The Emerging Role of RH Genotyping in Chronically Transfused Sickle Cell Disease Patients
Authors: Fasano Ross , Monaco Alessandro , Meier Emily Riehm , Otridge John , Lee-Stroka Hallie , Pary Philippe , Klein Harvey G. , Marincola Francesco M. , Luban Naomi L.C. , Stroncek David 
Published: April 2020

Abstract
Background: Individuals of African descent have different molecular RH variants but their clinical significance with respect to alloantibody formation to high-incidence RH antigens has not been studied in multiply transfused patients. Some rare Rh phenotypes occur exclusively in the black population, yet their association with alloimmunization remains unclear. Current serological methods are unable to detect these variants; however, recent advances in molecular genotyping permit the identification of polymorphisms which may contribute to alloimmunization.
Case: A 7-year-old Nigerian male with sickle cell disease (SCD) and with a silent stroke on routine MRI screening, was enrolled on the NHLBI Silent Infarct Transfusion Therapy (SITT) study and randomized to receive chronic transfusions.
His RBC serological phenotype was: B, RhD-C+c+E-e+, K-. After receiving his ninth leukoreduced, Type B, D-, E-, K- RBC transfusion, he was found to have an alloantibody with “e-like” specificity. Serological evaluation revealed positive reactions of patient’s sera with all commercially available Rhe+ RBCs, positive reactions with Rhe+(hrs-) and Rhe+(hrB-) in some but not all enhancement media, and no reaction to a South African-specific Rhe+(hrs-) RBC sample in all enhancement media. RBC molecular phenotyping through two sequence specific primer polymerase chain reaction (SSP-PCR) methodology platforms revealed a blood type B, RHD-C-c+E-e+, K-; in addition, the cdes genotype (r’s) was detected on one of the two platforms. RH DNA sequencing revealed the presence of known heterozygous single nucleotide polymorphisms (SNPs) on RHCE exons 1, 5, and 7 for W16C, L245V, and G336C respectively. With the use of D-specific primers, a homozygous SNP was identified on RHD exon 8 (T379M) which designates RHCE exon 8 from RHD; however, RHD exons 2, 4–7 failed to amplify. With the use of non-specific RH primers, exons 2, 4, and 7 were amplified and sequenced, revealing CE-specific exons 4, and 7 sequences with the G336C substitution. With non-specific amplification of exon 2, sequences from both RHD and RHCE, in addition to the L62F polymorphism, were identified, indicating the presence of both RHD and RHCE sequences. Sequencing data from RHD exon 3 identified a D/CE fusion point witihin exon 3, with a nucleotide substitution at position 410C&gt;T (A137V). Microarray results were consistent with the serologic phenotype and sequencing results, verified the presence of the cdes genotype, and confirmed the D/CE fusion point at exon 3. No signal was detected for RHD exons 2, and 4–7 indicating the large RHD deletion and RHCE insertion within the RHD gene.
Conclusions: This RH genotype is consistent with the previously reported (C)ces genotype. SCD patients should be comprehensively screened for RH variants prior to initiating transfusion therapy in order to determine alloimmunization risk. RBC genotyping provides additional resolution to serologic investigations for the identification of unique RH polymorphisms, which can aid in appropriate selection of RBCs to avoid alloimmunization, or purely identifying individuals who may benefit from alternative methods of treatment for their disease, such as hydroxyurea or stem cell transplant in SCD patients.