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

Abstract

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 (1,658 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.

Whole-exome sequencing for RH genotyping and alloimmunization risk in children with sickle cell anemia

“…15 RHD and RHCE alleles were assigned on the basis of either a single or a combination of genetic markers and haplotype associations based on reported alleles. 5,16,17 Among all 134 randomly assigned SWiTCH study participants, 27 children had documented Rh immunization at study entry. Controls were identified from nonimmunized SWiTCH participants and matched first for duration of transfusions (mean difference, 0.0 y; median difference, 0.0 y) and then for age at first stroke (mean difference, 0.6 y; median difference, 0.0 y).…”

“…RHD exon 8 had low sequence coverage (median coverage, ,63; Figure 3A), and therefore WES did not reliably detect the c.1138C.T change (p.Thr379Met) in exon 8 that has an allele frequency of 0.1019 to 0.1710 in African individuals (Table 2). 5,16 WES also did not detect the RHD 37-bp duplication that inactivates D antigen expression (D-phenotype) referred to as the RHD pseudogene (supplemental Table 2). However, the RHD pseudogene has the stop codon c.807T.G (p.Try269Stop) in exon 6 that was identified by WES.…”

Optimized Antigen-Matched in Sickle Cell Disease Patients: Chances and Challenges in Molecular Times - the Brazilian Way

“…RHD*weak D 4.2.2 was the most common RHD variant allele (50%), followed by RHD*weak partial D 4.0 (20%), RHD*DVII (10%), and RHD*DAU0.01 (10%); and the most prevalent RHCE -altered alleles were RHCE*ce48C (40%), RHCE*ceAR (20%), and RHCE*ce 48C,733G (8%). These alleles were also found in other cohorts of Afro-American and Afro-Caribbean SCD patients [28,29]. Identifying RH variants at the beginning of the transfusion protocol could have prevented RH alloimmunization by providing antigen-negative or haplotype-compatible RBC units, but RH genotyping is not currently available for all Brazilian centers that treat SCD patients.…”

Insights into <b>RHCE</b> Molecular Analysis in Samples with Partial <b>D</b> Variants: the Experience of Western France

“…1), including the recently reported RHCE*ce(c.48C, c.105T) allele [25], which is reminiscent to the RHCE*ce.01 allele with an additional silent c.105C>T variant, and a yet unreported RHCE*ce(c.712A, c.733G, c.787G, c.800A, c.916G) allele, which only differs from the RHCE*ce.04 allele by the c.48G>C transversion that is not found here. QMPSF analysis of the 47 samples genotyped as weak D type 4.2.2 exhibited aberrant results in five DNAs (data not shown), which is a typical feature of degraded genomic DNA [22].…”