RHCE Gene Research Articles

Next Generation DNA Sequencing of the RH Locus in 1135 Donors Implicates RHCE-RHD(2)-RHCE Hybrid Alleles As Causative of C+ Antigen

Background. The RHD and RHCE genes direct the expression of highly immunogenic antigens in the Rh system. These genes exhibit many types of genetic variation, including single nucleotide variants (SNVs), insertions and deletions, and larger structural variants (SVs) including RHD-RHCE hybrid alleles. Thus far, DNA-based testing methods, including next generation sequencing, have been confounded by the highly homologous nature of these genes in detecting some types of uncommon and complex variants at the RH locus. We applied BloodSeq, a new targeted next generation sequencing (NGS) method with custom genomic computational tools for the systematic, unbiased characterization of the RH locus and study of the causes of C expression.Methods. The BloodSeq NGS targeted panel captures a 269kb region spanning the RH locus. Captured DNA is multiplexed, sequenced on Illumina Hiseq machines using paired-end 100-bp sequencing, reads aligned to the human reference genome (hg19) using BWA-MEM, and SNVs assessed using GATK. An RH- customized read-depth based method detects SVs including hybrid alleles. Four WHO DNAs (NIBSC) were used as references; 1135 samples were selected from a parent study of blood donors self-identified to be of Asian American or Native American descent. Donors were previously tested for D and C by serology and for CcEe by SNVs. RH hybrid alleles predicted by NGS were validated by quantitative multiplex PCR of short fluorescent fragment (QMPSF) analysis. Gene-specific long range PCRs were developed to confirm RHD-RHCE exon 2 hybrid alleles.Results . Analysis of BloodSeq NGS data in these 1139 DNAs found that all C+ samples (by serology) have RHCE-RHD-RHCE gene conversion events involving RHD exon 2. In total there were 822 (72.17%) samples with these events, including 439 heterozygotes and 383 homozygotes. Predicted breakpoints were largely consistent across individuals, with the most common RHCE-RHD(2)-RHCE allele having a conversion region spanning a ~4.9kb genomic relative to RHCE . Other sized RHCE-RHD(2)-RHCE conversion events were detected ranging from 1kb to 6.8kb relative to RHCE . Three multi-exonic RHCE-RHD-RHCE SV events inclusive of RHD exon 2 were also identified. Notably, C+ associated hybrid alleles did not affect exon 1, and an RHCE*02/RHCE*04 (i.e. C+ allele)exon 1 SNV, c.48G>C, showed only moderate specificity (88.6%) in predicting C+ by serology in this minority study population.Gene-specific long range PCR confirmed the RHCE-RHD(2)-RHCE hybrid alleles identified by NGS. Informed by NGS predicted hybrid breakpoints, Sanger sequencing of cloned gene-specific long range PCR products was used to characterize the common C+ RHCE-RHD(2)-RHCE intron 1 and 2 breakpoints. C+ specific variation was identified at each breakpoint. At the intron 1 breakpoint, there is a previously unreported SNV 100% specific for C+ in this study. At the downstream hybrid intron 2 region breakpoint, there is 109bp of exogenous sequence consistent with the 109 bp intron 2 “RHCE insertion” known to predict C+ expression. In addition to these findings, numerous known and novel SNVs, indels, and SVs were identified in both RHD and RHCE.Conclusions. This work identifies RHCE-RHD(2)-RHCE hybrid alleles as the primary cause of C+ expression. Variation in the regions affected by these hybrid alleles provides fine mapping information for the minimal genomic region sufficient to confer C+ expression. We further characterized the common RHCE-RHD(2)-RHCE hybrid allele breakpoints and identified a previously unknown C+ diagnostic SNV. This work illuminates causes of C+ expression and provides the foundation for a DNA-based, high resolution Rh blood-typing method for the detection of clinically relevant RH locus genetic variation. DisclosuresNo relevant conflicts of interest to declare.

Risk Minimization of Hemolytic Disease of the Fetus and Newborn Using Droplet Digital PCR Method for Accurate Fetal Genotype Assessment of RHD, KEL, and RHCE from Cell-Free Fetal DNA of Maternal Plasma.

The molecular pathology of hemolytic disease of the fetus and newborn (HDFN) is determined by different RHD, RHCE, and KEL genotypes and by blood group incompatibility between the mother and fetus that is caused by erythrocyte antigen presence/absence on the cell surface. In the Czech Republic, clinically significant antierythrocyte alloantibodies include anti-D, anti-K, anti C/c, and anti-E. Deletion of the RHD gene and then three single nucleotide polymorphisms in the RHCE and KEL genes (rs676785, rs609320, and rs8176058) are the most common. The aim of this study is to develop effective and precise monitoring of fetal genotypes from maternal plasma of these polymorphisms using droplet digital (dd)PCR. Fifty-three plasma DNA samples (from 10 to 18 weeks of gestation) were analyzed (10 RHD, 33 RHCE, and 10 KEL). The ddPCR methodology was validated on the basis of the already elaborated and established method of minisequencing and real-time PCR and with newborn phenotype confirmation. The results of ddPCR were in 100% agreement with minisequencing and real-time PCR and also with newborn phenotype. ddPCR can fully replace the reliable but more time-consuming method of minisequencing and real-time PCR RHD examination. Accurate and rapid noninvasive fetal genotyping minimizes the possibility of HDFN developing.

Preliminary study on the function of TMEM50A and its correlation with the RH genes.

The purpose of this study was to investigate the association and impact of TMEM50A on RH genes activity and function. SMP1 is located on chromosome 1p36.11 in the RH gene locus, between the RHD and RHCE gene, where its position may be linked to RH haplotypes and contribute to selective pressures regarding certain RH haplotypes. TMEM50A is encoded by the SMP1 located in the intergenic region of RH, its influence on the function of the RH genes remains unclear. The expression of TMEM50A was regulated by transfection of plasmid and siRNA in K562 cell model. Western blot and real-time PCR were used to detect possible expression changes in the RH. The ammonium transport function of cells was monitored using pH-sensitive dye, while transcriptome sequencing was used to predict the potential function of TMEM50A. The overexpression of TMEM50A significantly up-regulated RHCE gene activity (63.56%). The inhibition of TMEM50A resulted in significantly decreased RHCE (41.82%) and RHD expression (27.35%). Compared to control group, there was no significant change in the NH4 + transport function of cells in the overexpressed TMEM50A group. Transcriptome analysis showed that TMEM50A not only affected the transcription of target gene through splicing activities, but also played a role in the development of embryonic nervous system. TMEM50A may regulate the expression of RH gene by affecting the stability of RH mRNA through splicing function. It speculates that TMEM50A may play an important role in the development of embryonic nervous system.

Variant genotypes associated with reduced expression of RhCE antigens among Brazilian blood donors.

The genetic diversity of the RHCE gene locus has been explored in diverse populations of different racial backgrounds. Data referring to the diversity of RHCE encoding weakened expression of C, c, E, and e in multiethnic populations is still incomplete. Samples from Brazilian blood donors presenting reduced expression of C, c, E, or e on gel method were selected for the study. All exons and flanking introns of RHCE were genotyped though direct Sanger sequencing for the included donors. Sixty-six donors were included: 23 with weak C, 22 with weak c, 6 with weak E, 14 with weak e, and 1 with weak c and E. Among the samples with weak C, the following altered RH*C were encountered: RHCE*CeMA (n=3), RHCE*Ce941C (n=1), and RHCE*CeVA (n=1). RHD*D-CE(4-7)-D was detected in six cases, RHCE*CE was presumably present in five cases, and seven cases were unexplained. Two altered alleles underlay the weak c phenotype: RHCE*ceJAL (n=20) and RHCE*ce340T (n=2), and two altered RHCE justified weak e: RHCE*ceMO (n=6) and RHCE*ceJAL (n=8). Three variant RHCE were associated with weak E: RHCE*cEJU (n=4), RHCE*cE382C (n=1), and RHCE*cEIV (n=1). The RHCE*cE905A justified one case of weak c and E. We describe the distribution of RHCE variants found in association with weak expression of C, c, E, and e in blood donors of multiethnic origin, which differs in comparison to that previously reported for people of African or Caucasian descent.

RHD and RHCE genes polymorphism: literature review

The article provides a literature review about RHD and RHCE polymorphisms which encode different RhD and RhC antigen variants. The data about genes RHD and RHCE polymorphisms, RhD weak types, RhD partial types and RhC variants in Russians is presented for the first time. The molecular and serological characteristics of rare RhD and RhC antigens are summarized. The role of serological and molecular methods in Rhesus system antigens identifying is shown.

Genetic Basis of Erythrocyte Alloimmunization Among Children with Sickle Cell Anemia in the Dominican Republic

Introduction: In low-resource settings, red blood cell (RBC) transfusions for children with sickle cell anemia (SCA) may be cross-matched for only ABO and RhD antigens, so RBC alloimmunization can develop due to differences in Rh and other antigens between donors and recipients. In the Rh system, these differences can include absence of the antigen or the presence of partial or weak antigens, the latter more common with African ancestry. RBC alloimmunization in SCA has not been described in the Dominican Republic, and the genetic diversity of the RHD and RHCE loci have not been reported in this Hispanic-Afro-Caribbean population. Methods: A pediatric stroke prevention trial in the Dominican Republic (SACRED, NCT02769845) provided serum and dried blood spots from children with SCA, collected at enrollment and stored at -80C. Sera were screened for alloantibodies using a modified test-tube agglutination identification method with commercially available Panocell-20 (Immucor) RBC suspensions of known antigen phenotypes. Reaction patterns identified antigen specificity using standard blood bank methods. Patterns reactive with all cells tested were reported as pan-reactive and reactions without patterns were deemed low frequency antibodies. For all samples with an alloantibody, a matched SACRED control (age, sex, and transfusion history) was identified. Genomic DNA was analyzed by PCR and direct sequencing of each RHD and RHCE exon for single nucleotide polymorphisms (SNPs) that confer weak, partial, or variant phenotypes. Copy number variant PCR techniques with gene-specific primers were used to detect exonic substitutions in the RHD and RHCE gene loci that lead to negative or partial phenotypes. Results: A total of 272 SACRED participants were screened for RBC alloantibodies; 179 (66%) had at least one transfusion prior to enrollment, of whom 37 (21%) had a positive alloantibody screening result. Children with RBC alloantibodies (17 males, 20 females, average age 9.0 ± 2.4 years) had variable transfusion exposure with 23 (62%) having >10 prior transfusions, while 7 (19%) had ≤2 previous transfusions. Among the 37 positive samples, 18 sera had reactivity against Rh antigens including anti-E (12), anti-D (6, including 3 females), and anti-C specificity (5); 8 samples were consistent with a low frequency antibody and 3 were pan-reactive. Other alloantibodies had specificity to K (3, 1 female), Fya (4), Fyb (1), Lea (1), Leb (1), S (1), s (1), Jka (1), and U (1). More than one alloantibody was identified in 7 samples, of which 5 had two Rh antibodies. Genomic DNA analysis revealed numerous RHD variants, including several with one or more exonic SNPs that predict weak or partial RhD phenotypes, as well as predicted D-negative or partial phenotypes with hybrid RHD-CE-D genes or an insert pseudogene (RHDψ). Comparing the alloimmunized patients and controls, there was an equivalent frequency of RHD SNP variants (15/37 = 41%) in both cohorts, but more children with hybrid RHD-CE-D genes affecting multiple RHD exons (10 versus 4, p=0.077). All samples had the homozygous ccee genotype in the RHCE locus. There was a high frequency of RHCE SNP variants in both alloimmunized (31/37 = 84%) and control cohorts (32/37 = 86%), and only one control sample with possible RHD insertion in RHCE exon 5. All six children with anti-D alloantibodies had hybrid RHD-CE-D genes, typically typing as D-negative in tube testing. Among 17 children with anti-C or anti-E alloantibodies, 16 had at least one RHCE SNP, most frequently RHCE*ce.01, but no SNPs were associated with the development of Rh alloantibodies. Conclusions: RBC alloimmunization in children with SCA is common and often unrecognized in low-resource settings. In the Dominican Republic, 21% of children with SCA who entered SACRED with previous transfusions had RBC alloimmunization, including 10% with antibodies against Rh antigens. DNA analysis confirms extensive genetic complexity in both RHD and RHCE genes, with numerous SNPs that alter Rh antigen expression. Large RHD-CE-D insertions may also confer increased risks of anti-D alloimmunization. Lack of typing beyond serologic tube testing of ABO/RhD, and the presence of weak/partial RhD phenotypes in blood donors, may further increase the risk. Extending phenotyping for RhCE, providing RhD-negative blood when available, and performing enhanced screening for RhD-negative donors, will help reduce RBC alloimmunization. Disclosures No relevant conflicts of interest to declare.

Genotyping for Auxiliary Identification of Anti-C Alloantibody with Anti-f Autoantibody Mimicking Alloantibody

To identify the difficult antibody specificity of 1 PNH patient with blood transfusion history by genotyping. RH typing of this patient was performed using gel card method, the antibody specificity was identified by panel cells, the RH-unrelated phenotype were excluded by genotyping method in difficult condition of serologic identification, furthmore different RH phenotype cells were used for adsorption-elution so as to re-examine the reactivity of antibodies in this patient's serum, and finally different RH phenotype cells were combined to exclude other unrelated antibodies. The RH phenotype presented as double population for C antigen, and positive agglutination for the other antigens. The results of RHD zygote, together with RHD and RHCE sequencing showed that the RHD genotype was homozygous RHD/RHD, and the c.122A＞G mutation did not found in RHCE gene, thus CW antigen was excluded; 48G lies in the 1st exon, the 5th exon showed nt676 G/C heterozygosity, the 2nd-4th, 6th-10th exons did not show mutations, however a new mutation was found in the 4th intron IVS4+29A＞C. It can be judged that the RH genotype was Dce/DcE, and its phenotype should be ccDEe. The anti-C alloantibody and rare anti-f autoantibody mimicking alloantibody in serum was determined by different RH phenotypic cell absorption-elution. Finally, cross-matched AB ccDEE blood units were selected for the patient and there was no adverse reactions of blood transfusion occurred. Genotyping can be auxiliarily applied to the identification of difficult antibodies in serum of a patient, thereby reducing the risk of blood transfusion.

A novel algorithm comprehensively characterizes human RH genes using whole-genome sequencing data

AbstractRHD and RHCE genes encode Rh blood group antigens and exhibit extensive single-nucleotide polymorphisms and chromosome structural changes in patients with sickle cell disease (SCD). RH variation can drive loss of antigen epitopes or expression of new epitopes, predisposing patients with SCD to Rh alloimmunization. Serologic antigen typing is limited to common Rh antigens, necessitating a genetic approach to detect variant antigen expression. We developed a novel algorithm termed RHtyper for RH genotyping from existing whole-genome sequencing (WGS) data. RHtyper determined RH genotypes in an average of 3.4 and 3.3 minutes per sample for RHD and RHCE, respectively. In a validation cohort consisting of 57 patients with SCD, RHtyper achieved 100% accuracy for RHD and 98.2% accuracy for RHCE, when compared with genotypes obtained by RH BeadChip and targeted molecular assays and after verification by Sanger sequencing and independent next-generation sequencing assays. RHtyper was next applied to WGS data from an additional 827 patients with SCD. In the total cohort of 884 patients, RHtyper identified 38 RHD and 28 RHCE distinct alleles, including a novel RHD DAU allele, RHD* 602G, 733C, 744T 1136T. RHtyper provides comprehensive and high-throughput RH genotyping from WGS data, facilitating deconvolution of the extensive RH genetic variation among patients with SCD. We have implemented RHtyper as a cloud-based public access application in DNAnexus (https://platform.dnanexus.com/app/RHtyper), enabling clinicians and researchers to perform RH genotyping with next-generation sequencing data.

Features of immunohematological and hematological parameters of a donor with a rare phenotype -D-

Background. This study is devoted to a rare variation of the -D- phenotype . The -D- phenotype was first discovered by R. Race, R. Sanger and J.G. Selwyn in 1951. In Russia, the phenotype -D- was first discovered by V. Morokov in 1985. Typically, the -D- phenotype is detected when physicians examine post-transfusion complications or hemolytic disease of the newborn, since such patients demonstrate high antibody titres to absent antigens. In the present study, the -D- phenotype was detected in a primary blood donor at the clinical laboratory of the Ulyanovsk Regional Blood Transfusion Station (Ulyanovsk, Russia). Aim. To study specific features of immunohematological and hematological blood parameters in a donor with a rare variation of the -D- phenotype.Materials and methods. The detection of the -D- phenotype by immunohematological methods was carried out using automatic analysers. Molecular DNA typing was used to confirm the -D- phenotype. The shape of erythrocytes of the donor with the -D- phenotype was evaluated using an atomic force microscope. The characteristics of the erythroid lineage were studied using an automatic hematological analyser.Results. The -D- phenotype was detected in a primary blood donor. Due to the extreme rarity of the -D- phenotype and the lack of programmed algorithms, the validation of the results by automatic analysers was incorrect. Of critical importance was the visual assessment of gel ID cards by the medical staff. Genotyping confirmed the lack of C, c, E, e, Cw specificities in the RHCE gene. The hematological parameters of the donor were within the age norm. An assessment of the image of a cytological blood preparation did not reveal changes in the shape of erythrocytes and their size.Conclusions. The primary determination of the -D- phenotype using automatic immunohematological analysers can be complicated by the impossibility of validating the results, the incorrect operation of the installed software and the need for expert evaluation of blood samples by the staff. The presented case of the -D- phenotype was not associated with changes in the shape of erythrocytes and blood hematological parameters.

Serological Detection of Rh-Del Phenotype among Rh-Negative Blood Donors at National Blood Center, Yangon, Myanmar.

Background Red cell Rhesus (Rh) antigen expression is influenced by the genetic polymorphism of RHD and RHCE genes and reveals serologically different reactions of RhD variants such as partial D, weak D, and Rh-Del. Serologically, Rh-Del type can only be detected by an adsorption-elution technique, and it might be mistyped as Rh-negative. The prevalence of Rh-Del has not been reported yet in Myanmar. Method A total of 222 Rh-negative blood donors in the National Blood Center were tested for weak D and Rh-Del by indirect antihuman globulin and adsorption-elution method, respectively. RhCE typing was performed among Rh-negative and Rh-Del. Results Of them, 75.2% (167/222) were Rh-negative, 15.8% (35/222) were Rh-Del, and 9% (20/222) were weak D. Of 202 blood donors (167 true Rh-negative and 35 Rh-Del), all of the Rh-Del positives were C-antigen-positive with 94.3% Ccee phenotype (33/35) and 5.7% CCee (2/35). Most of the Rh-negative donors (80.2%) were ccee phenotype (134/167). Conclusion About half of Rh-Del subjects were repeated donors, and attention was needed to avoid transfusion of truly Rh-negative patients to prevent alloimmunization. It is recommended to do Rh-Del typing of Rh-negative donors who are C-antigen-positive and consider moving them to the Rh-positive pool. Further study is needed to clarify the alloimmunization status for transfusion of Rh-Del blood to Rh-negative recipients. Molecular markers for RhD-negative and D variants should be established in the Myanmar population to improve selection of antisera for Rh typing and enhance safety of the transfusion services.

A novel silentRHCEallele in Chinese population

We aimed to analyse the molecular backgrounds of the family in which an eight-day-old baby was confirmed to have hemolytic disease of the newborn (HDN) and phenotype observed for the baby did not conform to the expected phenotype. The silent RHCE allele is rare in the Rh system. To determine the antibody specificity, her family members' blood samples were collected and tested using routine serological methods. The Rh C + c-e + E- phenotype observed for the baby did not conform to the expected phenotype based on the maternal RhC-c + E + e- phenotype. The RH genes of the family members were further analysed by sequencing. The Rh phenotypes of the baby, her brother, her mother and father were CCDee, CcDEe, ccDEE and CCDee, respectively. IgG anti-e was confirmed to cause the HDN in the case. A heterozygous silent RHCE * 03(c.1059G > A) mutation in exon 7 was found in the baby and her mother, which is a novel nonsense allele caused by a premature termination codon (Trp353stop). The silent RHCE * 03(c.1059G > A) variant was observed in a heterozygous state in mother and baby. We predict that, had this occurred in the homozygous state, it would give rise to the rare D-- phenotype. To enhance the safety of transfusion, considerable attention should be paid to the RHCE gene in the Chinese population.

Preimplantation genetic testing of pathogenic variants in genes SMN1, PKD1, RHD with high homologous copies

Introduction Preimplantation genetic testing for the monogenic disease (PGT-M) compared with conventional genetic diagnosis is complicated by extremely small amounts of biomaterial. This feature leads to increased allele dropout (ADO) rates, contamination significance, failed amplification events. Therefore, the development of PGT-M systems for pathogenic variants in the genes with homologous copies is more complicated challenge. PGT-M test-systems for the SMN1, RHD and PKD1 genes were developed in our laboratory and successfully applied in PGT-M cases. Material & methods The test-systems combine direct diagnosis of the pathogenic variants by PCR- RFLP or fragment analysis and linkage analysis of highly heterozygous STRs. The nested PCR was used for DNA amplification from different sources: single cells or whole genome amplification products (WGA) of embryo biopsy, total DNA. Results The test-system for PGT-M in the presence of homologous genes or pseudogene copies of the target gene has to exactly discriminate the allele with and without pathogenic variant and detect miss target amplification to evaluate the probability of misdiagnosis. PCR-RFLP was used to discriminate the single nucleotide difference between exon 7 of the SMN1 (NC_000005.9:g.70247773C) and SMN2 (NC_000005.9:g.69372353T) genes. This method cannot detect the number of the SMN1 copies, but confirms the presence of at least one copy. PGT-M for the SMN1 was performed in 7 cycles for 36 embryos. RHD detection was based on the difference of PCR products length from the RHD and RHCE intron 7 (NC_000001.10: 25636476-25636480GGGGT, 25639738-25639741GGCA in the RHD gene) and 8 (NC_000001.10: 25698440-25698442TAA in the RHCE gene). These nucleotide variations were performed for PGT-M previously, but we optimized this approach for WGA products. PGT for Rh- status was performed in 2 cycles for 11 embryos. Similarity of genes SMN1/SMN2 and RHD/RHCE sequences requires careful usage of STRs for indirect diagnostics. Among the STRs, selected for the test-system, we found some duplicate repeats, flanked by similar sequences and located in a similar way in each target/nontarget locus. D1S2561/ D1S2572 and D1S2564/D1S2569 for RHD/RHCE and D5S1556/ D5S6934 forSMN1/SMN2. The segregation analysis of such markers is not univocal and can lead to misdiagnosis. Exons 1–33 of all 46 exons of the PKD1have high similarity with six pseudogenes located on chromosome 16. Therefore, it is important to distinguish PCR product from the gene and pseudogenes as well as amplification of miss targets. Amplification of several miss targets is conjugated with high load of PCR reaction and can lead to increased ADO rate. The previously performed PGT test-system based on long-range PCR is not applicable with WGA products ( Zeevi et al. 2013 ). So, we developed new test-system and specified amplification by using “touchdown” amplification program and high- precision polymerase in combination with miss target PCR products detection by PCR-RFLP. PGT-M for PKD1 was performed in 2 cycles for 3 embryos. Conclusions The PGT-M test-system in the presence of homologous copies of target gene has to be able to distinguish target and nontarget pathogenic variant location. The reported test-systems for the SMN1, PKD1, RHD genes satisfy this condition, detect pathogenic variant with high efficacy and can be used with biomaterials of different types.

Red blood cell alloimmunization and minor red blood cell antigen phenotypes in transfused Ghanaian patients with sickle cell disease.

The routine pretransfusion investigations in Southern Ghana involve only ABO-D blood group typing and ABO compatibility testing without screening for irregular red blood cell (RBC) antibodies. The prevalence and specificities of RBC antibodies and frequencies of most minor blood group antigens in transfused patients with sickle cell disease (SCD) in Ghana are not known and are the objectives of this study. This was a cross-sectional study that investigated transfused patients with SCD for the presence of irregular RBC antibodies and Rhesus, Kell, Duffy, Kidd, and Ss antigens. From a total of 154 patients (median age, 9 years), 10 patients (6.5%) possessed 13 antibodies, predominantly against D, C, and E antigens. In three patients, the antibodies (anti-D, anti-D + C, and anti-C + e) were against antigens they possessed by serology. Genotyping showed that two of these patients had variant RHCE genes that encode for weak and partial e antigens and one patient had a partial RHC gene. Frequencies of most RBC antigens were comparable with frequencies established among the African American population; however, K-k- and Jk(a-b-) phenotypes were more frequent and were present in 21% and 17% of patients, respectively. The prevalence of RBC alloimmunization in transfused Ghanaian patients with SCD was 6.5% and the majority of antibodies were against antigens of the Rh system. Our findings stress the need to include pretransfusion testing for RBC antibodies in patients with SCD, to improve transfusion safety.

Comprehensive phenotypic and molecular investigation of RhD and RhCE variants in Moroccan blood donors.

To date, more than 650 (weak and partial) Rh variants have been reported. Nature and frequency of these variants are known to be ethnodependent. In transfusion medicine, their identification is important to ensure blood safety. The aim of this study is to investigate and describe the nature and estimate the frequency of Rh variants in blood donors in Morocco by serological tests and molecular analysis. Blood samples from 4,458 blood donors were collected and typed for Rh antigens (D, C, c, E and e) by an automated system with monoclonal antibodies. RhD-negative samples were tested for weak D expression by indirect antiglobulin test (IAT), as well as weak C, c, E, and e expression with monoclonal antibodies, by column agglutination technique. All samples exhibiting a weak D agglutination by the automated system and IAT were tested for partial D. RHD and RHCE genes were analysed by quantitative multiplex PCR of short fluorescent fragments (QMPSF) and/or Sanger sequencing. 4,038 (90.58%) and 420 (9.42%) samples were respectively typed serologically as D-positive and D-negative, including 23 (0.52%) presenting with a weak D phenotype. In 21 weak D samples investigated by molecular analysis, RHD*weak D type 4.0 was found to be the most prevalent variant allele (n=11), and a novel RHD(V270A) missense allele was found once. Variant Rh CcEe antigen expression was observed in 17 samples carrying 20 variant RHCE alleles, including a novel RHCE*ce(499G) missense allele (p.M167V). For the first time, molecular genetics of the Rh system was investigated in the Moroccan population. On the basis of our data and in order to optimise donor/recipient matching to prevent from a potential risk of alloimmunisation in recipients, we suggest that 1) quality control of serological reagents and screening strategies must be reviewed in Morocco, and 2) molecular analysis should be implemented and performed in blood donor centers.

Complete RHD next-generation sequencing: establishment of reference RHD alleles

AbstractThe Rh blood group system (ISBT004) is the second most important blood group after ABO and is the most polymorphic one, with 55 antigens encoded by 2 genes, RHD and RHCE. This research uses next-generation sequencing (NGS) to sequence the complete RHD gene by amplifying the whole gene using overlapping long-range polymerase chain reaction (LR-PCR) amplicons. The aim was to study different RHD alleles present in the population to establish reference RHD allele sequences by using the analysis of intronic single-nucleotide polymorphisms (SNPs) and their correlation to a specific Rh haplotype. Genomic DNA samples (n = 69) from blood donors of different serologically predicted genotypes including R1R1 (DCe/DCe), R2R2 (DcE/DcE), R1R2 (DCe/DcE), R2RZ (DcE/DCE), R1r (DCe/dce), R2r (DcE/dce), and R0r (Dce/dce) were sequenced and data were then mapped to the human genome reference sequence hg38. We focused on the analysis of hemizygous samples, as these by definition will only have a single copy of RHD. For the 69 samples sequenced, different exonic SNPs were detected that correlate with known variants. Multiple intronic SNPs were found in all samples: 21 intronic SNPs were present in all samples indicating their specificity to the RHD*DAU0 (RHD*10.00) haplotype which the hg38 reference sequence encodes. Twenty-three intronic SNPs were found to be R2 haplotype specific, and 15 were linked to R1, R0, and RZ haplotypes. In conclusion, intronic SNPs may represent a novel diagnostic approach to investigate known and novel variants of the RHD and RHCE genes, while being a useful approach to establish reference RHD allele sequences.

Transfusion strategy for weak D Type 4.0 based on RHD alleles and RH haplotypes in Tunisia

With more than 460 RHD alleles, this gene is the most complex and polymorphic among all blood group systems. The Tunisian population has the largest known prevalence of weak D Type 4.0 alleles, occurring in one of 105 RH haplotypes. We aimed to establish a rationale for the transfusion strategy of weak D Type 4.0 in Tunisia. Donors were randomly screened for the serologic weak D phenotype. The RHD coding sequence and parts of the introns were sequenced. To establish the RH haplotype, the RHCE gene was tested for characteristic single-nucleotide positions. We determined all RHD alleles and the RH haplotypes coding for the serologic weak D phenotype among 13,431 Tunisian donations. A serologic weak D phenotype was found in 67 individuals (0.50%). Among them, 60 carried a weak D Type 4 allele: 53 weak D Type 4.0, six weak D Type 4.2.2 (DAR), and one weak D Type 4.1. An additional four donors had one variant allele each: DVII, weak D Type 1, weak D Type 3, and weak D type 100, while three donors showed a normal RHD sequence. The weak D Type 4.0 was most often linked to RHCE*ceVS.04.01, weak D Type 4.2.2 to RHCE*ceAR, and weak D Type 4.1 to RHCE*ceVS.02, while the other RHD alleles were linked to one of the common RHCE alleles. Among the weak D phenotypes in Tunisia, no novel RHD allele was found and almost 90% were caused by alleles of the weak D Type 4 cluster, of which 88% represented the weak D Type 4.0 allele. Based on established RH haplotypes for variant RHD and RHCE alleles and the lack of adverse clinical reports, we recommend D+ transfusions for patients with weak D Type 4.0 in Tunisia.

Effect on gene expression of three allelic variants in GATA motifs of ABO, RHD, and RHCE regulatory elements.

Only a few genetic variants have been reported in regulatory elements of blood group genes. Most of them affect GATA motifs, binding sites for the GATA-1 transcription factor. Samples from two patients and one donor with unusual or discrepant serology for ABO, RhD, and RhCE antigens were analyzed by DNA sequencing. Analyzed regions included the coding sequence and portions of regulatory elements. The effect of some variants on gene expression was evaluated in reporter gene assays. Three new alleles were identified. Their key variants are located in the ABO Intron 1 enhancer, the RHD proximal promoter, and the RHCE proximal promoter. IVS1 + 5859A was found in an African American patient with a group O forward type and a group B reverse type. 5'UTR-115C was the only RHD variant sequence found in a mixed-race black and Caucasian prenatal patient showing mixed-field agglutination with anti-D. 5'UTR-83T was found in several black donors and patients in the context of the genetically related RHCE*ceBI and RHCE*ceSM alleles. Reporter assays of promoter constructs including 5'UTR-115C or 5'UTR-83T showed a significant reduction in RH gene expression. Three new alleles in the ABO, RHD, and RHCE genes consist of single-nucleotide changes within GATA motifs, emphasizing the key role of GATA transcription factors in the expression of blood group genes.

A novel splice-site mutation in RHD gene associated with RhD negative phenotype

Introduction: The Rh antigens are encoded by the highly polymorphic RHD and the RHCE genes. Individuals with D variants may make anti-D alloantibodies, which in pregnant women cause severe or fatal hemolytic disease of the fetus and newborn. Accurate D typing using reliable routine methods must be performed. A number of unusual RHD alleles are being currently reported due in large part to the growing implementation of molecular analyses. Case Report: A sample from a Caucasian male blood donor at Northern Italy was serologically classified as RhD negative (Ccee). Instead, the sample was genotyped with all SSP-PCR kits as RhD positive, Dd and the result obtained with Partial D-TYPE (Dva or Va like or Va associated or DBS) was consistent with a suggestion of a new variant. Through a microarray platform analysis (BLOODchip Reference), the sample was genotyped as DV/DBS [RHD-CE(S)-D hybrid], associated with a predicted partial D phenotype for the RhD group, due to the lack of a hybridization signal for RHD exon 5. The PCR analysis of the exon 5 of the RHD gene gave a positive amplification result and the sequencing revealed a polymorphism in hemizygous or homozygous state in position RHD*IVS5+1t. This novel splice-site mutation alters or completely abolishes the specific sequence where the splicing of RHD gene intron 5 takes place. Conclusion: The novel RHD mutation hereby described highlights the significance of using RHD genotyping to confirm and/or clarify D antigen uncertainties in order to prevent the unnecessary immunization of patients.

The DAU cluster: a comparative analysis of 18 RHD alleles, some forming partial D antigens.

The Rh system is the most complex and polymorphic blood group system in humans with more than 460 alleles known for the RHD gene. The DAU cluster of RHD alleles is characterized by the single-nucleotide change producing the p.Thr379Met amino acid substitution. It is called the DAU-0 allele and has been postulated to be the primordial allele, from which all other alleles of the DAU cluster have eventually evolved. For two novel DAU alleles, the nucleotide sequences of all 10 exons as well as adjacent intronic regions, including the 5' and 3' untranslated regions (UTR), were determined for the RHD and RHCE genes. A phylogenetic tree for all DAU alleles was established using the neighbor-joining method with Pan troglodytes as root. Standard hemagglutination and flow cytometry tests were performed. We characterized two DAU alleles, DAU-11 and DAU-5.1, closely related to DAU-3 and DAU-5, respectively. A phylogenetic analysis of the 18 known DAU alleles indicated point mutations and interallelic recombination contributing to diversification of the DAU cluster. The DAU alleles encode a group of RhD protein variants, some forming partial D antigens known to permit anti-D in carriers; all are expected to cause anti-D alloimmunization in recipients of red blood cell transfusions. The DAU alleles evolved through genomic point mutations and recombination. These results suggest that the cluster of DAU alleles represent a clade, which is concordant with our previous postulate that they derived from the primordial DAU-0 allele.

A D+ blood donor with a novel RHD*D-CE(5-6)-D gene variant exhibits the low-frequency antigen RH23 (D(W) ) characteristic of the partial DVa phenotype.

Blood donors whose red blood cells (RBCs) exhibit a partial RhD phenotype, lacking some D epitopes, present as D+ in routine screening. Such phenotypes can exhibit low-frequency antigens (LFAs) of clinical significance. The aim of this study was to describe the serologic and genetic profile for a blood donor with an apparent D+ phenotype carrying a variant RHD gene where D Exons 5 and 6 are replaced by RHCE Exon (5-6). Anti-D monoclonal antibodies were used to characterize the presentation of RhD epitopes on the RBCs. RHD exon scanning and DNA sequencing of short- and long-range polymerase chain reaction amplicons were used to determine the RHD structure and sequence. Extended phenotyping for LFAs RH23 (D(W) ) and Rh32 was performed. The donor serology profile was consistent with partial RhD epitope presentation. The donor was hemizygous for an RHD variant allele described as RHD*D-CE(5-6)-D hybrid. The RHCE gene insert is at least 3.868 kb with 5' and 3' breakpoints between IVS4 + 132-c.667 and IVS6 + 1960-IVS6 + 2099, respectively. The sequence for this hybrid was assigned GenBank Accession Number KT099190.2. The RBCs were RH23 (D(W) )+ and Rh32-. A novel RHD*D-CE(5-6)-D hybrid allele encodes a partial RhD epitope and carries the LFA RH23 (D(W) ). This and the epitope profile resemble the partial DVa phenotype. Given that RBCs from this individual lack some RhD epitopes, there is an alloimmunization risk if the donor is exposed to D+ RBCs. Conversely, transfusions of RH23 (D(W) )+ cells to RH23 (D(W) )- recipients also pose an alloimmunization risk.