Compound heterozygosity of two novel RHAG alleles leads to a considerable disruption of the Rh complex.

BACKGROUND: The Rhesus (Rh) complex consists of a core comprising the Rh proteins (RhD/RhCE) and the Rh-associated glycoprotein (RhAG) with accessory chains (GPB, LW, CD47). Molecular defects of the RHAG gene may cause a regulator Rhnull phenotype without Rh antigen expression or a Rhmod phenotype with decreased Rh antigen expression. STUDY DESIGN AND METHODS: Blood samples of a donor with strongly diminished Rh antigens and five family members were analyzed by serological phenotyping, flow cytometry, molecular testing, and gene expression analysis of Rh complex candidate genes. RESULTS: RHAG sequencing identified a missense mutation, c.241G>C (p.Gly81Arg) and a splice site mutation, c.640 + 3del14, among the cohort. Compound heterozygosity of these novel alleles identified in the propositus and two siblings gave rise to a strongly diminished expression of RhAG, Rh, and CD47 antigens on the RBC surface. CONCLUSION: The Rhmod phenotype was caused by a novel RHAG splice site mutation in association with a non-functional allele. The primary depression of RhAG is most likely due to posttranslational events that affect the interaction and processing of the RhAG glycoprotein and gave rise to a secondary depression of RhD, RhCE, and CD47, the major members of the Rh complex.

RHD variants in Polish blood donors routinely typed as D-.

BACKGROUND: Blood donors exhibiting a weak D or DEL phenotypical expression may be mistyped D- by standard serology hence permitting incompatible transfusion to D- recipients. Molecular methods may overcome these technical limits. Our aim was to estimate the frequency of RHD alleles among the apparently D- Polish donor population and to characterize its molecular background. STUDY DESIGN AND METHODS: Plasma pools collected from 31,200 consecutive Polish donors typed as D- were tested by real-time polymerase chain reaction (PCR) for the presence of RHD-specific markers located in Intron 4 and Exons 7 and 10. RHD+ individuals were characterized by PCR or cDNA sequencing and serology. RESULTS: Plasma cross-pool strategy revealed 63 RHD+ donors harboring RHD*01N.03 (n = 17), RHD*15 (n = 12), RHD*11 (n = 7), RHD*DEL8 (n = 3), RHD*01W.2 (n = 3), RHD-CE(10) (n = 3), RHD*01W.3, RHD*01W.9, RHD*01N.05, RHD*01N.07, RHD*01N.23, and RHD(IVS1-29G>C) and two novel alleles, RHD*(767C>G) (n = 3) and RHD*(1029C>A). Among 47 cases available for serology, 27 were shown to express the D antigen CONCLUSION: 1) Plasma cross-pool strategy is a reliable and cost-effective tool for RHD screening. 2) Only 0.2% of D- Polish donors carry some fragments of the RHD gene; all of them were C or E+. 3) Almost 60% of the detected RHD alleles may be potentially immunogenic when transfused to a D- recipient.

[RHD variant in RhD/-/ mother with anti-D makes noninvasive fetal RHD genotyping impossible]. / Obecnosc "niemego" genu RHD u RhD ujemnej kobiety ciezarnej z przeciwcialami anty-RhD uniemozliwia okreslenie genotypu RHD plodu metoda nieinwazyjna.

OBJECTIVES: Noninvasive fetal RHD genotyping from maternal plasma of RhD(/-) pregnant women of Caucasian race may be used for predicting the risk of hemolytic disease because the RHD gene is usually absent in such populations. If detected in plasma of such women, the RHD gene originates from the RhD(+) fetus. The number of fetal copies of the gene in maternal plasma is extremely small. In the presented case of the RhD(/-) pregnant woman with anti-D it was impossible to give a fetal RHD result due to mother's RHD(+) genotype. The fetal RHD was determined from amniocytes. AIM: to present the difficulties related to the interpretation of results of invasive and noninvasive procedures. MATERIAL AND METHODS: whole blood, plasma and amniotic fluid of the RhD(-) woman with anti-D (14 week of pregnancy) as well as whole blood of the newborn. RHD and RHCE*c were genotyped by real-time PCR in DNA isolated from maternal plasma and amniocytes and the RHD and d-genotypes were tested by SSP methods in DNA isolated from whole blood and amniocytes. RESULTS: RHD and RHCE*c were detected in DNA isolated from plasma. The high level of RHD suggested its origin from the mother's DNA therefore it was impossible to determine the fetal RHD. The d-little test identified a RHD(IVS3+ 1G>A) variant in the mother's genome. A weak signal of real-time PCR for the RHD was obtained in amniocytes but the RHD was not detected by SSP. The RHCE*c was detected by both methods. Results were inconclusive; the fetal RHD status remained unknown. The child was RhD(-) with RHD in its DNA undetected by either method. CONCLUSIONS: 1/The RHD(IVS3+ 1G>A) variant in the RhD(-) mother precluded formal noninvasive fetal RHD genotyping. 2/Real-time PCR is too sensitive for amniocyte testing and may lead to false results as it detects trace maternal DNA in amniotic fluid. 3/The frequency of RHD(IVS3+1G>A) occurrence in Poland requires further studies.

A preliminary evaluation of next-generation sequencing as a screening tool for targeted genotyping of erythrocyte and platelet antigens in blood donors.

BACKGROUND: Matching the compatibility of donor blood with the recipient's antigens prevents alloimmunisation. Next-generation sequencing (NGS) technology is a promising method for extensive blood group and platelet antigen genotyping of blood donors. It circumvents the limitations of detecting known alleles based on predefined polymorphisms and enables targeted sequencing on a massive scale. The aim of this study was to evaluate the NGS AmpliSeq application on the Ion Torrent platform as a screening tool for genotyping blood donors' erythrocyte/platelet antigens. MATERIALS AND METHODS: Primers for regions encoding antigens RhD (exons 5, 7), Rhc, RhE/e, Fya/b, Jka/b, M/N, S/s, HPA-1, 2, 3, 5, 15 were designed with Ion AmpliSeq Designer with manual inclusion of RHCE*C primers. DNA libraries of 57 regular blood donors with determined phenotype/genotype (prepared using the Ion AmpliSeq Library Kit and 14 primer pairs) were sequenced on the Ion Torrent PGM using 316v2 chips and 200 bp chemistry. RESULTS: Sequencing was successful in all but the MN and HPA-5 regions. Mean sequencing coverage in one experiment was 4,606 reads, except for the RHCE*C region (mean 568 reads). NGS results agreed with the known phenotype/genotype of donors except in one phenotypically Fy(a+b-) case in whom FY*A/FY*B alleles were found. Reading rates for homozygotes were 97-100%, while they were around 50% for heterozygotes. NGS of RHD regions led to identification of mutations in two RhD negative donors. DISCUSSION: NGS can be performed as a screening test to determine erythrocyte/platelet antigens in blood donors. This method allowed testing of 48 donors for 14 features (200 bp long) with the depth of a few thousand reads simultaneously, and the estimation of natural chimerism or hemi/homozygotic status. NGS screening can be adjusted to the genetic background of a given tested population.