McLeod syndrome: Five new pedigrees with novel mutations.

OBJECTIVE: To present five new McLeod Syndrome (MLS) pedigrees with novel XK gene mutations, review the literature of this disorder, and discuss the typical and atypical clinical features noted with these new mutations. METHODS: This is a multi-center retrospective review of five MLS cases with novel gene mutations. Genotypic and phenotypic information has been obtained from each center. RESULTS: Five novel mutations are reported in this Case series. New clinical findings include prolonged asymptomatic elevated creatine kinase (CK) levels, vocal tics, presence of obstructive sleep apnea (OSA), and one patient of Vietnamese ethnicity. CONCLUSIONS: We expand on the clinical and genetic spectrum of MLS demonstrating the clinical variability of MLS.

An AQP1 allele associated with co(a-b-) phenotype.

The Colton (CO) blood group system consists of four antigens, Co(a), Co(b), Co3, and Co4, located on aquaporin-1 (AQP1), with Co(a) highly prevalent in all populations (99.8%). The Colton null phenotype, Co(a-b-), is very rare, and individuals with this phenotype lack the high-prevalence antigen Co3. To date, only six Co(a-b-) probands have been reported and four silencing alleles characterized. We identified an AQP1-null allele in a white woman with anti-Co3 caused by deletion of a G at nucleotide 601 (nt601delG) that results in a frameshift and premature termination (Val201Stop). Available family members were tested for the allele. Although anti-Co3 has been associated with mild to severe hemolytic disease of the fetus and newborn, the antibody was not clinically significant as evidenced by a low titer and delivery of asymptomatic newborns with moderate to weakly positive direct antiglobulin tests for all four pregnancies.

Red cells from the original JAL+ proband are also DAK+ and STEM+.

BACKGROUND: The low-prevalence Rh antigen, JAL, was named after the index case, Mr. J. Allen. Based on reactivity of seven multi-specific sera with his RBCs, it was apparent that they express at least one additional low-prevalence antigen. The purpose of this study was to investigate the other low-prevalence antigen(s) on J. Allen's RBCs. METHODS: Blood samples and reagents were from our collections. Hemagglutination and DNA analyses were performed by standard methods. RESULTS: Our DNA analyses confirmed the presence of RHCE*ceS(340T) in J. Allen and revealed the presence of RHCE*ceBI (ce 48C, 712G, 818T, 1132G) and RHD*DOL (509T, 667T). RBCs from J. Allen were agglutinated by anti-JAL, anti-STEM, and anti-DAK. Two of the reactive multi-specific sera reported in the original paper reacted with RBCs from J. Allen, and with RBCs from four other people with RHCE*ceBI, including the original STEM+ index case (P. Stemper) but not with RBCs with the DIIIa, DAK+ phenotype. We conclude that they contain anti-STEM. CONCLUSION: J.Allen's RBCs express the low-prevalence Rh antigens, JAL, V/VS (extremely weakly), STEM, and DAK. The presence of JAL on the variant Rhce, RhceJAL (16Cys, 114Trp, 245Val), STEM on the variant Rhce, RhceBI (16Cys, 238Val, 273Val, 378Val), and DAK on the variant RhD (170Thr, 223Val), encoded by RHD*DOL in trans to RHCE*ceBI is consistent with expression of these antigens. When J. Allen RBCs are used to detect and identify an anti-JAL, it is important to remember that they also express STEM and DAK.

Consortium for Blood Group Genes (CBGG): 2009 report.

Consortium for Blood Group Genes is a worldwide organization whose goal is to have a vehicle to interact, establish guidelines, operate a proficiency program, and provide education for laboratories involved in DNA and RNA testing for the prediction of blood group, platelet, and neutrophil antigens. Currently, the consortium operates with representatives from Brazil, Canada, and the United States. Membership is voluntary with the expectation that members actively contribute to discussions involving blood group genetics. This year witnessed a change in the standing committee membership and the institution of a representative for the human platelet antigens group. Looking forward, the consortium sees challenges for the nomenclature of blood group alleles and user-required specifications for laboratory information systems to store genotype information.

Consortium for Blood Group Genes (CBGG): 2008 report.

The Consortium for Blood Group Genes is a worldwide organization whose goal is to have a vehicle to interact, establish guidelines, operate a proficiency program, and provide education for laboratories involved in DNA and RNA testing for the prediction of blood group, platelet, and neutrophil antigens.

RHD deletion in a patient with chronic myeloid leukemia.

Anomalous expression of the Rh antigen, D, has occasionally been observed in patients with certain myeloproliferative disorders. Indeed, this phenomenon led to the tentative assignment of RH to the short arm of chromosome 1. PCR-based analyses were performed on DNA from an 82-year-old D+ Caucasian patient with chronic myeloid leukemia after her RBCs became D-. For nearly 7 years, the patient's RBCs typed as strongly D+, but in March 2006, they typed weakly D+ and in August 2006 typed D- by both direct hemagglutination and the IAT. The D- typing persisted until the patient's death in September 2006. To study the underlying cause of the change in D type, PCR-based assays were performed on DNA extracted from peripheral WBCs from the patient's sample collected in August 2006. No amplification was obtained using primers designed to amplify RHD exons 5, 8, or 10, and intron 4. Very weak amplification was obtained using primers designed to amplify RHD exons 3, 4, or 7. Two assays that detect the hybrid Rhesus box showed deletion of RHD. Amplification of RHCE in the patient's DNA was as efficient as that of control samples, and multiplex and PCR-RFLP assays predicted her RBCs would be C-E-c+e+. Based on finding a hybrid Rhesus box and absence of D-specific exons, we conclude that DNA from the patient's WBCs carries a deleted RHD. This explains the molecular mechanism underlying the change from D+ to D-.

The potential of blood group genotyping for transfusion medicine practice.

Molecular diagnostics is the fastest growing area of clinical laboratory medicine. The ability to rapidly amplify genes of bacterial, viral, or human origin, and the development of DNA array platforms, are driving a technology revolution in the clinical laboratory. A DNA-based testing approach is particularly applicable to blood bank and transfusion medicine for rapid, cost-effective antigen typing. Experience with DNA-based methods during the past decade has shown that these assays are reproducible and highly correlated with the RBC phenotype. The recent availability of automated, high-throughput, DNA-array platforms now moves testing from the reference laboratory setting into hospital and donor testing centers. This approach has the potential to revolutionize the process of locating antigen-negative donor units by testing for all clinically significant blood group antigens in a single assay. When partnered with the same extended typing of the patient, electronic selection of units antigen-matched at multiple blood group loci is then possible. This paper discusses the potential of this approach to improve transfusion therapy by reducing or eliminating alloantibody production in specific patient populations. These include patients facing long-term transfusion therapy and at high risk for sensitization; patients with warm autoantibodies when compatibility cannot be demonstrated by standard methods; and women for whom the production of atypical antibodies carries a risk for hemolytic disease of the fetus and newborn, or at the very least, monitoring for an at-risk pregnancy.

Serologic and molecular genetic management of a pregnancy complicated by anti-Rh18.

Antibodies, such as anti-Rh18 (Hr/Hr(S)), that react with the common products of RHCE can cause HDN as well as severe hemolytic transfusion reactions. Individuals with anti-Rh18 antibodies can have different RHCE genetic backgrounds; therefore, sera and RBCs from these individuals may cross-react. In these situations, genotyping may be the best method to determine compatibility. We report a 26-year-old pregnant Puerto Rican woman who presented at 31 weeks' gestation with anti-E and anti-Rh18 in her serum. No potential donors were identified among family members or within the American Rare Donor Program; therefore, a unit of the patient's RBCs was collected one week before her planned caesarian section. To improve our ability to supply blood for this patient in the future, molecular testing was performed. The patient was found to be homozygous for an RH haplotype in which a variant RHD*DAR, is linked to a variant RHCE*ceAR. The DAR-ceAR haplotype has been described in Dutch-African populations, but this is the first report of an individual self-identified of Hispanic ethnicity. This case report demonstrates the clinical importance of molecular testing of patients with rare Rh phenotypes.

Molecular characterization of GYPB and RH in donors in the American Rare Donor Program.

Transfusion of patients with sickle cell disease (SCD) has been a challenge in clinical transfusion medicine, especially when the required donor RBCs must be U- and negative for high-prevalence Rh phenotypes (hr(B), hr(S)). It is now possible to genotype donors to identify or confirm Uvar and U- phenotypes, as well as Rh hr(B)- and hrS- phenotypes, and to characterize the different RH backgrounds found in these donors. In a preliminary study of donors registered in the American Rare Donor Program, twelve different RH backgrounds were identified in eighteen hr(B)- or hr(S)- donors. These results, summarized in the current report, confirm the heterogeneous nature of these phenotypes and are relevant for selection of donor units for patients with antibodies to high-prevalence Rh antigens. Not all phenotypically similar units will be compatible, and matching the Rh genotype of the donor to the patient is important to prevent further Rh sensitization. Most donors referred were hr(B)- and carry at least one hybrid RHD-CE(3-7)-D gene that encodes a variant C antigen linked to RHCE*ceS that encodes the VS+V- phenotype. Surprisingly, the majority of donors were heterozygous, some even carrying conventional alleles, suggesting that the loss of expression of the hr(B) epitopes on RBCs is a dominant phenotype. Although antigen-matching of patients with SCD with donors for C, E, and K antigens has decreased the incidence of alloimmunization, some patients still become immunized to Rh antigens, indicating the units were not truly matched. RH genotyping can identify those patients with SCD who carry RH alleles that encode altered C, e, or D who are at risk for production of "apparent auto" and alloantibodies to Rh antigens. RH genotyping of alloimmunized patients with SCD, partnered with genotyping of donors, can identify compatible units that would also eliminate the risk of further Rh alloimmunization.

Transport characteristics of mammalian Rh and Rh glycoproteins expressed in heterologous systems.

The development and use of heterologous expression systems is critical for deciphering the function of mammalian Rh and Rh-glycoproteins. The studies here use Xenopus oocytes, well known for their ability to readily traffic and express difficult membrane proteins, and S. cerevisiae wild-type strains and mutants that are defective in ammonium transport. Data obtained in both of these expression systems revealed that mammalian Rh-glycoprotein-mediated transport (RhAG, RhBG, and RhCG) is an electroneutral process that is driven by the NH4+ concentration and the transmembrane H+ gradient, effectively exchanging NH4+ for H+ in a process that results in transport of net NH3. Homology modeling and functional studies suggest that the more recently evolved erythrocyte blood group proteins, RhCE and RhD, may not function directly in ammonia transport and may be evolving a new function in the RBC membrane. The relationship of Rh and Rh-glycoproteins to the Amt/Mep ammonium transporters is substantiated with functional transport data and structural modeling.

Review: the Kell, Duffy, and Kidd blood group systems.

After the discovery (over 50 years ago) that the IAT could be applied to the detection of antibodies to blood group antigens, there was a rapid increase in the identification of alloantibodies that caused transfusion reactions or HDN. After Rh, antibodies in the Kell, Duffy, and Kidd blood group systems were the next in clinically significant antibodies to be revealed. Much of what has been learned about these blood groups since the journal Immunohematology issued its first edition has to do with the proteins, the genes, and the molecular basis for the antigens. What has not changed is that, after ABO and Rh, antibodies to antigens in these three systems are still the most clinically significant. They will form the basis of this review.

DNA analysis for donor screening of Dombrock blood group antigens.

Due to the scarcity of reliable antibodies, RBC typing for Doa and Dob is notoriously difficult. Inaccurate typing can place patients at risk for hemolytic transfusion reactions. The molecular basis of the DOA/DOB polymorphism is associated with three nucleotide changes:378 C>T, 624 T>C,and 793 A>G of DO. While the 378 C>T and 624 T>C are silent mutations, the 793A>G polymorphism in codon 265 encodes asparagine for Doa and aspartic acid for Dob. We describe here the use of a PCR-RFLP assay as an alternative to traditional hemagglutination for typing donor blood for Dombrock. Primers were designed to amplify the region of DO containing the 793A>G polymorphism. DNA samples from blood donors were amplified and subjected to RFLP analysis. A total of 613 samples were tested for the Dombrock polymorphism (793 A>G) by PCRRFLP. PCR-RFLP can be used to screen for Do(a-) or Do(b-) donors. This approach overcomes the scarcity of the reagents required for testing by hemagglutination.

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.

Evidence supporting the requirement for two proline residues for expression of c.

BACKGROUND: In humans, c antigen expression is associated with a proline residue at amino acid position 103 in the second extracellular loop of the CE protein. Comparison of nonhuman primate Rh proteins suggested that c reactivity might actually involve two proline residues. It has been shown that the RBCs of New World capuchin monkeys (Cebus apella) react with anti-c. To further define the amino acid residues involved in c expression, Rh cDNA from the capuchin was analyzed. STUDY DESIGN AND METHODS: Rh transcripts were amplified by reverse transcription PCR from RNA isolated from the reticulocytes of a capuchin monkey and were cloned and sequenced. RESULTS: Rh transcripts from the capuchin monkey, whose RBCs react with anti-c, were found to encode adjacent proline residues at 102 and 103. CONCLUSION: Sequencing of Rh transcripts from the capuchin monkey supports the hypothesis that the expression of c requires two adjacent proline residues. Proline causes bends or loops in proteins, which, in this case, might form a unique, stable structure resistant to perturbations induced by changes in upstream or downstream residues. This would explain the scarcity in humans of c variants as compared to the other major Rh antigen variants, and the preservation of c reactivity despite 24-percent divergence between the human and capuchin Rh proteins.