusion reactions and haemolytic disease of the newborn (HDN).
JK antigens on erythrocytes are carried by glycoproteins with the capacity to transport urea through cell membranes. cDNA complementary to mRNA transcribed at the JK locus was cloned in 1994. The molecular basis of the Jk^a/Jk^b blood group polymorphism was recently shown to be a single nucleotide substitution predicting an amino acid change (Asp280Asn) in an extracellular loop of the JK glycoprotein.
After confirmation of the JK gene polymorphism we developed a rapid and robust technique for JK genotyping with allele-specific primers in a single-tube PCR. In addition, a 217 bp intron located at nucleotides 811–812 in the JK gene was found and sequenced. The genotyping test was validated with samples from 106 Caucasian Swedish and 13 Black South African random blood donors. Complete phenotype–genotype correlations were obtained. However, four Jk(a−b−) samples of Polynesian and Finnish origin typed as Jk^bJk^b .
Potential use of the presented method can be predicted in clinical transfusion medicine including prenatal determination of the JK genotype in a fetus at risk for HDN caused by JK antibodies.     

Blood group genotype analysis for the quality improvement of reagent test red blood cells

BACKGROUND AND OBJECTIVES: Reagent red blood cells (RBCs) for antibody detection should express certain important antigens as a double dose, that is, the donors must be homozygous for the corresponding alleles. Traditionally, dose is determined by serological typing and known allele frequencies. However, RHD zygosity cannot be predicted serologically owing to the absence of an antithetical antigen, and FY zygosity is confounded by two variant haplotypes, FY*0 and FY*X. Furthermore, lack of reagents hampers our ability to type for some clinically important antigen pairs such as Do(a)/Do(b). MATERIALS AND METHODS: Genomic DNA was isolated from reagent RBC samples. Established, validated methods were used to determine the RHD, FY, and DO genotypes. RESULTS: Three of 52 D+ samples gave results that differed from the predicted genotype: two presumed R(1)R(1) samples and an R(2)R(2) sample were shown to be R(1)r' and R(2)r', respectively. Five of 59 samples that were from presumed homozygotes for either FY*A or FY*B were heterozygous, together with either FY*X (three samples) or FY*0 (two samples). Seventy-five samples tested for DO were DO*A/A (n = 14), DO*A/B (n = 39), or DO*B/B (n = 22). CONCLUSIONS: The results show that serologically determined RhD and Duffy phenotypes of reagent RBCs are unreliable and that antigens we thought were represented as a double dose were single dose. The addition of Dombrock genotyping provides information which is useful in antibody identification. We conclude that selected genotype analyses are a valuable quality assurance measure to ensure that reagent RBCs comply with national and international recommendations for test sensitivity.     

Rh phenotype,allele and haplotype frequencies among 51,857 blood donors in North India

Background

This study was performed to provide information on the frequencies of Rh antigens, alleles, phenotypes, and haplotypes from our region in India and to compare them with those from other races.

Materials and methods

This observational study was conducted on blood donors from March 2009 to August 2011 using a fully automated system for Rh typing of blood cells. The data were collected and calculations done to determine the antigen, phenotypes, allele and haplotype frequencies. The chi square test was used for comparisons between the results of our study and those of other studies.

Results

A total of 51,857 donors were included in this study. The most common Rh antigen found was “e”. DCCee was the most prevalent phenotype in our study with the phenotype distribution being significantly different between our study and other studies from different regions of the world.

Discussion

We have determined the prevalence of Rh antigens and Rh phenotypes in the North Indian blood donor population and derived the allele and haplotype frequencies in the same population. The Rh blood group distribution in this population was different from that in other populations.     

骨髓增生异常综合征ABO血型鉴定观察

目的:研究46例骨髓增生异常综合征(MDS)病程中ABO血型检定及抗体的变化。方法:采用血型血清学试验检测ABO血型抗原强度变化。结果:血型抗原减弱或丢失者占17例(37.0%),A抗原最容易减弱或丢失,ABO抗体也可以减弱。结论:吸收放散试验、血型物质检测及家系调查可以防止误定ABO血型。     

Rh血型抗原分析在建立实用型稀有血型库中的应用探讨

目的：从临床输血应用的角度,指导对稀有Rh血型的保存与实用型稀有血型库的建立。方法：对2000年～2006年的Rh阴性配血样本进行抗体分析,对1999年4月～2006年9月检出RhD阴性无偿献血者进行D抗原检测和D^Del血型的筛选,及对其他Rh血型抗原检测。结果：在364例Rh阴性配血样本中,有抗-D抗体36例,抗-E20例,抗-C14例,抗-C、E9例,抗-c4例,抗-e10例,抗-D、E2例,抗-D、c4例;在1215名RhD阴性的A、B、O和AB血型献血者中,Rh血型抗原表现型以dccee（51．60％）和dCcee（31．52％）居多,其次为dccEe和dCCee表现型,未见dCCEE和dCcEE表现型。1999年4月～2006年8月共查出D^u50人;在2005年5月～2006年1月的225名初筛RhD阴性献血者中共检出D“型13人,所占比例为5．8％。结论：把0型的dccEE和dC—Cee表型红细胞,在工作中发现后及时冻存并在临床对应使用,在建立实用型稀有血型库、解决临床对特殊血型的需求具有实际的应用意义。     

Genetic basis of blood group diversity

In the last 18 years the genes that encode all but one of the 29 blood group systems present on red blood cells (RBCs) have been identified. This body of knowledge has permitted the application of molecular techniques to characterize the common blood group antigens and to elucidate the background for some of the variant phenotypes. Just as the RBC was used as a model for the biochemical characterization of cell membranes, so the genes encoding blood groups provide a readily accessible model for the study of gene expression and diversity. The application of genotyping techniques to identify fetuses at risk of haemolytic disease of the newborn is now the standard of care, and the expansion of nucleic acid testing platforms to include both disease testing and blood typing in the blood centre is on the horizon. This review summarizes the molecular basis of blood groups and illustrates the mechanisms that generate diversity through specific examples.     

Molecular typing for the Indian blood group associated 252G>C single nucleotide polymorphism in a selected cohort of Australian blood donors

Background

The Indian blood group antigens, In^a and In^b, are clinically significant in transfusion medicine. However, antisera to type these antigens are difficult to obtain. The In^b antigen is a high frequency antigen present in all populations, while the frequency of the antithetical In^a ranges from 0.1% in Caucasians up to 11% in Middle Eastern groups. This antigen polymorphism is encoded by the single nucleotide polymorphism (SNP) 252G>C in CD44. The aim of this study was to establish and compare two genotyping methods to measure the frequency of the IN*A and IN*B alleles in a blood donor cohort.

Materials and methods

Donor blood samples (n=151) were genotyped by a novel real-time polymerase chain reaction (PCR) high-resolution meltcurve (HRM) analysis and a custom matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) assay. Samples with the rare IN*A allele were further investigated by nucleotide sequencing, red cell agglutination, and flow cytometry techniques.

Results

In this study group, 149 IN*B homozygous and 2 IN*A/B heterozygous samples were detected with 100% concordance between HRM and MALDI-TOF MS methods. For PCR HRM, amplicon melting alone did not differentiate IN*A and IN*B alleles (class 3 SNP), however, the introduction of an unlabelled probe (UP) increased the resolution of the assay. Sequencing confirmed that the two non-homozygous samples were IN*A/B heterozygous and phenotyping by red cell agglutination, and flow cytometry confirmed both In^a and In^b antigens were present as predicted.

Discussion

Genotyping permits conservation of rare antisera to predict blood group antigen phenotype. In PCR UP-HRM the IN*A and IN*B alleles were discriminated on the basis of their melting properties. The In^a frequency in this selected donor population was 1.3%. Application of genotyping methods such as these assists in identifying donors with rare blood group phenotypes of potential clinical significance.     

Sequence-specific primers for MNS blood group genotyping

Background

Various techniques of genotyping the MNSs blood group have been described, but none of them enables the complete detection of all MNS antigens.

Materials and Methods.

Blood samples were obtained from blood donors. Primers were created using the published DNA sequences for glycophorins A and B. Genotyping was performed using polymerase chain reaction sequence-specific primers (PCR-SSP).

Results

A total of seven primers were found to specifically amplify the most common MNS antigens. The use of these primers has enabled us to correctly genotype all blood samples tested so far (n=116).

Discussion.

Specifically created primers enable genotyping of the MNS antigens in a single PCR-SSP run. The method is reliable, easy to perform, and can be used in routine practice.     

The serological and genetic basis of the cis-AB blood group in Korea

BACKGROUND AND OBJECTIVES: The cis-AB blood group is rare, although relatively common amongst Koreans. The serological characteristics and genetic basis of Korean cis-AB blood donors were investigated. MATERIALS AND METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), designed to detect the cis-AB01 allele, was performed on 194 AB samples which demonstrated weak or unusual expression of either or both of the A or B antigens. RESULTS AND CONCLUSIONS: Sixty cis-AB01 donors were identified. cis-AB01/O01 or O02 were the most common genotypes (36/60) detected only in A(2)B(3) donors, and cis-AB01/B101 (nine of 60) was the least common genotype identified only in A(2)B donors. Surprisingly cis-AB01/A102 (15/60) was identified in a variety of phenotypes (A(1)B(3), A(1)B(x) or el, A(int)B(3)).     

The rare Kell blood group phenotype KO in a Japanese family

Abstract. The rare Kell blood group phenotype K° was detected in a Japanese male when his red cells failed to agglutinate in either anti-K or anti-k. Further testing showed that his cells, and those of a brother, did not react with anti-Kp^a, Kp^b, Ku, Js^s or Js^b, but did react with Claasen serum from a rare Dutch phenotype in the Kell system. The sera of the propositus and his brother had no antibodies to any of the Kell antigens. The observed frequency of the K° phenotype in over 14,000 Japanese is 0.000069, similar to that in Caucasoid populations.     

Genomic characterisation of the Jk(a-b-) phenotype in Thai blood donors

Background

The Kidd (JK) blood group antigens are encoded by the JK gene. The rare Jk(a−b−) phenotype can be caused by homozygosity for a silent JK allele. Currently, JK^null alleles have been identified among different populations; however, information on its presence among Thais is not available.

Materials and methods

Screening for the Jk(a−b−) phenotype by the urea lysis test was performed in 25,340 blood samples from Thai blood donors. The Jk(a−b−) phenotypes were confirmed by an indirect antiglobulin test (IAT). Additionally, polymerase chain reaction amplification and sequence analysis of the JK gene were performed using previously described methods.

Results

Five samples were confirmed as having a Jk(a−b−) phenotype by a urea lysis test and IAT; four of these samples were investigated. Two samples of JK*02 alleles were homozygous for a g>a mutation at the 3′ acceptor splice site of intron 5 of the JK gene, as in previous studies in Asians and Polynesians. Moreover, one sample of JK*02 alleles was homozygous for an 896G>A mutation at exon 9 (Gly299Glu), as in a previous study in Polynesians. Interestingly, missense dual mutations of JK*01 alleles from a female blood donor were identified. The first mutation was 956C>T (Thr319Met) in exon 10, as in a recent study in African-Americans. The second mutation was 130G>A (Glu44Lys) at exon 4, as in previous studies among Caucasians.

Conclusion

There are various different molecular bases of the Jk(a−b−) phenotype. This is the first report of JK^null alleles among Thais. The information presented in this study could be beneficial in planning genotyping strategies for blood donors and patients.     

人红细胞RhD血型抗原的甲氧基聚乙二醇修饰研究

目的：探讨甲氧基聚乙二醇（mPEG）遮蔽修饰的红细胞RhD血型抗原的有效性和稳定性以及被修饰红细胞结构和功能的变化。方法：用mPEG-SPA修饰RhD血型阳性人红细胞,观测修饰和未修饰红细胞的抗-D凝集反应性、mPEG结合产物稳定性、电镜下细胞形态、变形指数、渗透脆性、自身溶血率、2,3-二磷酸甘油酸含量、膜Na^＋,K^＋ATP酶、乙酰胆碱酯酶活性和胆固醇含量。结果：0．5mmol／L、1．0mmol／L、2．Ommol／L、2．5mmol／L和5．0mmol／L的mPEG-SPA修饰红细胞与抗D的凝集反应情况依次为50％凝集、可见凝集、无凝集、无凝集、无凝集,在4℃保存14d和在37℃保存2d的修饰红细胞与抗D均无凝集反应,而末修饰红细胞则全凝集。电镜下修饰和未修饰红细胞均为双凹圆盘状形态．细胞大小均一。修饰和未修饰红细胞的各观测指标分别为：2,3-二磷酸甘油酸含量（71．00±12．88）mmol／L和（65．13±13．98）mmol／L,红细胞沉降率（2．75±2．05）mmjh和（8．00±3．82）mm／h,细胞变形指数（0．98土0．18）和（0．98士0．29）,自身溶血率（2．27±0．28）％和（1．32±0．32）％,渗透脆性（0．44±0．06）％和（0．44±0．03）％,膜胆同醇含量（0．10±0．03）g／i．和（0．10±0．06）g／L,Na^-,K^-ATP酶活性（4．834±1．27）U／mg和（5．41±1．32）U／mg,乙酰胆碱酯酶活性（27．88±5．09）U／mg和（29．68±4．165U／mg。修饰红细胞的沉降速率低于未修饰红细胞,自身溶血率高于未修饰红细胞（P＜0．05）,但是修饰红细胞沉降速率和自身溶血率都在参考值范围。结论：2．0mmol／L的mPEG-SPA能够有效并且稳定地遮蔽红细胞RhD血型抗原。修饰红细胞具有未修饰红细胞的双凹圆盘状形态、细胞膜结构、变形性和运送氧气能力．     

Heterozygosity for two novel null alleles of the KEL gene causes the Kell-null phenotype in a Japanese woman

The Kell-null (Ko) phenotype is rare and it does not express the Kell antigens on erythrocyte membranes. Recently, several distinct missense and nonsense base substitutions in the coding region and the donor splice site of intron 3 were identified in the KEL gene in individuals with the Ko phenotype. We analysed both genomic DNA and cDNA sequences of the KEL gene in a Japanese woman with the Ko phenotype. She was found to be heterozygous for two novel null KEL alleles. One allele contained an A to G substitution in intron 5 that changes the 3'-splice site of intron 5 from AAG to AGG, resulting in a reading frameshift by a single guanine insertion in KEL mRNA, and the other allele contained a single G to A substitution in exon 12 (codon 459) creating a termination codon. Neither mutation was found in 114 randomly selected Japanese individuals. The results suggested that the Ko blood group phenotype might be owing to several distinct non-functional alleles without any prevalent allele.     

无偿献血者初筛血型错误原因分析及预防措施

目的:对无偿献血者初筛血型错误进行总结分析,提出预防和改进措施。方法:统计无偿献血者标本182 184人次中初筛与检验科血型不符的例数及原因。结果:182 184例无偿献血标本,共检出错误血型92(0.05%)例,其中人为错误41例,技术性错误22例,标本原因29例。结论:通过本地区初筛血型错误原因的分析,制定预防措施后有明显改善,从而减少初筛错误,保证临床患者安全输血。     

恶性肿瘤与Rh血型系统各表型的相关性分析

目的：分析恶性肿瘤患者Rh血型表型的分布特点,并与正常对照组进行比较,查找恶性肿瘤与Rh血型表型相关性。方法：对120例恶性肿瘤患者（患者组）及100例正常健康人群（对照组）进行Rh血型表型测定,对其表型分布进行统计分析,采用χ2检验和相对危险度OR值来估计相对危险性。结果：恶性肿瘤患者组与对照组Rh血型表型CcDEe分布之间的差异有统计学意义（χ2=4.51,P〈0.05）。恶性肿瘤患者组Rh血型表型分布从高到低分别是CcDEe54例（45.00%）,CCDee39例（32.50%）,CcDee14例（11.67%）,ccDEE9例（7.50%）,ccDEe3例（2.50%）,CCDEe1例（0.83%）;对照组Rh血型表型分布从高到低分别是CCDee47例（47.00%）,CcDEe32例（32.00%）,ccDEE9例（9.00%）,CcDee6例（6.00%）,ccDEe5例（5.00%）,CCDEe1例（1.00%）;结论：恶性肿瘤的发生与Rh血型表型的分布有关,CcDEe（OR=1.82）、CcDee（OR=2.07）表型发病率明显高于其他表型。     

A DNA-based immunization protocol to produce monoclonal antibodies to blood group antigens

A major challenge facing transfusion medicine is the establishment of immunological methods to produce specific and avid blood group typing reagents to the many polymorphic blood group antigens. This is especially true when sources of human antibody are limited. Based on the knowledge that inoculation with plasmid DNA can induce a humoral response in the host animal, we inoculated mice with plasmid DNA followed by a single boost injection with plasmid-transfected cells that have a high level of expression of the same target protein. Using this method, several hybridoma clones that produced strongly reactive antibodies specific for the Kell polymorphic antigens (anti-K, anti-k, anti-Kp(a)) were isolated. The monoclonal antibodies that were produced with this method have potential clinical utility for identifying a patient's blood type and for screening for antigen-negative donor blood.