Lack of G blood group antigen in DIIIb erythrocytes is associated with segmental DNA exchange between RH genes

Summary. The Rh blood group antigens D, Cc and Ee are encoded by two highly related genes, RHD and RHCE. Almost all red cells which carry D and all cells which carry C also express the G (Rh12) antigen. In this report we have determined the molecular basis of the D^IIIb category phenotype which represents a very rare condition characterized by the presence of most of the D epitopes and the total absence of the antigen G. mRNA sequencing and Southern blot analysis of two unrelated samples indicated that the D^IIIb category phenotype is associated with a segmental DNA exchange between exon 2 of the RHD and RHCE genes resulting in three D c amino acid substitutions (Ile60Leu, Ser68Asn Ser103Pro).     

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。     

Variants of RhD – current testing and clinical consequences

Anti‐D (‐RH1) of the Rh blood group system is clinically important as it causes haemolytic transfusion reactions and haemolytic disease of the fetus and newborn. Although most people are either D+ or D−, there is a plethora of D variants, often categorized as either weak D or partial D. These two types are inadequately defined and the dichotomy is potentially misleading. DVI is the D variant most commonly associated with anti‐D production and UK guidelines recommend that patients are tested with anti‐D reagents that do not react with DVI. Weak D types 1, 2, and 3 are seldom, if ever, associated with alloanti‐D production, so a policy recommendation would be to treat patients with those D variants as D+, to preserve D− stocks, whereas patients with all other D variants would be treated as D−. All donors with D variant red cells, including DVI, should be treated as D+.     

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.     

Molecular analysis of blood group Rh transcripts from a rGr variant

The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE . The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the r^Gr phenotype. Nucleotide sequence analysis revealed that the r  ^G allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid r  ^G gene ( c-C-e or c-D-e ) carries Ile60, Ser68 and Ser103 (as C and D polypeptides); any of these positions appear to be critical in the formation of the G antigen. In addition, Cys16 was found to be important in the phenotypic expression of C.     

A Leu55 to Pro substitution in the integrin alphaIIb is responsible for a case of Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. A new mutation, a T to C substitution at base 258 in the alphaIIb gene, leading to the replacement of Leu55 with Pro, was found by sequence analysis of a patient's alphaIIb cDNA. In transfection experiments using COS7 cells, the cells co-transfected with the mutated alphaIIb cDNA containing C258 and wild-type beta3 cDNA scarcely expressed the alphaIIbbeta3 complex. The Leu55 to Pro substitution in the alphaIIb gene was found to be responsible for this case of Glanzmann's thrombasthenia.     

Rh抗原分型及血型不规则抗体筛检在精准输血中的应用效果研究

［摘要］　目的　探讨Rh抗原分型及血型不规则抗体筛检在精准输血中的应用效果。方法　选择2019年1月1日至2021年9月30日在青龙满族自治县医院住院且需临床输血治疗的患者200例,年龄12～71（48.43±1.45）岁。以2019年1月1日至2020年8月31日未实施Rh(D、C、c、E和e）5种血型抗原检测的100例输血病例作为对照组;以2020年9月1日至2021年9月30日经Rh血型抗原检测行相容性配血的100例输血病例作为试验组。在血制品入库前,应用抗人球蛋白卡式法对标本进行ABO血型和Rh(D、C、c、E和e）5种血型抗原及不规则抗体进行检测,并将结果录入输血信息管理系统。在使用时,应用输血信息管理系统为试验组受血者自动匹配ABO血型和Rh(D、C、c、E和e）5种血型抗原相合的血袋,采用盐水法及抗人球蛋白卡式法进行交叉配血试验,实现临床精准化输血。结果　对照组检出不规则抗体11例（11.00%）,试验组检出2例（2.00%）,比较差异有统计学意义（χ²=6.664,P=0.010）。试验组中未发生输血不良反应病例。对照组中发生输血不良反应1例,系患者产生了血型不规则抗-E抗体,经疑难配血、输注相合性血液4 U后,患者血红蛋白浓度升至预期水平,康复出院。与对照组相比,试验组疑难配血用时、住院时间更短,输注去白悬浮红细胞量更少,差异有统计学意义（P<0.05）。结论　开展Rh血型抗原检测有助于实现临床精准化输血,有效避免不规则抗体的产生,减少临床输血不良反应,提高临床输血治疗的安全性,值得临床推广。     

16Cys encoded by the RHce gene is associated with altered expression of the e antigen and is frequent in the R0 haplotype

Serological observations have suggested that numerous D, many e (especially in Blacks), several E, and rare c variants exist within the Rh blood group system. The molecular basis for expression of many of these variants has been elucidated. This study describes five unrelated Caucasians whose red blood cells reacted with polyclonal anti-e but did not react with some monoclonal anti-e, which suggested that they carried a variant e antigen. Molecular investigation revealed the presence of a 48G-->C change (encoding cysteine instead of tryptophan at amino acid 16) in their RHce gene. No other differences were found, which suggests that amino acid residues located in the first transmembrane region can affect expression of the e antigen, whose critical residues are on the predicted fourth external loop of the protein. This polymorphism has not previously been observed because polyclonal anti-e does not distinguish this variant from wild type. This position is polymorphic in RHce alleles and the presence of the 48C nucleotide is often found in the R0 (Dce) haplotype.     

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.     

The ABCB6 mutation p.Arg192Trp is a recessive mutation causing the Lan− blood type

Background and Objective The membrane transporter ABCB6 has recently been shown to carry the high‐frequency red‐blood‐cell (RBC) antigen Lan. All the Lan? individuals genotyped so far have inherited two recessive null mutations in ABCB6. The finding of a family with the Lan? blood type occurring in two successive generations prompted this study. Methods Mutations in ABCB6 were searched by Sanger sequencing of exons and flanking intronic regions. Expression analysis of the Lan antigen was carried out by serology and flow cytometry. PCR‐RFLP genotyping and Western blot analysis were also applied. Results All the Lan? members of this family were homozygous for c.574C>T, p.Arg192Trp in ABCB6 while the Lan+ members were heterozygous for this missense mutation encoded by the SNP rs149202834. Homozygosity for p.Arg192Trp was associated not only with absence of the Lan antigen, but also of the ABCB6 transporter in RBC membrane. The complete absence of Lan expression resulting from p.Arg192Trp homozygosity was confirmed by the subsequent identification of five unrelated Lan? individuals who were homozygous for this mutation and who developed an anti‐Lan. We also provide evidence that three other single amino acid mutations in ABCB6 (c.826C >T, p.Arg276Trp; c.85_87delTTC, p.Phe29del; c.1762G >A, p.Gly588Ser) may also define ABCB6 null alleles. Conclusion p.Arg192Trp is the first ABCB6 missense mutation causing the Lan? blood type and appears to be a relatively frequent cause of this rare blood type. Like the previously reported frameshift, nonsense and essential splice‐site mutations in ABCB6, this missense mutation is recessive and defines an ABCB6 null allele. Other single amino acid mutations in ABCB6 may also cause the Lan? blood type.     

LU21: a new high-frequency antigen in the Lutheran blood group system

BACKGROUND AND OBJECTIVES: The Lutheran blood group system comprises 18 antigens numbered LU1 to LU20, with two numbers obsolete. Thirteen antigens are of high frequency. MATERIALS AND METHODS: Serological tests were performed by conventional methods. The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay was carried out with monoclonal antibodies to Lutheran glycoprotein. All exons of the LU gene were amplified by the polymerase chain reaction (PCR) and directly sequenced from genomic DNA. RESULTS: A patient was found to have an antibody to a high-frequency red cell antigen during her second pregnancy. The antibody was shown to be Lutheran-related and was distinguished from all reported Lutheran antibodies. MAIEA suggested the antibody was defining a novel epitope in domain 1 of the Lu-glycoprotein. Sequencing of the LU gene revealed a new homozygous single-point mutation, C282G, in exon 3, encoding an Asp94Glu change in the first domain of the Lu-glycoprotein. CONCLUSIONS: The antibody detected a new high-frequency Lutheran antigen, numbered LU21, that appears to result from an Asp94Glu substitution in the first, N-terminal domain of the Lu-glycoprotein.     

Molecular basis of the rare gene complex, DIVa(C)-, which encodes four low-prevalence antigens in the Rh blood group system

Background Over 40 years ago, an unusual Rh phenotype denoted DIVa(C)‐ was identified in a case of fatal haemolytic disease of the newborn in the third child of Madame Nou. Her RBCs expressed a partial D, weak C and four low‐prevalence Rh antigens: Go^a (RH30), Rh33 (RH33), Riv (RH45) and FPTT (RH50). The purpose of this study was to determine the molecular basis associated with this rare DIVa(C)‐ complex. Material and Methods Blood samples were from three donors previously identified as carrying the DIVa(C)‐ haplotype. Molecular analyses were performed by standard methods. Results The three donors were heterozygous for RHD and RHD*DIVa.2, and all carried a compound hybrid allele at the RHCE locus. This hybrid RHCE allele contained exons 2 and 3 from RHD*DIVa.2 and exon 5 from RHD [RHCE*CE‐DIVa.2(2‐3)‐CE‐D(5)‐CE] and is in cis to RHD*DIVa.2. The RHCE allele on the in trans chromosome differs between the donors and is RHCE*cE in donor 1, RHCE*ce (254C, 733G) in donor 2 and RHCE*ce in donor 3. Conclusions The RHD*DIVa.2 encodes the Go^a antigen, whereas the compound hybrid allele most likely encodes Rh33, Riv and FPTT. The weakly expressed C antigen on RBCs with the DIVa(C)‐ phenotype could be encoded by exons 2 and 3 from RHD*DIVa.2 in the compound hybrid. This is the first report of RHD*DIVa.2 being involved in a hybrid gene at the RHCE locus. As only one example of anti‐Riv has been described, our molecular analysis and findings provide a tool by which to predict Riv expression.     

ABO blood group is a risk factor for coronary artery disease in patients with poor blood pressure control

Background: Few studies had examined the role of ABO blood groups on CAD in hypertensive patients with different blood pressure (BP) controls. Methods: A total of 2708 patients with primary hypertension (HTN) were consecutively enrolled and underwent coronary angiography (CAG) due to angina-like chest pain. The severity of coronary artery stenosis was assessed by Gensini score (GS). Patients were divided into two groups due to results of CAG: HTN with CAD (n = 2185) and HTN without CAD (n = 523). Poor BP control was defined as systolic BP (SBP) ≥ mean in the study. Multivariable regression analysis was used to determine the potential impact of ABO blood groups on risk of the presence and severity of CAD. Results: Compared to HTN without CAD group, the percentage of A blood group was statistically higher and O blood group was significantly lower in HTN with CAD group. Moreover, percentage of the angiography-proven CAD was higher in A blood group than that in non-A blood group (p < 0.05). After adjusting for confounding factors, A blood group was independently associated with CAD (odds ratio (OR): 1.422; 95% confidence interval (CI): 1.017–1.987; p = 0.039) and GS (β = 0.055, p = 0.046) in patients with poor BP control. Conclusions: A blood group was an independent risk factor for the presence and severity of CAD in hypertensive patients with poor BP control.     

Expression of the Rh/RhAG complex is reduced in Mi.III erythrocytes

Background and Objectives Miltenberger blood group antigen subtype III (Mi.III) is characterized by expression of a glycophorin B‐A‐B hybrid (Gp.Mur) on the erythrocyte surface. The two alleles of glycophorin B are substituted with the B–A–B hybrid alleles in homozygous Mi.III (Mi.III^+/+), and thus, Mi.III^+/+ erythrocytes lack glycophorin B (GPB) and express Gp.Mur only. Because GPB is a major component of the Rh complex on RBCs, in this study, we explored how the absence of GPB might affect Rh expression in Mi.III RBCs. Materials and Methods (1) Mi.III+ RBCs were serologically identified and further differentiated their homozygosity or heterozygosity by immunoblot or direct sequencing. (2) RhD and RhCcEe mRNA was cloned, and their sequences analysed. (3) The expression levels of Rh antigen, Rh‐associated glycoprotein (RhAG) and the U antigen in MI.III vs. non‐Mi.III RBCs were assessed by flow cytometry and Western blot. Results Compared with the non‐Mi.III samples, the surface expression of the Rh antigen was reduced to 76·4% in Mi.III^+/+ RBCs and 93·6% in Mi.III^+/?. RhAG expression was also significantly reduced in Mi.III^+/+, but not in Mi.III^+/?. The U antigen expression in Mi.III^+/? was only 14·9% relative to the control RBCs, while GPB was half the level of the controls. The mRNA sequences of Rh polypeptides from Mi.III+ samples were identical to the NCBI reference sequences. Conclusion Substitution of GPB with Gp.Mur significantly reduced the expression of Rh antigen and RhAG on the Mi.III^+/+ erythrocyte membrane. The Mi.III phenotype is predicted to induce considerable structural variations within the band 3/Rh‐associated macrocomplexes.     

A new blood group system,RHAG: three antigens resulting from amino acid substitutions in the Rh‐associated glycoprotein

Background and Objectives Rh‐associated glycoprotein (RhAG) is closely associated with the Rh proteins in the red cell membrane. Two high frequency antigens (Duclos and DSLK) and one low frequency antigen (Ol^a) have serological characteristics suggestive of expression on RhAG. Materials and Methods RHAG was sequenced from the DNA of one Duclos‐negative, one DSLK‐negative, and two Ol(a+) individuals. Recombinant protein was expressed in HEK 293 cells. Protein models with RhAG subunits were constructed. Results The original Duclos‐negative patient was homozygous for RHAG 316C>G, encoding Gln106Glu. HEK 293 cells expressing Gln106Glu mutant RhAG did not react with anti‐Duclos. An individual with DSLK‐negative red cells was homozygous for 490A>C, encoding Lys164Gln. Two Ol(a+) members of the original Norwegian family were heterozygous for 680C>T, encoding Ser227Leu. A Japanese donor with Rh_mod phenotype had Ol(a+) red cells and was homozygous for 680C>T. Conclusion The three red cell antigens encoded by RHAG form the RHAG blood group system: Duclos is RHAG1 (030001); Ol^a is RHAG2 (030002); and DSLK is provisionally RHAG3 (030003).