CD59 gene: 143 haplotypes of 22,718 nucleotides length by computational phasing in 113 individuals from different ethnicities.

BACKGROUND: CD59 deficiency due to rare germline variants in the CD59 gene causes disabilities, ischemic strokes, neuropathy, and hemolysis. CD59 deficiency due to common somatic variants in the PIG-A gene in hematopoietic stem cells causes paroxysmal nocturnal hemoglobinuria. The ISBT database lists one nonsense and three missense germline variants that are associated with the CD59-null phenotype. To analyze the genetic diversity of the CD59 gene, we determined long-range CD59 haplotypes among individuals from different ethnicities. METHODS: We determined a 22.7 kb genomic fragment of the CD59 gene in 113 individuals using next-generation sequencing (NGS), which covered the whole NM_203330.2 mRNA transcript of 7796 base pairs. Samples came from an FDA reference repository and our Ethiopia study cohorts. The raw genotype data were computationally phased into individual haplotype sequences. RESULTS: Nucleotide sequencing of the CD59 gene of 226 chromosomes identified 216 positions with single nucleotide variants. Only three haplotypes were observed in homozygous form, which allowed us to assign them unambiguously as experimentally verified CD59 haplotypes. They were also the most frequent haplotypes among both cohorts. An additional 140 haplotypes were imputed computationally. DISCUSSION: We provided a large set of haplotypes and proposed three verified long-range CD59 reference sequences, based on a population approach, using a generalizable rationale for our choice. Correct long-range haplotypes are useful as template sequences for allele calling in high-throughput NGS and precision medicine approaches, thus enhancing the reliability of clinical diagnostics. Long-range haplotypes can also be used to evaluate the influence of genetic variation on the risk of transfusion reactions or diseases.

Neutrophil antigen antibodies affect engraftment and secondary graft failure in hematopoietic progenitor cell transplantation.

BACKGROUND: Research is limited on the role of antibodies against human neutrophil antigen (HNA) in hematopoietic progenitor cell (HPC) transplantation outcomes. STUDY DESIGN AND METHODS: A retrospective review was conducted on medical records of patients at the NIH Clinical Center enrolled in six research protocols. This case-control study included 21 patients tested for HNA antibodies from January 2010 to March 2022 who underwent HPC transplantation. In addition, 42 patients following the same research protocols were randomly selected as a control group. RESULTS: The cumulative incidence of time to neutrophil engraftment was significantly impacted by the patients' anti-HNA status (p = .042), with the patients with anti-HNA experiencing delayed engraftment. Secondary graft failure occurred in 4 out of 42 patients (9.52%; 95% confidence interval [CI]: 3.7-22.1) of the control group, while 5 out of 9 patients (55.5%; 95% CI: 26.7-81.1) with anti-HNA experienced secondary graft failure (p = .005). Furthermore, patients with anti-HNA had a lower proportion (p = .008 for full and p = .002 for partial chimerism) and cumulative incidence (p = .016 for full and p = .010 for partial chimerism) of achieving donor chimerism compared to the control group. DISCUSSION: The study reveals a potential link between anti-HNA and HPC transplantation outcomes not previously reported. Patients with anti-HNA had a lower proportion and cumulative incidence of achieving donor chimerism. Additionally, anti-HNA status affected the time for neutrophil engraftment, with a slower rate of neutrophil engraftment and increased risk of secondary failure in patients with anti-HNA.

Rh flow cytometry: An updated methodology for D antigen density applied to weak D types 164 and 165.

BACKGROUND: An original methodology for determining the D antigen density on red cells was published in 2000 and has been applied in many publications since. This flow cytometry-based assay remained largely unrevised utilizing monoclonal anti-Ds that are not readily available anymore. We updated the methodology to quantify erythrocyte D antigen sites using microspheres and monoclonal anti-Ds that are commercially available today. METHODS: The absolute D antigen density of a frozen standard CcDEe cell, drawn in 2003, a fresh blood donation from the same individual, drawn in 2022, and an internal control CcDEe cell, was quantified by flow cytometry using fluorescence-labeled microspheres. The internal control CcDEe cell was used in conjunction with 9 commercial anti-Ds to determine D antigen densities of 7 normal D, 4 partial D, and 11 weak D type samples, including 2 novel alleles. RESULTS: The reproducibility of the updated assay was evaluated with red cells of published D antigen densities. The current results matched the known ones closely. The new weak D types 164 and 165 carried 4500 and 1505 D antigens/red cell, respectively. The absolute D antigen density decreased from 27,231 to 26,037 in an individual over 19 years. DISCUSSION: The updated assay gave highly reproducible results for the D antigen densities of Rh phenotypes. Readily available anti-Ds allowed for the determination of the D antigen densities of 7 weak D types. The assay is suitable to evaluate the effects of distinct amino acid substitutions on the RhD phenotype.

Asian-type DEL (RHD*DEL1) with an allo-anti-D: A paradoxical observation in a healthy multiparous woman.

BACKGROUND: The DEL phenotype is the D variant expressing the least amounts of D antigen per red cell. Asian-type DEL (RHD:c:1227G > A) is the most prevalent DEL in East Asia without any anti-D alloimmunization reported before. We investigated the first observation of an anti-D in any DEL phenotype, reported in the Japanese language at a 1987 conference, only 3 years after the discovery of DEL. METHODS: We contacted the proband 35 years after the initial report. Standard hemagglutination, adsorption/elution, and flow cytometry tests were performed, as was nucleotide sequencing for the RHD, RHCE, and HLA class I and class II genes. RESULTS: The healthy multiparous Japanese woman, a regular blood donor, still had the anti-D of titer 8 representing an alloantibody by standard serologic methods. Unexpectedly, she carried an Asian-type DEL without any additional RHD gene variation. All 12 HLA alleles identified were known in the Japanese population. Interestingly, one of her HLA-DRB1 and a variant of her HLA-DQB1 alleles had previously been associated with anti-D immunization. CONCLUSION: We described an allo-anti-D, maintained for more than three decades, in an Asian-type DEL. The combination of two implicated HLA alleles were rare and could have contributed to the anti-D immunization. Continued monitoring of anti-D immunization events in patients with DEL is warranted, and we discuss possible mechanisms for further study. As only this single observation has been recognized in the last 35 years, the current recommendation is affirmed: Individuals with Asian-type DEL should be treated as Rh D-positive for transfusion and Rh immune prophylaxis purposes.

Patients with Asian-type DEL can safely be transfused with RhD-positive blood.

Red blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; none of the recipients (0/42; 95% confidence interval, 0-8.40) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4045 serologic D- pregnant women throughout China; alloanti-D was found only in individuals with true D- (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

BACKGROUND AND OBJECTIVES: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. MATERIALS AND METHODS: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. RESULTS: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. CONCLUSION: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

DEL in China: the D antigen among serologic RhD-negative individuals.

BACKGROUND: Providing RhD-negative red cell transfusions is a challenge in East Asia, represented by China, Korea, and Japan, where the frequency of RhD-negative is the lowest in the world. FINDINGS: Among 56 ethnic groups in China, the RhD-negative frequency in Han, the prevalent ethnicity, is 0.5% or less, similar to most other ethnic groups. The Uyghur ethnic group has the highest reported RhD-negative frequency of up to 4.7%, as compared to 13.9% in the US. However, an estimated 7.15 million RhD-negative people live in China. The RhD-negative phenotype typically results from a loss of the entire RHD gene, causing the lack of the RhD protein and D antigen. The DEL phenotype carries a low amount of the D antigen and types as RhD-negative in routine serology. The DEL prevalence in RhD-negative individuals averages 23.3% in the Han, 17% in the Hui and 2.4% in the Uyghur ethnicities. The Asian type DEL, also known as RHD*DEL1 and RHD:c.1227G > A allele, is by far the most prevalent among the 13 DEL alleles observed in China. CONCLUSION: The purpose of this review is to summarize the data on DEL and to provide a basis for practical strategy decisions in managing patients and donors with DEL alleles in East Asia using molecular assays.

Cataloguing experimentally confirmed 80.7 kb-long ACKR1 haplotypes from the 1000 Genomes Project database.

BACKGROUND: Clinically effective and safe genotyping relies on correct reference sequences, often represented by haplotypes. The 1000 Genomes Project recorded individual genotypes across 26 different populations and, using computerized genotype phasing, reported haplotype data. In contrast, we identified long reference sequences by analyzing the homozygous genomic regions in this online database, a concept that has rarely been reported since next generation sequencing data became available. STUDY DESIGN AND METHODS: Phased genotype data for a 80.6 kb region of chromosome 1 was downloaded for all 2,504 unrelated individuals of the 1000 Genome Project Phase 3 cohort. The data was centered on the ACKR1 gene and bordered by the CADM3 and FCER1A genes. Individuals with heterozygosity at a single site or with complete homozygosity allowed unambiguous assignment of an ACKR1 haplotype. A computer algorithm was developed for extracting these haplotypes from the 1000 Genome Project in an automated fashion. A manual analysis validated the data extracted by the algorithm. RESULTS: We confirmed 902 ACKR1 haplotypes of varying lengths, the longest at 80,584 nucleotides and shortest at 1,901 nucleotides. The combined length of haplotype sequences comprised 19,895,388 nucleotides with a median of 16,014 nucleotides. Based on our approach, all haplotypes can be considered experimentally confirmed and not affected by the known errors of computerized genotype phasing. CONCLUSIONS: Tracts of homozygosity can provide definitive reference sequences for any gene. They are particularly useful when observed in unrelated individuals of large scale sequence databases. As a proof of principle, we explored the 1000 Genomes Project database for ACKR1 gene data and mined long haplotypes. These haplotypes are useful for high throughput analysis with next generation sequencing. Our approach is scalable, using automated bioinformatics tools, and can be applied to any gene.

Pharmacogenomics with red cells: a model to study protein variants of drug transporter genes.

The PharmacoScan pharmacogenomics platform screens for variation in genes that affect drug absorption, distribution, metabolism, elimination, immune adverse reactions and targets. Among the 1,191 genes tested on the platform, 12 genes are expressed in the red cell membrane: ABCC1, ABCC4, ABCC5, ABCG2, CFTR, SLC16A1, SLC19A1, SLC29A1, ATP7A, CYP4F3, EPHX1 and FLOT1. These genes represent 5 ATP-binding cassette proteins, 3 solute carrier proteins, 1 ATP transport protein and 3 genes associated with drug metabolism and adverse drug reactions. Only ABCG2 and SLC29A1 encode blood group systems, JR and AUG, respectively. We propose red cells as an ex vivo model system to study the effect of heritable variants in genes encoding the transport proteins on the pharmacokinetics of drugs. Altered pharmacodynamics in red cells could also cause adverse reactions, such as haemolysis, hitherto unexplained by other mechanisms.

Long-range haplotype analysis of the malaria parasite receptor gene ACKR1 in an East-African population.

The human ACKR1 gene encodes a glycoprotein expressing the Duffy blood group antigens (Fy). The Duffy protein acts as a receptor for distinct pro-inflammatory cytokines and malaria parasites. We determined the haplotypes of the ACKR1 gene in a population inhabiting a malaria-endemic area. We collected blood samples from 60 healthy volunteers in Ethiopia's southwestern low-altitude tropical region. An assay was devised to amplify the ACKR1 gene as a single amplicon and determine its genomic sequence. All haplotypes were resolved at 5178 nucleotides each, covering the coding sequence (CDS) of the ACKR1 gene and including the 5'- and 3'-untranslated regions (UTR), intron 1, and the 5'- and 3'-flanking regions. When necessary, allele-specific PCR with nucleotide sequencing or length polymorphism analysis was applied. Among the 120 chromosomes analyzed, 18 ACKR1 alleles were confirmed without ambiguity. We found 18 single-nucleotide polymorphisms (SNPs); only one SNP was novel. The non-coding sequences harbored 14 SNPs. No SNP, other than c.-67T>C, indicative of a non-functional allele, was detected. We described haplotypes of the ACKR1 gene in an autochthonous East-African population and found 18 distinct ACKR1 alleles. These long-range alleles are useful as templates to phase and analyze next-generation sequencing data, thus enhancing the reliability of clinical diagnostics.