Genomic analyses of RH alleles to improve transfusion therapy in patients with sickle cell disease.

BACKGROUND: Red cell (RBC) blood group alloimmunization remains a major problem in transfusion medicine. Patients with sickle cell disease (SCD) are at particularly high risk for developing alloantibodies to RBC antigens compared to other multiply transfused patient populations. Hemagglutination is the classical method used to test for blood group antigens, but depending on the typing methods and reagents used may result in discrepancies that preclude interpretation based on serologic reactivity alone. Molecular methods, including customized DNA microarrays, are increasingly used to complement serologic methods in predicting blood type. The purpose of this study was to determine the diversity and frequency of RH alleles in African Americans and to assess the performance of a DNA microarray for RH allele determination. MATERIAL AND METHODS: Two sets of samples were tested: (i) individuals with known variant Rh types and (ii) randomly selected African American donors and patients with SCD. Standard hemagglutination tests were used to establish the Rh phenotype, and cDNA- and gDNA-based analyses (sequencing, PCR-RFLP, and customized RHD and RHCE microarrays were used to predict the genotype). RESULTS: In a total of 829 samples (1658 alleles), 72 different alleles (40 RHD and 32 RHCE) were identified, 22 of which are novel. DNA microarrays detected all nucleotides probed, allowing for characterization of over 900 alleles. CONCLUSIONS: High-throughput DNA testing platforms provide a means to test a relatively large number of donors and potentially prevent immunization by changing the way antigen-negative blood is provided to patients. Because of the high RH allelic diversity found in the African American population, determination of an accurate Rh phenotype often requires DNA testing, in conjunction with serologic testing. Allele-specific microarrays offer a means to perform high-throughput donor Rh typing and serve as a valuable adjunct to serologic methods to predict Rh type. Because DNA microarrays test for only a fixed panel of allelic polymorphisms and cannot determine haplotype phase, alternative methods such as Next Generation Sequencing hold the greatest potential to accurately characterize blood group phenotypes and ameliorate the clinical course of multiply-transfused patients with sickle cell disease.

RHCE*ceMO is frequently in cis to RHD*DAU0 and encodes a hr(S) -, hr(B) -, RH:-61 phenotype in black persons: clinical significance.

BACKGROUND: RHCE*ceMO has nucleotide changes 48G>C and 667G>T, which encode, respectively, 16Cys and 223Phe associated with altered expression of e antigen. RHD*DAU0 has Nucleotide 1136C>T, which encodes 379Met associated with normal levels of D. We compiled serologic and DNA testing data on samples with RHCE*ceMO to determine the red blood cell (RBC) antigen expression, antibody specificity, RHD association, and the prevalence in African-American persons. STUDY DESIGN AND METHODS: Serologic testing was performed by standard methods. Genomic DNA was used for polymerase chain reaction-restriction fragment length polymorphism and RH-exon sequencing, and for some, Rh-cDNA was sequenced. Seventy-seven (50 donor and 27 patient) samples with RHCE*ceMO were studied, and 350 African-American persons were screened for allele prevalence. RESULTS: RBCs from RHCE*ceMO homozygotes (or heterozygotes with RHCE*cE in trans) were weak or nonreactive with some anti-e and were nonreactive with polyclonal anti-hr(S) and anti-hr(B) . Twenty-three transfused patients homozygous for RHCE*ceMO/ceMO or with RHCE*ceMO in trans to RHCE*cE or *ce had alloanti-e, anti-f, anti-hr(S) /hr(B) , or an antibody to a high-prevalence Rh antigen. Three patients with alloanti-c had RHCE*ceMO in trans to RHCE*Ce. RHD*DAU0 was present in 30% of African-American persons tested and in 69 of 77 (90%) of samples with RHCE*ceMO. CONCLUSIONS: RHCE*ceMO encodes partial e, as previously reported, and also encodes partial c, a hr(S) - and hr(B) - phenotype, and the absence of a high-prevalence antigen (RH61). The antibody in transfused patients depended on the RHCE allele in trans. RHCE*ceMO was present in one in 50 African-American persons with an allele frequency of 0.01, is often linked to RHD*DAU0, and is potentially of clinical significance for transfusion.

RHCE*ceTI encodes partial c and partial e and is often in cis to RHD*DIVa.

BACKGROUND: In the Rh blood group system, variant RhD and RhCE express several partial antigens. We investigated RH in samples with partial DIVa that demonstrated weak and variable reactivity with anti-C. STUDY DESIGN AND METHODS: Standard hemagglutination techniques, polymerase chain reaction-based assays, and RH sequencing were used. RESULTS: DNA analysis showed that six red blood cell (RBC) samples with weak and inconsistent reactivity with anti-C lacked RHCE*C, but all had RHD*DIVa, which encodes partial D and Go(a) . We then tested RBCs from 19 Go(a+) cryopreserved samples (confirmed to have RHD*DIVa) with four anti-C and observed weak variable reactions. RHCE genotyping found all but one of the samples with RHD*DIVa also had RHCE nt 48G>C and 1025C>T, named RHCE*ceTI. Lookback of samples referred for workup and found to have either allele revealed 47 of 55 had both RHD*DIVa and RHCE*ceTI, four had RHD*DIVa without RHCE*ceTI, and four had RHCE*ceTI without RHD*DIVa. Alloanti-c was found in a patient with c+ RBCs and RHCE*ceTI in trans to RHCE*Ce, and alloanti-e was found in a patient with e+ RBC and RHCE*ceTI in trans to RHCE*cE. RHD*DIVa in trans to RHD erroneously tested as RHD hemizygous. CONCLUSIONS: RHD*DIVa and RHCE*ceTI almost always, but not invariably, travel together. This haplotype is found in people of African ancestry and the RBCs can demonstrate aberrant reactivity with anti-C. RHCE*ceTI encodes partial c and e antigens. We confirm that RHD zygosity assays are unreliable in samples with RHD*DIVa.

The low-prevalence Rh antigen STEM (RH49) is encoded by two different RHCE*ce818T alleles that are often in cis to RHD*DOL.

BACKGROUND: STEM (RH49) is a low-prevalence antigen in the Rh blood group system. A scarcity of anti-STEM has precluded extensive study of this antigen. We report that two alleles with a RHCE*ce818C>T change encode a partial e, and a hr(S) -, hr(B) +, STEM+ phenotype and that both alleles are frequently in cis to RHD*DOL1 or RHD*DOL2. STUDY DESIGN AND METHODS: Blood samples were from donors and patients in our collections. Hemagglutination, DNA, and RNA testing was performed by standard techniques. RESULTS: Fourteen STEM+ samples were heterozygous RHCE*ce818C/T: six had RHCE*ceBI and eight had a novel allele, RHCE*ceSM. Eleven were heterozygous for RHD*DOL1 or RHD*DOL2. Eleven samples, previously typed STEM-, had RHCE*ce818C/C (consensus nucleotide). RBCs from informative STEM+ samples were e+/- hr(S) - hr(B) +. One person who was heterozygous RHCE*ceBI and RHCE*cE had an anti-e-like antibody in her plasma, and one person, who was hemizygous for RHD*DOL2, had anti-D in her plasma. CONCLUSIONS: We show that two alleles with a RHCE*ce818C>T change (RHCE*ceBI and RHCE*ceSM) encode a hr(S) - hr(B) + STEM+ phenotype. In addition, both alleles are frequently in cis to RHD*DOL1 or RHD*DOL2 and RHCE*ceBI encodes a partial e antigen. In the small cohort of samples tested, RHD*DOL invariably traveled with RHCE*ce818T. Our study also confirmed the presumption that RHD*DOL2, like RHD*DOL1, encodes a partial D antigen and the low-prevalence antigen DAK.

DIII Type 7 is likely the original serologically defined DIIIb.

BACKGROUND: Due to their homology, close proximity, and opposite orientation, RHD and RHCE can exchange nucleotides giving rise to variant alleles. Some of these variants encode the so-called partial phenotypes. The DIII partial D category has been subdivided into DIIIa, DIIIb, DIIIc, DIII Type 4, DIII Type 6, and DIII Type 7. During DNA-based screening tests, we identified a second example of DIII Type 7 in a Dce donor from South Africa. Our study describes hemagglutination tests on this sample and raises a question regarding the molecular basis of the originally defined DIIIb category. STUDY DESIGN AND METHODS: Hemagglutination and DNA testing were performed by standard techniques. RESULTS: Red blood cells from this DIII Type 7 donor typed D+C-E-c+e+G-, DAK+ and did not react with anti-D made by people with the DIII phenotype. The allele is RHD*DIII 150C, 178C, 201A, 203A, 307C, 410T, 455C, 602G, 667G. CONCLUSIONS: Based on the serotype and ethnicity (black African), it is likely that DIII Type 7 is the originally defined DIIIb category.

DIIIa and DIII Type 5 are encoded by the same allele and are associated with altered RHCE*ce alleles: clinical implications.

BACKGROUND: The partial D phenotype DIIIa was originally reported to be associated with 455A>C in Exon 3, 602C>G in Exon 4, and 667T>G in Exon 5. Other alleles with these changes were subsequently identified and designated DIII Types 5, 6, and 7, as they had additional alterations. The observation that DNA samples associated with the DIIIa phenotype had more changes than those originally reported motivated us to reanalyze the DIIIa probands (BP and DJ) from the original study. We also studied additional DIIIa samples to clarify the RHD background and establish the associated RHCE. STUDY DESIGN AND METHODS: Hemagglutination testing was performed by standard methods. RHD and RHCE were analyzed by combinations of polymerase chain reaction-restriction fragment length polymorphism, exon-specific sequencing, cloning, or direct sequencing of Rh-cDNAs. RESULTS: The RHD alleles from BP, DJ, and 58 additional DIIIa samples had the three reported nucleotide changes as well as 186G>T, 410C>T, and 819G>A. The DIIIa allele was associated with several altered RHCE*ce-alleles, the prominent one being ceS (48C, 733G, 1006T). CONCLUSION: The DIIIa phenotype is associated with six RHD changes, five of which encode amino acid changes, and partial DIIIa and DIII Type 5 are encoded by the same RHD allele. In all samples, RHD*DIIIa was inherited with altered RHCE*ce. Patients with partial DIIIa are at risk for production of alloanti-D, but they are also at risk for alloanti-e, -c, or antibodies to high-prevalence Rh antigens if there is no conventional RHCE*ce in trans. Among 39 patients studied, 16 had alloanti-D and 27 had alloanti-e or anti-hrB.