Systematic RH genotyping and variant identification in French donors of African origin.

BACKGROUND: RH molecular analysis has enabled the documentation of numerous variants of RHD and RHCE alleles, especially in individuals of African origin. The aim of the present study was to determine the type and frequency of D and/or RhCE variants among blood donors of African origin in France, by performing a systematic RH molecular analysis, in order to evaluate the implications for blood transfusion of patients of African origin. MATERIALS AND METHODS: Samples from 316 African blood donors, whose origin was established by their Fy(a-b-) phenotype, were first analysed using the RHD and RHCE BeadChips Kit (BioArray Solutions, Immucor, Warren, NJ, USA). Sequencing was performed when necessary. RESULTS: RHD molecular analysis showed that 26.2% of donors had a variant RHD allele. It allowed the prediction of a partial D in 11% of cases. RHCE molecular analysis showed that 14.2% of donors had a variant RHCE allele or RH [RN or (C)ces] haplotype. A rare Rh phenotype associated with the loss of a high-prevalence antigen or partial RhCE antigens were predicted from RHCE molecular analysis in 1 (0.3%) and 17 (5%) cases, respectively. DISCUSSION: Systematic RHD and RHCE molecular analysis performed in blood donors of African origin provides transfusion-relevant information for individuals of African origin because of the frequency of variant RH alleles. RH molecular analysis may improve transfusion therapy of patients by allowing better donor and recipient matching, based not only on phenotypically matched red blood cell units, but also on units that are genetically matched with regards to RhCE variants.

Molecular analysis of patients with weak D and serologic analysis of those with anti-D (excluding type 1 and type 2).

Whether or not patients whose red blood cells (RBCs) carry certain weak D types produce anti-D, and if they do whether it is allo- or auto anti-D, remains controversial. The aim of this study was to determine the serologic features of anti-D in individuals expressing a weak D other than type 1 or type 2 and to assess whether the anti-D was an allo- or autoantibody. Serologic D typing and molecular analyses were performed on 748 individuals.Serologic characterization of anti-D included autologous controls,direct antiglobulin test, elution, and titration of anti-D before and after adsorption of serum onto autologous RBCs. From molecular analyses, 459 individuals exhibited a weak D type. We described seven novel RHD variant alleles. The most frequent types of weak D were type 1 (30.1%), type 2 (23.7%), type 4.0 (10.2%), type 4.2.2(20.3%), type 11 (3.9%), and type 15 (3.7%). Anti-D was identified in the sera of 9 of 47 individuals with weak D type 4.0, in 14 of 93 with weak D type 4.2.2, in 1 of 18 with weak D type 11, in 1 of 17 with weak D type 15, and in 1 weak D type 33 individual.Anti-D was demonstrated to be an alloantibody in weak D type 4.0, type 4.2.2, and type 15 individuals, but an autoantibody in weak D type 11 and type 33 individuals. In conclusion, only a complete serologic investigation of individuals with a given weak D type identified by molecular analysis allows concluding on the nature of the antibody. Transfusing weak D type 4.2.2 and type 15 patients with D- RBC units and proposing anti-D immunoprophylaxis to women with these weak D types should be considered.

Identification of novel silent KEL alleles causing KEL:-5 (Ko) phenotype or discordance between KEL:1,-2 phenotype/KEL*01/02 genotype.

BACKGROUND: The Kell system, encoded by the KEL gene, is one of the most clinically important blood group systems. Molecular defects may lead to the absence of Kell antigen expression. The very rare KEL:5 results from silent KEL genes, also called KELnull alleles. In a few cases, the rare KEL:1,-2 phenotype may be associated with silent KEL*02 alleles. STUDY DESIGN AND METHODS: The aim of this study was to perform DNA investigations to identify silent KEL alleles among 10 KEL:-5 patients and 121 individuals presenting the rare KEL:1,-2 phenotype. Serologic investigations were performed on patients' red blood cells and serum. The KEL gene analysis was done by using a BeadChip assay (HEA Version, 1.2, Immucor), real-time polymerase chain reaction, and/or sequencing of all 19 exons of the KEL gene. RESULTS: In KEL:-5 patients, two novel KELnull alleles were described: 821G>A being the second described KELnull allele on a KEL*01 backbone and 184Tdel. In the 121 KEL:1,-2 individuals, nine (7.4%) were found to display a discordant KEL:1,-2 phenotype and KEL*01/KEL*02 genotype. Three novel silent KEL*02 alleles were described: 1084C>A, 1708G>A, and IVS11+5g>a. CONCLUSION: The number of silent KEL alleles and the notion that KEL null alleles are on a KEL*02 background may evolve in the coming years. Systematic DNA analysis showed that the number of discordant phenotype/genotype results, related to silent KEL*02 alleles was higher than expected in France. These data emphasize that clinical practice based on DNA analysis for blood group antigens requires caution and should improve the performance of the blood group phenotype prediction.

RHD*DOL1 and RHD*DOL2 encode a partial D antigen and are in cis with the rare RHCE*ceBI allele in people of African descent.

BACKGROUND: Several studies showed in people of African descent the existence of a genetic linkage between RHD alleles encoding a variant D antigen and a given altered RHCE*ce allele. RHCE*ceBI is a rare allele encountered in people of African descent, that encodes a Hr- hr(S) - Rhce protein. Our study shows that RHCE*ceBI appears to be genetically linked to two very similar variant RHD alleles, RHD*DOL1 and RHD*DOL2, and demonstrates for the first time that DOL-2 is a partial D antigen. STUDY DESIGN AND METHODS: After finding out an individual with both RHCE*ceBI and RHD*DOL presumed to be in cis, we hypothesized a genetic linkage between those two genes. All individuals (n = 7) known to carry RHCE*ceBI in our laboratory, including the index case, were fully investigated at the serologic and molecular level. RESULTS: One individual with alloanti-D, being homozygous for RHCE*ceBI and RHD*DOL2, allowed us to confirm the genetic linkage between those two genes, as well as the partial D status of DOL-2. In the six RHCE*ceBI remaining individuals, three were found with RHD*DOL2 and 3 with RHD*DOL1, likely in cis. Three of them made an alloanti-D; one was DOL-1 and two were DOL-2. CONCLUSION: The rare RHCE*ceBI allele appears to be in cis either with RHD*DOL1 or with RHD*DOL2 in people of African descent. DOL-1 and DOL-2 must be considered as partial D antigens. We recommend a systematic search for RHD*DOL1 and RHD*DOL2 in people found to carry RHCE*ceBI and vice versa, especially in patients with sickle cell disease.

Molecular background of novel silent RHCE alleles.

BACKGROUND: The absence of expression of C/c and E/e antigens has been associated with rare variant RHCE alleles, referred to as silent RHCE alleles, classically identified among individuals with a rare D- - or Rhnull phenotype. This work reports on different molecular mechanisms identified in three novel silent RHCE alleles. STUDY DESIGN AND METHODS: Samples from D- - or Rhnull individuals and their family members, from families for whom Rh phenotype and/or serologic data were unexplained by inheritance of conventional RH alleles, were analyzed. Genomic DNA and transcripts were tested by sequencing analysis. RESULTS: The first silent allele was a RHCE*cE allele carrying an intronic IVS3+5G>A mutation. The second was a RHCE*ce allele carrying an intronic IVS7-2A>G mutation, whereas the third was a silent RHCE*ce allele carrying a 5-bp deletion (Nucleotides 679-683) in Exon 5. CONCLUSION: In addition to hybrid alleles and nucleotide deletion, intronic mutations may be associated with the nonexpression of RhCE antigens. Regarding the RH system, silent alleles may not be investigated among D- - or Rhnull individuals only. Rh phenotype and/or serologic data unexplained by inheritance of conventional RH alleles should lead to molecular investigations.

Null alleles of ABCG2 encoding the breast cancer resistance protein define the new blood group system Junior.

The breast cancer resistance protein, also known as ABCG2, is one of the most highly studied ATP-binding cassette (ABC) transporters because of its ability to confer multidrug resistance. The lack of information on the physiological role of ABCG2 in humans severely limits cancer chemotherapeutic approaches targeting this transporter. We report here that ABCG2 comprises the molecular basis of a new blood group system (Junior, Jr) and that individuals of the Jr(a-) blood type have inherited two null alleles of ABCG2. We identified five frameshift and three nonsense mutations in ABCG2. We also show that the prevalence of the Jr(a-) blood type in the Japanese and European Gypsy populations is related to the p.Gln126* and p.Arg236* protein alterations, respectively. The identification of ABCG2(-/-) (Jr(a-)) individuals who appear phenotypically normal is an essential step toward targeting ABCG2 in cancer and also in understanding the physiological and pharmacological roles of this promiscuous transporter in humans.

ABCB6 is dispensable for erythropoiesis and specifies the new blood group system Langereis.

The human ATP-binding cassette (ABC) transporter ABCB6 has been described as a mitochondrial porphyrin transporter essential for heme biosynthesis, but it is also suspected to contribute to anticancer drug resistance, as do other ABC transporters located at the plasma membrane. We identified ABCB6 as the genetic basis of the Lan blood group antigen expressed on red blood cells but also at the plasma membrane of hepatocellular carcinoma (HCC) cells, and we established that ABCB6 encodes a new blood group system (Langereis, Lan). Targeted sequencing of ABCB6 in 12 unrelated individuals of the Lan(-) blood type identified 10 different ABCB6 null mutations. This is the first report of deficient alleles of this human ABC transporter gene. Of note, Lan(-) (ABCB6(-/-)) individuals do not suffer any clinical consequences, although their deficiency in ABCB6 may place them at risk when determining drug dosage.

Antibodies to co-trimoxazole (trimethoprim and/or sulfamethoxazole) related to the presence of the drug in a commercial low-ionic-strength solution.

BACKGROUND: Drug-dependent antibodies have been associated with approximately 10% of acquired immune hemolytic anemia cases. These antibodies are a rare cause of interference in pretransfusion red blood cell (RBC) serologic testing. The aim of this work was to report three cases of subjects developing antibodies against co-trimoxazole, a combination of trimethoprim (TMP) and sulfamethoxazole (SMX). CASE REPORT AND METHODS: Blood samples of donor/patients were referred to our laboratory for the exploration of a positive antibody detection test. There was no recent history of drug taking. Antibody identification was performed by gel test using an indirect antiglobulin test, with reagent RBCs in low-ionic-strength solutions (LISS) containing co-trimoxazole or not. RESULTS: All three sera showed positive reactions when RBCs were resuspended in LISS containing co-trimoxazole, but negative reactions when RBCs were resuspended in LISS without antibiotic. We detected antibodies against co-trimoxazole showing three different antibody patterns: anti-TMP plus anti-SMX, anti-TMP alone, or anti-SMX alone. Anti-TMP showed an apparent anti-Ku specificity in the two cases where it was present. Anti-SMX showed an apparent anti-H specificity in one of the two cases described. The drug-dependent antibodies were not associated with acquired hemolytic anemia or other pathologies. CONCLUSION: Antibodies against co-trimoxazole may only be detected when using a diluent for reagent RBCs containing the drug in question. Antibody pattern (anti-TMP and/or anti-SMX) may vary according to individuals' immune response. Drug-dependent antibodies may react as antibodies against a high-prevalence antigen, supporting the hypothesis of antibodies to drug and membrane components. Drug-dependent antibodies such as anti-co-trimoxazole may be a serologic finding without clinical features.

Proof of principle for transfusion of in vitro-generated red blood cells.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34(+) HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 10(10) cRBCs generated under good manufacturing practice conditions and labeled with (51)Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro-generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.

Anti-D investigations in individuals expressing weak D Type 1 or weak D Type 2: allo- or autoantibodies?

BACKGROUND: Whether anti-D produced by individuals with a weak D phenotype are allo- or autoantibodies remains a matter of debate even though blood transfusion practice is impacted. The aim of our study was to determine the serologic features of anti-D in individuals expressing the most frequent weak D type in Caucasians that are weak D Type 1 or weak D Type 2, to assess whether anti-D were allo- or autoantibodies. STUDY DESIGN AND METHODS: Serologic D typing and molecular analysis enabled the including of 121 weak D Type 1 individuals and 99 weak D Type 2 individuals in our study. Serologic features of anti-D included autologous controls, direct antiglobulin test, elution, and titration of anti-D before and after adsorption of serum on autologous red blood cells (RBCs). RESULTS: Serologic D typing showed a variable reactivity of RBCs expressing weak D Type 1 or weak D Type 2 (4+ to 0). Anti-D was identified in six weak D Type 1 and six weak D Type 2 individuals, respectively. The serologic data were in favor of autoantibodies. CONCLUSION: A complete anti-D investigation in individuals with a D variant (weak D or partial D identified by molecular analysis) should be systematically performed before any valid conclusion on the nature of the antibody. Transfusing weak D Type 1 or weak D Type 2 patients with D+ RBC units should be recommended. Weak D Type 1 or weak D Type 2 pregnant women do not need anti-D immunoprophylaxis.

Analysis of RhCE variants among 806 individuals in France: considerations for transfusion safety, with emphasis on patients with sickle cell disease.

BACKGROUND: DNA testing has enabled the documenting of numerous variants of RHCE alleles, especially in individuals of African origin. The risk for production of clinically significant alloantibodies to Rh antigens of patients carrying variant RHCE alleles has led us to analyze the different RhCE variants investigated by molecular biology. Alloimmunization was analyzed regarding the RHCE genetic profile. STUDY DESIGN AND METHODS: Samples from 806 individuals with altered expression of RhCE antigens and/or producing anti-RhCE in the presence of the corresponding antigen were analyzed. RESULTS: A total of 572 individuals were shown to express RhCE variants. Variant RHCE*ce alleles and RH haplotypes were identified in 83% of cases, the most frequent ones being the R(N) haplotype, the ceMO allele, the (C)ce(s) haplotype/ce(s) 1006 allele, and the ceAR allele identified in 36, 23, 20, and 17% of the tested samples, respectively. The absence of a high-prevalence Rh antigen was documented in 93 individuals. Partial C and partial e were expressed by 53% of individuals with RhCE variants. Rh antibodies were identified in 127 (20%) of 623 patients. They were found to be alloantibodies in 48 (38%) of these 127 patients. Alloimmunization against a high-prevalence Rh antigen was detected in 25% of cases. CONCLUSION: The challenge in clinical red blood cell (RBC) transfusion of patients with sickle cell disease, notably, would be to provide not only phenotypically matched, but also genetically matched, RBC units regarding RhCE variants.

Blood group genotyping by high-throughput DNA analysis applied to 356 reagent red blood cell samples.

BACKGROUND: DNA analysis for the prediction of blood group antigen expression has broad implications in transfusion medicine. It may be of particular interest especially to detect variants, when antigen expression is weak or altered. The use of high-throughput DNA analysis has never been applied to donors whose red blood cells (RBCs) are selected for reagent RBCs. The aim of this study was to analyze the concordance between the serologic phenotype and that predicted from DNA analysis in panel donors, to determine the benefit of the use of DNA analysis in reagent RBC selection strategy. STUDY DESIGN AND METHODS: The "Panel National de Référence du Centre National de Référence sur les Groupes Sanguins" is a reference reagent mainly used for antibody identification. DNA genotyping of 356 panel donors was performed with BeadChips (human erythrocyte antigen v1.2 BeadChips, BioArray Solutions). The comparison between serologic phenotype and that predicted from DNA analysis held on 8876 paired results obtained from 10 blood group systems and 25 antigens. RESULTS: A 99.95% concordance was observed. Discrepancies in four cases (RH, KEL, LU, and DO systems) were analyzed. Genotyping precisions on the Duffy system were of particular interest. No new rare blood group was observed. CONCLUSION: Systematic DNA analysis of panel donors should unquestionably change the management of reagent RBC selection. The notion of "antigens in double dose" should evolve regarding data obtained from DNA analysis, allowing an improved quality of reagent RBCs for antibody screening and identification.

A functional AQP1 allele producing a Co(a-b-) phenotype revises and extends the Colton blood group system.

BACKGROUND: The Colton blood group system currently comprises three antigens, Co(a) , Co(b) , and Co3. The latter is only absent in the extremely rare individuals of the Colton "null" phenotype, usually referred to as Co(a-b-), which lack the water channel AQP1 that carries the Colton antigens. The discovery of a Co(a-b-) individual with no AQP1 deficiency suggested another molecular basis for the Co(a-b-) phenotype. STUDY DESIGN AND METHODS: Red blood cells were analyzed by stopped-flow light scattering and Western blotting and typed by hemagglutination and flow cytometry. Genotyping by sequencing and polymerase chain reaction-restriction fragment length polymorphism was applied. An expression system for Colton antigens was developed in mammalian cells. RESULTS: Although Co(a-b-), the proband expressed fully functional AQP1 and had developed a novel Colton alloantibody. Sequencing of AQP1 revealed a homozygous nucleotide change (140A>G) encoding the single-amino-acid substitution Q47R. A second case was identified due to the presence of this novel Colton alloantibody. By generating an expression system for Colton antigens in K-562 cells, the Q47R substitution was shown to inhibit the expression of both Co(a) and Co(b) antigens. Other naturally occurring single-amino-acid substitutions, that is, A45T, P38L, and N192K, were also studied in this Colton antigen expression system. CONCLUSIONS: The Co(a-b-) phenotype can be generated by a functional AQP1 allele, that is, AQP1 140G encoding AQP1 (Q47R) and allowing the development of a novel Colton alloantibody. This study also shows that the Co(b) antigen can be produced by at least two different substitutions at Amino Acid Position 45, that is, A45V and A45T.

Analysis of complement receptor type 1 expression on red blood cells in negative phenotypes of the Knops blood group system, according to CR1 gene allotype polymorphisms.

BACKGROUND: The KN blood group system, which consists of nine antigen specificities, is located on complement receptor Type 1 (CR1/CD35). CR1, a complement regulatory protein, acts as a vehicle for immune complex clearance. CR1 exhibits a red blood cell (RBC) density polymorphism. CR1 sites on RBCs in normal individuals range from 150 to 1200 molecules per cell. CR1 density polymorphism is regulated by HindIII restriction fragment length polymorphism and Q981H and P1786R polymorphisms in Caucasians. Yet, the role of the different polymorphisms in determining the CR1 density on RBCs remains unknown. The "null" serologic KN phenotype, known as Helgeson phenotype, was reported to be related with a very low CR1 density, less than 150 molecules per cell. STUDY DESIGN AND METHODS: The aim of this work was to investigate whether the KN-negative phenotype displayed by 60 individuals was related to the CR1 density by performing the phenotypic and genetic analysis of CR1 and to investigate the molecular background associated with the KN system. RESULTS: We showed that the Helgeson-like phenotype had a prevalence of 12% in this population. The overall genotype/phenotype concordance was 90%. Among individuals with a KN-negative phenotype, the prevalences of Kn(a-), McC(a-), Sl1-negative, Sl3-negative, and KCAM-negative deduced phenotype were 37, 12, 29, 7, and 24%, respectively. CONCLUSION: From our data, we suggest that the definition of the Helgeson phenotype must be revised, since the latter may be due not only to a very low CR1 density on RBCs, but also to the absence of expression of a high-prevalence KN antigen.

Alloanti-c/ce in a c+ceAR/Ce patient suggests that the rare RHCE ceAR allele (ceAR) encodes a partial c antigen.

BACKGROUND: ceAR (RHCE ceAR) is a rare RH allele encountered in people of African/Caribbean ancestry, known to encode a partial e antigen. The homozygous ceAR/ceAR genotype encodes the rare blood group Hr-. This study describes alloanti-c/ce in a ceAR/Ce patient, suggesting that ceAR also encodes a partial c antigen. CASE REPORT: A 21-year-old patient suffering from intermediate beta-thalassemia, with transfusion history, was hospitalized for severe anemia. Blood samples were referred to the National Reference Laboratory for suspicion of a mixture of alloantibodies or an alloantibody to a high-prevalence antigen. MATERIALS AND METHODS: Standard hemagglutination methods were performed to investigate the patient's RBCs and serum. A molecular analysis of RHD and RHCE was carried out by allele-specific polymerase chain reaction and DNA sequencing. RESULTS: Blood type performed by the referring laboratory was B, D+C+E-c+e+, K-. Several antibodies were identified: anti-c/ce, anti-Fy(b), anti-Jk(a), and anti-S. Full serologic investigations showed that anti-c/ce could be very likely considered as an alloantibody. The patient's genotype was ceAR/Ce. Anti-c/ce reacted with ceAR/ceEK, ceEK/ceEK, and ceAR/ceBI but not with ceAR/ceAR, ceMO/ceMO, and ce(s)(340)/ce(s)(340) RBCs. CONCLUSION: This is the first case of alloanti-c/ce related to ceAR, suggesting that this rare RHCE allele encodes a partial c antigen. The presence of the C antigen in the patient allowed for the partial expression of the c antigen encoded by ceAR. The c antigen encoded by ceAR appeared to be different than that encoded by ceEK and ceBI and may share common lacking epitopes with the c antigens encoded by ceMO and ce(s)(340).

Anti-HrB and anti-hrb revisited.

BACKGROUND: Since their description in the 1970s, anti-Hr(B) (antibody against a high-prevalence Rh antigen) and anti-hr(B) (anti-e-like antibody) are still a subject of debate about representing two aspects of a global immune response or being two independent antibodies. STUDY DESIGN AND METHODS: The aim of this study was to evaluate the immune response against the antigens of Rh system of 30 individuals presenting a hr(B)(RH31)- phenotype. Genomic analysis of RH genes was performed in all individuals. RESULTS: Among the 30 individuals, 27 had a Hr(B)(RH34)- phenotype. No immunization against Rh antigens was found in 16 individuals. Three individuals made anti-D only, whereas six individuals made anti-Hr(B) (four with anti-hr(B) and two without anti-hr(B)) and two individuals made anti-hr(B) without anti-Hr(B). Among the 30 individuals, three had a Hr(B)+ phenotype. No immunization against Rh antigens was found in one individual, whereas two individuals made anti-hr(B); the genomic analysis of selected individuals showed the presence of a (C)ce(s) haplotype, either Type 1 or Type 2, and a DIII Type 5 ce(s) haplotype, in the homozygous state, in compound heterozygosity with each other or in heterozygosity with a DcE haplotype. Genomic data were in accordance with serologic data. CONCLUSION: Our data provide the evidence that anti-Hr(B) and anti-hr(B) are independent antibodies, defining two different specificities. These antibodies may be produced by individuals expressing variants of RhCE protein. Serologic and molecular data indicate that e antigen encoded by the (C)ce(s) haplotype is a partial antigen. In individuals carrying a (C)ce(s) haplotype, the risk and the type of alloimmunization to Rh antigens are related to the second Rh haplotype.

Alloanti-c (RH4) revealing that the (C)ce s haplotype encodes a partial c antigen.

BACKGROUND: In the Rh blood group system, published observations showed that the c antigen has the fewest variant forms of the principal antigens in this system. The partial nature of the c antigen was only reported in c+ Rh:-26 persons and to be associated with the ce(s)(340) allele. This study reports the first case of alloanti-c related to a (C)ce(s) haplotype. STUDY DESIGN AND METHODS: Serologic and genetic studies were performed on blood samples of a multitransfused 40-year-old African patient with sickle cell disease displaying a DCcee phenotype. RESULTS: Red blood cells (RBCs) of the patient displayed normal expression of C, c, e, ce antigens either with routine reagents or with monoclonal antibodies. Analyses of DNA and Rh transcripts showed that the patient carried a (C)ce(s)/DCe genotype. The patient's serum contained anti-D, anti-c, anti-E, anti-e, anti-V, anti-Js(a), and anti-S. Anti-c was isolated from the mixture of antibodies by using absorption and adsorption-elution techniques. Anti-c provided consistent reactions with c+ RBCs. Reactions were stronger with c+ ce+ RBCs than with c+ ce- RBCs. No agglutination of RBCs from individuals carrying a homozygous (C)ce(s) genotype was observed. CONCLUSION: These data provide the evidence that anti-c in our patient was an alloanti-c and, consequently, that (C)ce(s) haplotype encodes a partial c antigen. The clinical significance of anti-c related to this haplotype should be evaluated in the future.

Heterogeneous molecular background of the weak C, VS+, hr B-, Hr B- phenotype in black persons.

BACKGROUND: The rare Hr(B)- phenotype is encoded by the (C)ce(s) haplotype when present at the homozygous state. This haplotype contains two altered genes: a hybrid RHD-CE-D(s) gene segregated with a ce(s) allele of RHCE (733C>G and 1006G>T substitutions in Exon 5 and Exon 7 respectively). The aim of this study was to further investigate the molecular background of the (C)ce(s) haplotype. STUDY DESIGN AND METHODS: Twelve individuals with depressed C and/or depressed e phenotype were selected from their genomic DNA analysis showing both 733C>G and 1006G>T substitutions. Phenotypic expression of low- and high-prevalence Rh antigens was studied. Complete sequences of RHD and RHCE transcripts were analyzed when obtained. RESULTS: A new hybrid RHD-CE-D(s) gene (Exons 1 and 2; complete Exon 3; Exons 8, 9, and 10 from RHD; and Exons 4 through 7 from RHCE) segregated with a ce(s) allele, which genomic organization was almost identical to that of the classical (C)ce(s) haplotype, is described. The two different (C)ce(s) haplotypes encoded two different patterns of Rh antigen expression. Although both encoded weak e, VS, and did not produce D, V, hr(B), or Hr(B) antigens, the new haplotype encoded a much weaker C antigen and red blood cells lacked expression of Rh42, in contrast to the classic (C)ce(s) haplotype. CONCLUSION: The study showed the heterogeneity of the molecular background of the weak C, VS+, hr(B)-, Hr(B)- phenotype in the black population. The screening of blood donors in this population for hr(B)- or Hr(B)- phenotype should implement the molecular characterization of Rh genes.

Identification and characterization of a novel XK splice site mutation in a patient with McLeod syndrome.

BACKGROUND: McLeod syndrome is a rare X-linked neuroacanthocytosis syndrome with hematologic, muscular, and neurologic manifestations. McLeod syndrome is caused by mutations in the XK gene whose product is expressed at the red blood cell (RBC) surface but whose function is currently unknown. A variety of XK mutations has been reported but no clear phenotype-genotype correlation has been found, especially for the point mutations affecting splicing sites. STUDY DESIGN AND METHODS: A man suspected of neuroacanthocytosis was evaluated by neurologic examination, electromyography, muscle biopsy, muscle computed tomography, and cerebral magnetic resonance imaging. The McLeod RBC phenotype was disclosed by blood smear and immunohematology analyses and then confirmed at the biochemical level by Western blot analysis. The responsible XK mutation was characterized at the mRNA level by reverse transcription-polymerase chain reaction (PCR), identified by genomic DNA sequencing, and verified by allele-specific PCR. RESULTS: A novel XK splice site mutation (IVS1-1G>A) has been identified in a McLeod patient who has developed hematologic, neuromuscular, and neurologic symptoms. This is the first reported example of a XK point mutation affecting the 3' acceptor splice site of Intron 1, and it was demonstrated that this mutation indeed induces aberrant splicing of XK RNA and lack of XK protein at the RBC membrane. CONCLUSION: The detailed characterization at the molecular biology level of this novel XK splice site mutation associated with the clinical description of the patient contributes to a better understanding of the phenotype-genotype correlation in the McLeod syndrome.

Fatal hemolytic disease of the fetus and newborn associated with anti-Jr.

BACKGROUND: Jr(a) is a high-prevalence red cell (RBC) antigen. The clinical significance of anti-Jr(a) is controversial. When hemolytic disease of the fetus and newborn (HDFN) occurred, most reported cases were clinically mild. We report the first case of fatal HDFN due to anti-Jr(a). CASE REPORT: A 28-year-old Caucasian woman with transfusion history was monitored at the 29th week of pregnancy (G4P1). An ultrasound scan showed fetal cardiomegaly and hepatomegaly. An antibody directed against a high-prevalence antigen was detected, but without conclusive identification. An emergency cesarean section was performed at the 36th week. The newborn was hydropic and showed severe anemia. Death occurred 30 hours after birth. MATERIALS AND METHODS: Serologic methods were performed to investigate the mother's RBCs and serum. An in vitro functional cellular assay and semiquantitative measurement of anti-Jr(a) were used to determine the clinical significance of the antibody. RESULTS: Anti-Jr(a) was identified in the serum and Jr(a-) phenotype was confirmed. The anti-Jr(a) titer was 1024, with predominant immunoglobulin (Ig)G1 and minor IgG4 subclasses. The functional cellular assay was consistent with an antibody unlikely to cause HDFN. Semiquantitative measurement of anti-Jr(a) showed a reactivity equivalent to a 25 IU per mL (5 microg/mL) concentration of anti-D, a value associated with a significant risk of HDFN. CONCLUSION: This is the first documented case of fatal HDFN due to anti-Jr(a). Therefore, we recommend close monitoring of pregnant women with a high-titer anti-Jr(a), especially those with an incompatible transfusion history and/or multiple pregnancies. This case report provides new arguments about the clinical significance of anti-Jr(a) in the transfusion setting.