Rh Gene Research Articles

Identification of a novel RHCE*Ce (829G > A) allele associated with absence of C and e antigens expression.

The Rh blood group antigens are encoded by the RHD and RHCE genes, which possess a remarkable degree of polymorphism owing to their high homologous structures. These variants of the RH genes can lead to absence or weak expression of antigens. Analysis of RHCE genotyping by Polymerase Chain Reaction (PCR-SSP) method specific to detect c.48G, c.48C, 109 bp insertion of IVS2, c.201A and c.307C and RhCE phenotyping, were conducted in 316 Chinese patients in previous study. One patient with discrepancy typing result was collected for further RhCE serologic typing using microcolumn gel method and tube method in saline using monoclonal antibodies. PacBio sequencing was performed for RHCE, RHD and RHAG complete sequence analysis. 3D molecular models of the protein with the wild-type and mutant residue were generated using the DynaMut web server. The effect of the mutation on the protein function was predicted by PolyPhen-2 software. One male patient of Chinese Han was detected with RHCE*C allele showed by PCR-SSP method but ccEE phenotype. Further PacBio sequencing identified one normal RHCE*cE allele and one RHCE*Ce allele carried a novel c.829G > A (p.Gly277Arg) variant, which the encoded amino acid located in the ninth transmembrane segment of RhCE protein. Crystallisation analysis of 3D molecular models revealed that the substitution at Arg277 leads to the formation of additional hydrogen bonds, including weak hydrogen bonds between multiple atoms. It also results in hydrophobic ion interactions between Arg277 and Ala244. This mutation is predicted to have a damaging effect on protein function. One novel RHCE*Ce allele with c.829G > A (p.Gly277Arg) variant was identified to resulting in the absence or weak expression of C and e antigens.

First investigation of RH gene polymorphism in patients with sickle cell disease and associated blood donors in Cameroon, Central Africa.

Although genetic polymorphism of the RH blood group system is well known in sub-Saharan Africa, national/regional specificities still remain to be described precisely. For the first time in Cameroon, Central Africa, and in order to better characterize the molecular basis driving RH phenotype variability, as well as to identify the main antigens that may be potentially responsible for alloimmunization, we sought 1) to study the RH genes in a cohort of 109 patients with sickle cell disease; 2) to study the same genes in the corresponding donors whose red blood cells (RBCs) were transfused to the patients (108 donors in 98 patients); 3) to predict RH phenotype on the basis of the molecular data and compare the results with serologic testing; and 4) to identify retrospectively patients at risk for alloimmunization. In order to generate an exhaustive dataset, the RH genes of all patient and donor samples were systematically investigated 1) by quantitative multiplex PCR of short fluorescent fragments (QMPSF) for characterization of RHD gene zygosity and potential structural variants (SVs), and 2) by Sanger sequencing for identification of single nucleotide variants (SNVs). Subsequent to molecular analysis, the genotypes and RH phenotype were deduced and predicted, respectively, from reference databases. In a total of 217 Cameroonian individuals, as many as 24 and up to 22 variant alleles were identified in the RHD and RHCE genes, respectively, in addition to the reference alleles. Interestingly, 65 patients with SCD (66.3%) were assumed to be exposed to one or more undesirable RH antigen(s) with varying degrees of clinical relevance. Beyond the comprehensive report of the nature and distribution of RH variant alleles in a subset of Cameroonian patients treated by transfusion therapy, this work highlights the need for an extensive review of current practice, including routine serologic typing procedures, preferably in the near future.

Challenges in blood transfusion caused by anti-Hr0: A rare case of D-- Phenotype in Asia Abstract

Introduction D–Phenotype is linked to abnormal expression of RHCE gene. Consequently, individuals with this condition may develop antibodies against high-prevalence Rh antigens when exposed to a normal Rh phenotype, leading to challenges in blood matching.Case presentationA 90-year-old male was admitted to the hospital due to prostatitis and acute retention of urinary. Surgery was planned after evaluation by urologist. A request was made for 2 units of red blood cells (RBC). The result for unexpected antibody screening was positive, and the antibody was identified as anti-Hr0 alloantibody , a rare RHCE * CE-D (5) – CE was discovered by genetic testing. The patient has only one history of blood transfusion, which may result in the production of the anti-Hr0 antibody. Compatible blood donors for this patient were not found, even after extensive screening.ConclusionThis report records a rare case of a D-- phenotype individual who produced anti-Hr0 alloantibody. Subsequent analysis of RH gene sequencing revealed a genotype that has not been previously reported in Asia.

Integrated analyses reveal unexpected complex inversion and recombination in RH genes

AbstractPhenotype D-- is associated with severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. It is typically caused by defective RHCE genes. In this study, we identified a D-- phenotype proband and verified Rh phenotypes of other 6 family members. However, inconsistent results between the phenotypic analysis and Sanger sequencing revealed intact RHCE exons with no mutations in the D-- proband, but the protein was not expressed. Subsequent whole-genome sequencing by Oxford Nanopore Technologies of the proband revealed an inversion with ambiguous breakpoints in intron 2 and intron 7 and copy number variation loss in the RHCE gene region. Given that the RHCE gene is highly homologous to the RHD gene, we conducted a comprehensive analysis using Pacific Biosciences long-read target sequencing, Bionano optical genome mapping, and targeted next-generation sequencing. Our findings revealed that the proband had 2 novel recombinant RHCE haplotypes, RHCE∗Ce(1-2)-D(3-10) and RHCE∗Ce(1-2)-D(3-10)-Ce(10-8)-Ce(3-10), with clear-cut breakpoints identified. Furthermore, the RH haplotypes of the family members were identified and verified. In summary, we made, to our knowledge, a novel discovery of hereditary large inversion and recombination events occurring between the RHD and RHCE genes, leading to a lack of RhCE expression. This highlights the advantages of using integrated genetic analyses and also provides new insights into RH genotyping.

Transfusing children with sickle cell disease using blood group genotyping when the pool of Black donors is limited.

Red blood cell transfusion is an effective treatment for patients with sickle cell disease (SCD). Alloimmunization can occur after a single transfusion, limiting further usage of blood transfusion. It is recommended to match for the ABO, D, C, E, and K antigens to reduce risks of alloimmunization. However, availability of compatible blood units can be challenging for blood providers with a limited number of Black donors. A prospective cohort of 205 pediatric patients with SCD was genotyped for the RH and FY genes. Transfusion and alloimmunization history were collected. Our capacity to find RhCE-matched donors was evaluated using a database of genotyped donors. Nearly 9.8% of patients carried a partial D variant and 5.9% were D-. Only 45.9% of RHCE alleles were normal, with the majority of variants affecting the RH5 (e) antigen. We found an alloimmunization prevalence of 20.7% and a Rh alloimmunization prevalence of 7.1%. Since Black donors represented only 1.40% of all blood donors in our province, D- Caucasian donors were mostly used to provide phenotype matched products. Compatible blood for patients with rare Rh variants was found only in Black donors. A donor with compatible RhCE could be identified for all patients. Although Rh-compatible donors were identified, blood units might not be available when needed and/or the extended phenotype or ABO group might not match the patient. A greater effort has to be made for the recruitment of Black donors to accommodate patients with SCD.

Determination of Rh type and gender using circulating cell-free fetal DNA in early pregnancy of Rh negative women in turkey.

Choosing the right clinical approach for early and reliable diagnosis/screening is becoming more important day by day. The aim of the study was to determine the early RhD type with cff-DNA obtained from maternal plasma, especially in the light of recent developments. In this way, it is aimed to apply Rh Ig only to mothers who are determined to have RhD (+) fetuses and to prevent unnecessary further tests that may possess a risk for RhD (-) fetuses. Prediction of fetal gender and RH genotype was performed by using RT-qPCR method. With simultaneous amplification of sequences of SRY, DYS14 and RH genes (exon 7 and exon 10). Fetal gender and RhD were determined in 30 RHD (-) pregnant women with cfDNA. As a result of genotyping, the gender of 67% (20/30) fetuses was determined as male; the gender of 33% (10/30) fetuses was determined as female in a sample group of 30 pregnancies. It was determined that the DYS14 100% (20/20) gene was more sensitive than the SRY 97% (18/20) gene in gender determination after examining prenatal and postnatal results. As a result of the analysis, the presence of 17% (5/30) RhD (-) fetuses and 83% (25/30) RhD (+) fetuses were determined which is 100% compatible with postnatal results. Detecting fetal RhD gene in maternal plasma made an important contribution to its use in non-invasive prenatal screening. This study shows that unnecessary intervention and cost can be avoided with successful genotyping analysis performed with RT-qPCR.

Hybrids and microconversions in RH genes: investigation and implication in transfusion therapy

: The discovery and characterization of the RH genes, i.e., RHD and RHCE, in the early 90s have been milestones in the field of blood group genetics. In addition to the single nucleotide variants (SNVs), which are commonly considered as the predominant molecular actors resulting in the variability of antigen expression, genetic exchange between both RH genes has been shown to be a frequent mutational mechanism. Indeed, dozens of hybrid alleles and microconversions associated with variant RH phenotype have been reported so far. Interestingly, these alleles display population-specific patterns of distribution. Also, they are directly involved in the qualitative and quantitative alteration of RH antigen expression, which has immediate consequences for diagnostics and transfusion management. Overall, hybrid alleles and microconversions contribute largely to the complexity of the RH blood group system. In the current state of knowledge, this narrative review: (I) defines and introduces the molecular determinants of the genetic exchange in the RH genes; (II) describes how these variant alleles may alter the expression of polymorphic and high-frequency antigens (HFAs) and/or result in the expression of low-frequency antigens (LFAs); (III) reports on their distribution at a low-resolution population scale; and (IV) discusses their relevance and identification in routine clinical practice for transfusion management at the laboratory level.

Characterisation of the Rh50 protein from the ammonia-oxidising bacterium Nitrosomonas europaea

The transport of ammonium through the cell membranes, an essential process in all kingdoms of life, is accomplished by the ubiquitous Amt/Mep/Rh superfamily of proteins. The functional context of Amt/Mep and Rh transporters is diverse: bacteria, fungi, and plants use Amt/Mep proteins to scavenge ammonium for biosynthetic assimilation, whereas mammals use the Rh proteins for ammonium detoxification in erythrocytes, kidney, and liver tissues. While RH50 genes are widespread in eukaryotes they are present in some prokaryotes: an example is a chemolithoautotroph Nitrosomonas europaeawhich gains all its energy from the oxidation of ammonia to nitrate. While Amt/Mep/Rh proteins have divergent physiological functions, they are structurally very similar, which raises the important question about the universality of the transport mechanism. We have recently proposed an elegant new model for the mechanism of electrogenic ammonium transport in bacteria Amt protein: after deprotonation of NH4+ at the periplasmic side of the transporter, a previously undiscovered polar conduction route enables H+ transfer into the cytoplasm. A parallel pathway, lined by hydrophobic groups within the protein core, facilitates the simultaneous transfer of uncharged NH3. In this context, we propose to elucidate at the molecular level the mechanism of ammonium translocation through rhesus protein from Nitrosomonas europaeaand establish whether there is a universal mechanism for biological ammonium transport. Beyond the elucidation of a central biological process, this work has important medical implications, as some Rh mutations have been associated with human pathologies. We propose to demonstrate how specific Rh mutations affect the activity of the protein to establish the relationship between Rh malfunction and the associated diseases.

RHD Genotyping of Rh-Negative and Weak D Phenotype among Blood Donors in Southeast Iran

Background: The D antigen is a subset of Rh blood group antigens involved in the hemolytic disease of the newborn [HDFN] and hemolytic transfusion reaction [HTR]. The hybrid Rhesus box that was created after RH gene deletion, was known as a mechanism of the Rh-negative phenotype. Hybrid marker identification is used to confirm the deletion of the RHD gene and to determine zygosity. This study aims to detect this marker in Rh-negative and weak D phenotype blood donors of the southeast of Iran. Materials and Methods: The molecular analysis of the hybrid Rhesus box was performed on the 200 Rh-negative blood donors in Sistan and Baluchestan province, southeast Iran. The presence of alleles responsible for the D variants was assessed by DNA sequencing in 26 weak D phenotype donors. Results: Of the 200 Rh-negative blood samples, 198 samples were homozygous (99%), and two samples were heterozygous (1%). Heterozygous samples had RHD*01N.73 allele and the RHD*01N.18 allele. Of the 26 samples with weak D phenotype, 16 partial DLO (61%), 4 partial DBT1 (15.3%), 2 partial DV type 2 (7.7%), 1 weak D type 1, 1 weak D type 4.2.3, 1weak D type 105 and 1 RHD (S103P) (4%) were determined. Conclusion: Since RHD gene deletion is the main mechanism of the Rh-negativity in Sistan and Baluchestan provinces, a hybrid Rhesus box marker can be used in resolving RhD typing discrepancies by RHD genotyping methods.

Association of the blood groups with mostly public Diseases

The ABO blood group system is identified in 1900. Its principle that antigens as sugars physically exposed on the exterior of red blood cells *RBCs* differ between individuals, who have immunological tolerance only toward what occurs in their own bodies. As a result, many humans express isoantibodies-antibodies against isoantigens, natural components present in the bodies of other members of the same species but not themselves. Isoantibodies may be present against the A and/or B antigens in people who do not themselves have the same antigens in their own blood. These antibodies act as haemagglutinins, which cause blood cells to clump and break apart if they carry the foreign antigens, because A and B antigens are chemically modified of a precursor of that is also present in type O individuals, people with type A and B antigens can accept blood of type O individuals; identification of ABO and Rh gene frequencies among human populations has various benefits in transfusion medicine, transplantation and disease risk. The food, bacterial, viral, or plant antigens have epitopes similar enough to A and B glycoprotein antigens lead to create antibodies in the first years of life, Anti-A antibodies are originate of immune response towards influenza virus, Anti-B antibodies are originate of antibodies produced against Gram-negative bacteria.The ABO antigen is expressed on the von Willebrand factor (vWF) glycoprotein which participates in control of bleeding; individuals with group O predisposes to bleeding as 30% of the total genetic variation due to this group normally have significantly lower plasma levels of vWF (and Factor VIII) than other blood groups. The individuals with A1 AB, and B blood groups have a 14% reduced risk of squamous cell carcinoma; B antigen links with increased risk of ovarian cancer; type O blood is associated with a reduced risk of pancreatic cancer but increased risk of peptic ulcer and infection with cholera while gastric cancer has reported to be more common in blood group A especially in patient with Helicobacter pylori infection. The blood types A and AB are associated with an increased risk of nasopharyngeal carcinoma; in 2019, a recently evidence showed a higher risk for renal cell cancer was found in non-O blood group women, but not in men. Many studies indicate the individuals with group A and B are less likely to have Diabetes Mellitus than group AB.

Customized Induced Pluripotent Stem Cell-Derived Red Cell Reagents

Red blood cell transfusion is an essential therapy for patients with sickle cell disease (SCD). A major problem is the complexity of antibodies found in patients with SCD, in large part due to RH gene variation. Rh antibodies are common despite provision of serologic Rh-matched transfusions. The antibodies may be mistaken for autoantibodies when in fact they are alloantibodies to high prevalence Rh antigens that are absent or have altered Rh epitopes on the patient's RBCs. Finding compatible red cell units is often complicated by a lack of reagent red cells to properly identify the fine antibody specificity. This can delay patient care, increase costs, and make transfusion therapy impossible for some patients. We hypothesized that human induced pluripotent stem cells (iPSCs) could be reprogrammed from rare donors or genetically engineered to produce standard and reliable red cell reagents to aid identification for patients with complex antibodies. However, most human iPSC-derived red blood cells (iRBCs) are developmentally primitive red cells that differ from donor-derived red cells with high levels of embryonic globin expression, are larger in size and fail to enucleate in culture. We aimed to improve iPSC hematopoietic differentiation protocols to generate iRBCs compatible with standard blood bank assays.We designed a panel of customized iPSCs that included 1) Rh null cells, 2) cells lacking the high prevalence Rh antigen termed hrS, 3) cells expressing a partial C antigen and lacking the high prevalence Rh antigen termed hrB and, 4) cells expressing the low prevalence Rh antigens V, VS and lacking hrB. For the Rh null line, we identified wild type iPSC lines that were group O, RhD- by homozygous RHD deletion. We used CRISPR/Cas9 genetic engineering to disrupt the RHCE alleles either by a large deletion from exon 1 to exon 2 and/or via insertion-deletion mutations resulting in a frameshift and early stop codon. For lines lacking high prevalence Rh antigens, we reprogrammed group O donor cells whose genotypes were heterozygous for RHD*DAU0/RHCE*ceMO and RHD*DOL/RHCE*ceBI, homozygous for RHD*DIIIa-CE(4-7)-D/RHCE*ceS, and homozygous for RHD/RHCE*ce733G, respectively. At least 2 independent clones from each customized line were characterized for pluripotency by standard flow cytometry and gene expression markers, and a normal karyotype confirmed.We performed hematopoietic differentiation of customized iPSCs by a 3-step embryoid body protocol in defined, serum-free media with combinations of hematopoietic cytokines. We obtained CD41+235+ primitive hematopoietic progenitors on day 8 and cultured these cells in liquid culture with erythroid specific cytokines to generate iRBCs. We assessed erythroid cell maturation by morphology on cytospin preparations and by flow cytometry. We found an almost pure population of CD71+235+ iRBCs with erythroblast morphology by day 6 of culture, and progressive maturation to Band 3 high, alpha integrin low iRBCs that were smaller and had condensed nuclei that resemble erythroid cultures of primary CD34+ cells. Comparison of total Rh protein expression by flow cytometry on untargeted group O, D+ iRBCs compared to donor-derived RBCs demonstrated comparable cell surface Rh expression. As proof of principal, we assessed RBC agglutination by gel card assay of untargeted group O, D+, E-, e+ and the genetically engineered Rh null iRBCs using standard anti-D, -E, and -e reagents for Rh typing (Ortho Bioclone). As expected, the untargeted O, D+ iRBCs agglutinated with anti-D and anti-e while the engineered Rh-null iRBCs showed no agglutination with all 3 antibodies. Only 0.5 e6 iRBCs were required per gel assay.We have designed a panel of customized iPSCs reprogrammed from rare donors or genetically engineered to express rare blood group antigen phenotypes or combinations that are difficult or impossible to find as donor red cells. Any number of combinations not found in natural populations can be produced and generated in quantities sufficient for reagents. iRBC produced from these customized iPSCs can be used with standard blood bank assays and potentially provide the means to streamline and standardize antibody identification in alloimmunized patients with complex antibody specificities. In the future, when technology for scale-up is available, Rh null iRBCs could be used as “universal” donor cells for future therapeutic applications. DisclosuresNo relevant conflicts of interest to declare.

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.

Ammonia Stress Coping Strategy in a Highly Invasive Ascidian

The outbreak of invasive ascidianMolgula manhattensishas negatively affected marine and coastal ecosystems and caused huge economic loss in various industries such as aquaculture. In mariculture systems usually characterized by high ammonia nitrogen, the capacity ofM. manhattensisto defend against drastic ammonia elevation plays a crucial role in its survival and subsequent invasions. However, ammonia coping strategies and associated genes/proteins remain largely unknown. Here we investigated rhesus glycoproteins (Rh)-mediated ammonia transport by identifying all Rh proteins and exploring their mRNA expression regulations under ammonia stress. Three types of primitive Rh proteins were identified, and all contained conserved amino acid residues and functional domains. Ammonia stress largely suppressed the expression of immune-related genes, but rapidly induced the increased expression of Rh genes. Ammonia was converted into glutamine as indicated by the increased expression of glutamine synthetase gene, rather than urea as illustrated by the stable expression of arginase gene. Collectively,M. manhattensismitigates ammonia challenge by enhancing ammonia excretion through Rh channels and detoxifying ammonia into glutamine. Our results provide insights into the molecular mechanisms underlying high tolerance and invasion success to high ammonia environments by invasive ascidians.

MYB30 and ETHYLENE INSENSITIVE3 antagonistically modulate root hair growth in Arabidopsis.

Root hair (RH) is essential for plant nutrient acquisition and the plant-environment communication. Here we report that transcription factors MYB30 and ETHYLENE INSENSITIVE3 (EIN3) modulate RH growth/elongation in Arabidopsis in an antagonistic way. The MYB30 loss-of-function mutant displays enhanced RH length, whereas the RH elongation in MYB30-overexpressing plants is highly repressed. MYB30 physically interacts with EIN3, a master transcription factor in ethylene signaling. MYB30 directly binds the promoter region of ROOT HAIR DEFECTIVE SIX-LIKE4 (RSL4) and represses its transcription. RSL4 loss-of-function suppresses the enhanced RH growth in myb30 mutant plants. Ethylene enhances MYB30-EIN3 complex formation, and reduces the association between MYB30 and RSL4 promotor via the action of EIN3. MYB30 and EIN3 antagonistically regulate the expression of RSL4 and a subset of core RH genes in a genome-wide way. Taken together, our work revealed a novel transcriptional network that modulates RH growth in plants.

Blood group systems

Blood group systems

Серологічно слабкий D-фенотип: огляд та інтерпретація групи крові RhD

The Rhesus system is the second most important erythrocyte system for transfusion after ABO. Accurate determination of the Rhesus status of the donor, recipient, pregnant allows to prevent the development of post-transfusion hemolytic complications of the fetus or newborn associated with incompatibility of the blood of the mother and the fetus by D antigen. Generally, determination of the Rhesus affiliation of a person is performed by serological methods using anti-Rhesus reagents with full or incomplete antibodies. However, the results of serological studies are not always clear. Mutations and other effects of the RH gene locus disrupt the production of the normal D antigen and lead to the emergence of numerous varieties of antigen D. The variant of antigen D was described in 1946 and was designated as Du. The study showed that the differences between Du antigen and normal D were quantitative rather than qualitative. The Du antigen was later designated as Dweak - a weak D-antigen or a weak D-phenotype. In the early 1950s, anti-D antibodies were detected in recipients with a weak D-antigen after transfusion with Rh-positive blood and in pregnant women with the Du phenotype during pregnancy and at the birth of a D+ baby. It was suggested that the D antigen was not homogeneous and consisted of numerous partial variants: D1, D2, D3, etc. A complete set of partial variants corresponds to a complete D-antigen. The absence of any of one or more partial factors leads to the appearance of attenuated forms of the D antigen, denoted as Dpartial. People lacking certain partial antigens can produce anti-D antibodies against them. Differentiation of weak D phenotypes has great clinical importance, because transfusion of Rh-erythrocytes to recipients with Dweak and who are actually Rh-positive, has no scientific justification and leads to unjustified consumption of deficient blood and unnecessary immunoprophylaxis of anti-Rh immunoglobulin to pregnant women. International laboratory practice has no unified policy regarding the diagnosis of weak variants of D antigen and the interpretation of the results. Polymerase chain reaction allows to accurately define the Rh status of an individual and to avoid unreasonable transfusions of Rh-negative blood and unnecessary immunoprophylaxis

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.

An Interesting Case of Blood Group Switch after Breast Cancer Therapy

Blood groups are determined by specific antigens attached to red blood cells that are either carbohydrate or protein in nature. Changes in gene expression encoding for various blood types are rare, with the ABO blood group antigen most commonly altered. We report a female patient whose blood group changed from A, Rh positive to A, Rh negative, over the period of 10 years (1999-2009). During this time period, she was diagnosed with breast carcinoma and a lumpectomy was performed, followed by chemotherapy (with anthracycline and docetaxel) along with local irradiation. There has been no remission after recovery. It was noticed that the patient’s blood group had changed. A possible reason may be Rh mosaicism and a mutation in the Rh gene, resulting in the alteration of Rh antigen expression. The reason causing this mutation is unclear. However, in some cases changes in blood groups are related to patients’ chemotherapy treatment and their remission status.

The Rhesus glycoprotein Rhcgb is expendable for ammonia excretion and Na+ uptake in zebrafish (Danio rerio)

In zebrafish (Danio rerio), the ammonia-transporting Rhesus glycoprotein Rhcgb is implicated in mechanisms of ammonia excretion and Na+ uptake. In particular, Rhcgb is thought to play an important role in maintaining ammonia excretion in response to alkaline conditions and high external ammonia (HEA) exposure, in addition to facilitating Na+ uptake via a functional metabolon with the Na+/H+-exchanger Nhe3b, specifically under low Na+ conditions. In the present study, we hypothesized that CRISPR/Cas9 knockout of rhcgb would reduce ammonia excretion and Na+ uptake capacity, particularly under the conditions listed above that have elicited increases in Rhcgb-mediated ammonia excretion and/or Na+ uptake. Contrary to this hypothesis, however, larval and juvenile rhcgb knockout (KO) mutants showed no reductions in ammonia excretion or Na+ uptake under any of the conditions tested in our study. In fact, under control conditions, rhcgb KO mutants generally displayed an increase in ammonia excretion, potentially due to increased transcript abundance of another rh gene, rhbg. Under alkaline conditions, rhcgb KO mutants were also able to maintain ammonia excretion, similar to wild-type fish, and stimulation of ammonia excretion after HEA exposure also was not affected by rhcgb KO. Surprisingly, ammonia excretion and Na+ uptake were unaffected by rhcgb or nhe3b KO in juvenile zebrafish acclimated to normal (800 μmol/L) or low (10 μmol/L) Na+ conditions. These results demonstrate that Rhcgb is expendable for ammonia excretion and Na+ uptake in zebrafish, highlighting the plasticity and flexibility of these physiological systems in this species.

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.