Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion.

Next-generation sequencing (NGS) technologies have transformed medical genetics. However, short-read lengths pose a limitation on identification of structural variants, sequencing repetitive regions, phasing of distant nucleotide changes, and distinguishing highly homologous genomic regions. Long-read sequencing technologies may offer improvements in the characterization of genes that are currently difficult to assess. We used a combination of targeted DNA capture, long-read sequencing, and a customized bioinformatics pipeline to fully assemble the RH region, which harbors variation relevant to red cell donor-recipient mismatch, particularly among patients with sickle cell disease. RHD and RHCE are a pair of duplicated genes located within an ∼175 kb region on human chromosome 1 that have high sequence similarity and frequent structural variations. To achieve the assembly, we utilized palindrome repeats in PacBio SMRT reads to obtain consensus sequences of 2.1 to 2.9 kb average length with over 99% accuracy. We used these long consensus sequences to identify 771 assembly markers and to phase the RHD-RHCE region with high confidence. The dataset enabled direct linkage between coding and intronic variants, phasing of distant SNPs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints. A limiting factor in phasing is the frequency of heterozygous assembly markers and therefore was most successful in samples from African Black individuals with increased heterogeneity at the RH locus. Overall, this approach allows RH genotyping and de novo assembly in an unbiased and comprehensive manner that is necessary to expand application of NGS technology to high-resolution RH typing.

RH genotypes and red cell alloimmunization rates in chronically transfused patients with sickle cell disease: A multisite study in the USA.

BACKGROUND: Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part, due to a high frequency of variant RH alleles in patients with SCD and Black blood donors. STUDY DESIGN AND METHODS: We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the USA. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays. RESULTS: Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5% to 41% (p = .0035) and 5%-33% (p = .0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p = .0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens. DISCUSSION: These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.

ABO Genotyping finds more A2 to B kidney transplant opportunities than lectin-based subtyping.

ABO compatibility is important for kidney transplantation, with longer waitlist times for blood group B kidney transplant candidates. However, kidneys from non-A1 (eg, A2) subtype donors, which express less A antigen, can be safely transplanted into group B recipients. ABO subtyping is routinely performed using anti-A1 lectin, but DNA-based genotyping is also possible. Here, we compare lectin and genotyping testing. Lectin and genotype subtyping was performed on 554 group A deceased donor samples at 2 transplant laboratories. The findings were supported by 2 additional data sets of 210 group A living kidney donors and 124 samples with unclear lectin testing sent to a reference laboratory. In deceased donors, genotyping found 65% more A2 donors than lectin testing, most with weak lectin reactivity, a finding supported in living donors and samples sent for reference testing. DNA sequencing and flow cytometry showed that the discordances were because of several factors, including transfusion, small variability in A antigen levels, and rare ABO∗A2.06 and ABO∗A2.16 sequences. Although lectin testing is the current standard for transplantation subtyping, genotyping is accurate and could increase A2 kidney transplant opportunities for group B candidates, a difference that should reduce group B wait times and improve transplant equity.

Variant RHD alleles and Rh immunization in patients with sickle cell disease.

RH diversity among patients and donors contributes to Rh immunization despite serologic Rh-matched red cell transfusions. Anti-D can occur in D+ patients with RHD variants that encode partial D antigens. Anti-D has also been reported in patients with conventional RHD transfused primarily with units from Black donors who frequently have variant RHD. We report 48 anti-D in 690 D+ transfused individuals with sickle cell disease, categorized here as expressing conventional D, partial D or D antigen encoded by RHD*DAU0. Anti-D formed in a greater proportion of individuals with partial D, occurred after fewer D+ unit exposures, and remained detectable for longer than for those in the other categories. Among all anti-D, 13 had clinical or laboratory evidence of poor transfused red cell survival. Most individuals with anti-D were chronically transfused, including 32 with conventional RHD who required an average of 62 D- units/year following anti-D. Our findings suggest that patients with partial D may benefit from prophylactic D- or RH genotype-matched transfusions to prevent anti-D. Future studies should investigate whether RH genotype-matched transfusions can improve use of valuable donations from Black donors, reduce D immunization and minimize transfusion of D- units to D+ individuals with conventional RHD or DAU0 alleles.

A novel high-prevalence antigen in the Lutheran system, LUGA (LU24), and an updated, full-length 3D BCAM model.

BACKGROUND: The basal cell adhesion molecule (BCAM) carries the antigens of the Lutheran (LU, ISBT005) system. We report a novel Lutheran antigen and propose an updated, full-length 3D model of BCAM. STUDY DESIGN AND METHODS: Red blood cell testing, antibody identification, and BCAM genomic DNA sequencing were done by standard methods. Multi-template homology modeling of BCAM used structural templates selected for coverage, highest sequence identity, and protein domain family. All variants causing the loss or gain of a Lutheran antigen were analyzed for residue accessibility and intraprotein interactions. RESULTS: An antibody to a high-prevalence antigen in the plasma of a pregnant woman was determined to be directed at a novel Lutheran antigen. Sequencing of BCAM found three homozygous changes: c.212G > A (p.Arg71His) and two silent, c.711C > T and c.714C > T. The model was built from the first two immunoglobulin crystallized domains of BCAM (D1, D2), three other templates (for D3, D4 and D5 with a higher sequence identity with the target than those used for the model proposed by Burton and Brady in 2008, and for the transmembrane region) and RaptorX (for the intracellular domain). All residues associated with a Lutheran antigen were found to be exposed in wild-type or variant proteins, except p.447 associated with loss of Lu13 expression. CONCLUSION: The c.212G > A change results in the loss of LUGA (LU24) antigen. Whole genome sequencing continues to reveal polymorphisms with uncertain immunogenicity. This model and demonstration that nearly all residues associated with the expression of a Lutheran antigen are exposed will help evaluate the significance of new polymorphisms.

Evidence that donors with variant RH genotypes are associated with unexpected Rh antibodies.

BACKGROUND: We previously reported unexpected Rh antibodies in the plasma of patients with sickle cell disease (SCD) that demonstrated common Rh specificities in the absence of transfusion of RBCs positive for that antigen. We hypothesize that these antibodies might result from transfusion of antigen-negative donor units with variant RH genotypes. METHODS: Plasma testing by tube and IgG gel, extended RBC phenotyping, and HEA and RH genotyping were by standard methods. CASE: A 6-year-old female with SCD, phenotype D + C-c + E-e + K- undergoing exchange transfusion with CEK- and Fy(a-) units, presented with anti-C in the plasma, a + DAT and warm autoantibody (WAA) in the eluate. Her RH genotype was unremarkable: RHD*D/DAU0 and RHCE*ce/ce(48C). Units (n = 10) transfused over the prior 6 months were confirmed CEK- by serology and DNA testing. Most (n = 7) were Rh-negative. A unit with variant RH, RHD*DIIIa/weak partial 4.0, RHCE*ceVS.03/ceVS.02, was transfused 5 weeks prior. Anti-C and + DAT continued to demonstrate for 25 weeks. Total hemoglobin and % Hgb S did not deviate from her established baseline. CONCLUSION: We show direct association of plasma anti-C with transfusion of a C-negative unit with variant RH encoding partial D and uncommon V/VS+ hrB - phenotype. The antibody was transient, without evidence of compromised survival of transfused RBCs. The +DAT and WAA complicated workups and selection of units, and it is uncertain whether donors of the same genotype should be avoided. Minority donors are important for CEK-matching to avoid depleting Rh-negative supplies. Consideration of patient and donor RH genotypes may avoid unexpected antibodies and improve allocation of rare donations.

Two new Scianna variants causing loss of high prevalence antigens: ERMAP model and 3D analysis of the antigens.

BACKGROUND: Scianna (Sc) antigens, seven high and two of low prevalence, are expressed on erythrocyte membrane-associated protein (ERMAP). We investigated SC (ERMAP) in individuals who made antibodies to high prevalence Scianna antigens, and propose a 3D model for ERMAP to precisely localize the residues associated with the known antigens. METHODS: Serological testing and DNA sequencing was performed by standard methods. A 3D structural model was built using a multi-template homology approach. Protein structures representing missense variants associated with the loss or gain of an antigen were generated. Residue accessibility and intraprotein interactions were compared with the wild-type protein. RESULTS: Two new SC alleles, one with c.349C > T (p.Arg117Cys) in a woman from South India with anti-Sc3 in her plasma, and a c.217_219delinsTGT (p.Arg73Cys) in an African-American woman with an antibody to a new high prevalence antigen, termed SCAC, were identified. Six structural templates were used to model ERMAP. 3D analysis showed that residues key for Scianna antigen expression were all exposed at the surface of the extracellular domain. The p.Arg117Cys change was predicted to abolish interactions between residues 93 and 117, with no compensating interactions. CONCLUSION: We confirm the extracellular location of Scianna residues responsible for antigen expression which predicts direct accessibility to antibodies. Loss of intraprotein interactions appear to be responsible for a Sc null and production of anti-Sc3 with p.117Cys, SC*01 N.03, and for loss of a high prevalence antigen with p.73Cys, termed SCAC for Sc Arg to Cys. Comparative modeling aids our understanding of new alleles and Scianna antigen expression.

YTGT: A new high-prevalence antigen in the Yt blood group system in two unrelated Native Americans and transfusion management.

BACKGROUND: The Yt system consists of five antigens: antithetical Yta /Ytb and the high-prevalence antigens YTEG, YTLI, and YTOT. We investigated a sample from a Native American (NA) female with post-operative anemia and an unidentified antibody who developed rigors, tachycardia, and hypotension on transfusion of incompatible RBCs. METHODS AND MATERIALS: Serologic testing methods included LISS, PEG, and IgG gel. Test RBCs were treated with papain, trypsin, alpha-chymotrypsin, 2-amino-ethylisothiouronium, and dithiothreitol. Rare RBCs were tested, and inhibition studies were performed. DNA extracted from WBCs was used for Sanger sequencing. RESULTS: Initial testing showed strong 3-4+ plasma reactivity with all panel cells at LISS IAT; auto control was negative. Positive reactions were observed with numerous rare RBCs except for PNH-III, which lack GPI-linked DO, Yt, CROM, JMH, and Emm. Enzyme sensitivity patterns suggest Yt specificity, and soluble recombinant srYt neutralized reactivity. ACHE sequencing revealed YT*A/A genotype but with a homozygous change in exon 2, c.290A>G (p.Gln97Arg). Antibody reactivity was reminiscent of that seen in an unrelated NA male investigated previously. His RBCs were nonreactive with her plasma. ACHE carried the same c.290G/G change. CONCLUSION: Two unrelated NA patients were found to have an antibody to a new high-prevalence Yt antigen, designated YTGT (YT6), associated with a clinically significant transfusion reaction. Identification of the specificity relied on enzyme sensitivity, use of PNH-III RBCs, neutralization using soluble recombinant Yt, and the finding of a novel change in ACHE, c.290A>G (p.Gln97Arg), designated YT*01.-06. IVIG and steroids were used to mitigate further reactions to transfusion.

Screening of blood donors for sickle cell trait using a DNA-based approach: Frequency in a multiethnic donor population.

BACKGROUND: Minority RBC donors are important to support the transfusion needs of patients with sickle cell disease. Testing of donors for sickle cell trait (SCT) is performed to avoid transfusion of hemoglobin S+ (HbS+) RBCs to specific patient groups and to investigate leukoreduction failures. A screening assay based on hemoglobin solubility is commonly used. The purpose of this study was to validate a DNA approach for HbS screening. METHODS: Hemoglobin solubility screening (Pacific Hemostasis or SICKLEDEX) and PreciseType human erythrocyte antigen (HEA)-HbS (Immucor) targeting c.20A>T in the ß-globin gene were performed according to manufacturer's directions. Resolution of differences in results included gene sequencing and high-performance liquid chromatography (HPLC). RESULTS: Initial validation of HEA-HbS performed by testing 60 known samples, 20 HbS/A, A/A, and S/S, gave expected results. However, in the subsequent parallel testing phase, 4/58 samples HbS+ by solubility assay tested negative by HEA-HbS; the negative results were confirmed by ß-globin gene sequencing. Samples from donors self-identifying as White testing HbS+ by solubility assay (n = 60) were retested by HEA-HbS and HPLC. The HEA-HbS assay was concordant with HPLC which is recognized as the gold standard for hemoglobin variation. CONCLUSION: A DNA-based approach is an alternative to screen donors for SCT, found in approximately 7% of Black and 1.7% of our random donors. HEA-HbS correlated with HPLC results in all samples tested, supporting the use of HEA-HbS as the test of record. The method allows higher throughput screening and testing at the donor center allows association of the screening result with the donor record to avoid repeat testing.

Impact of RHD genotyping on transfusion practice in Denmark and the United States and identification of novel RHD alleles.

BACKGROUND: Reduced D antigen on red blood cells (RBCs) may be due to "partial" D phenotypes associated with loss of epitope(s) and risk for alloimmunization or "weak" D phenotypes that do not lack major epitopes with absence of clinical complications. Genotyping of samples with weak and discrepant D typing is recommended to guide transfusion and RhIG prophylaxis. The goal was to compare the impact of RHD genotyping on transfusion practice in two centers serving different populations. STUDY DESIGN AND METHODS: Fifty-seven samples from Denmark and 353 from the United States with weak or discrepant D typing were genotyped. RBC typing was by multiple methods and reagents. DNA isolated from white blood cells was tested with RBC-Ready Gene D weak or CDE in Denmark or RHD BeadChip in the United States. RHD was sequenced for those unresolved. RESULTS: Of Caucasian samples from Denmark, 90% (n = 51) had weak D types 1, 2, or 3; two had other weak D, two partial D, and two new alleles. In diverse ethnic U.S. samples, 44% (n = 155) had weak D types 1, 2, or 3 and 56% (n = 198) had other alleles: uncommon weak D (n = 13), weak 4.0 (n = 62), partial D (n = 107), no RHD (n = 9), and new alleles (n = 7). CONCLUSION: Most samples with weak or variable D typing from Denmark had alleles without risk for anti-D. In U.S. samples, 48% could safely be treated as D+, 18% may require consideration if pregnancy possible, and 34% could potentially benefit from being treated as D-. Black and multiracial ethnicities were overrepresented relative to population.

ABO maternal-child discordance: Evidence of variable allelic expression and considerations for investigation.

BACKGROUND: We report a case of apparent mother-child ABO group noninheritance. A Caucasian mother initially typed as group O and her infant group AB. Investigation ruled out preanalytical causes such as mislabeled samples and in vitro fertilization. MATERIALS AND METHODS: Red blood cells were characterized by routine serologic testing. Genomic data were analyzed by targeted polymerase chain reaction-restriction fragment length polymorphism and Sanger sequencing. Transferase structures were modeled using PyMOL molecular visualization software. RESULTS: Serologic testing initially demonstrated the mother was group O, father group AB, and infant group AB. Further testing of the maternal sample with anti-A,B demonstrated weak A expression. Molecular testing revealed the maternal sample had an ABO*O.01.01 allele in trans to an A allele, ABO*AW.29 (c.311T>A, p.Ile104Asn), determined by gene sequencing. The sample from the infant carried the same ABO*AW.29 allele in trans to a B allele, ABO*B.01. CONCLUSION: ABO genotyping revealed an A transferase encoded by ABO*AW.29, with apparent variable activity. Although A antigen expression is well known to be weak in newborns, it was robust on the red blood cells (RBCs) of the AB infant and undetectable with anti-A on the mother. Variable expression of weak subgroups may reflect competition or enhancement by a codominant allele, as well as glycan chain maturation on red cells. Previous examples in group AB mothers with Aweak infants suggested that the decreased expression is primarily due to glycan immaturity. To our knowledge, this is the first reported case of the ABO*AW.29 allele presenting with weak A expression in a group Aweak mother and robust A expression in a group AB infant, suggesting the in trans allele is an important factor in determining transferase activity and may override age-related effects.

RH genotype matching for transfusion support in sickle cell disease.

Rh alloimmunization remains a challenge for patients with sickle cell disease (SCD) despite transfusion of serologic Rh C, E, and K antigen-matched red cells. Inheritance of altered RH alleles contributes to the prevalence of Rh antibodies after blood transfusion in patients with SCD and explains approximately one-third of cases. The remainder seem to be stimulated by altered Rh proteins on African American donor red cells. Matching patients with donors on the basis of RH genotype may mitigate Rh alloimmunization, but the feasibility and resources required are not known. We compared RH allele frequencies between patients with SCD (n = 857) and African American donors (n = 587) and showed that RH allele frequencies are similar. Overall, 29% of RHD and 53% of RHCE alleles are altered in patients and African American donors. We modeled RH genotype matching compared with serologic Rh D, C, and E, along with K antigen matching, and found that approximately twice the number of African American donors would be required for RH genotype vs Rh serologic matching at our institution. We demonstrated that African American donor recruitment is necessary to maintain an adequate supply of C-, E-, and K-negative donor units to avoid depleting the Rh-negative (RhD-) blood supply. Our results suggest that prophylactic RH genetic matching for patients with SCD is feasible with a donor pool comprised primarily of African-Americans and would optimize the use of our existing minority donor inventory. The current cost of RH genotyping all minority donors and management of the data remain limiting factors.

Reliability of labeling red cell units with minor antigen historical results and process considerations.

BACKGROUND: Several approaches are used by blood centers when providing minor (non-ABO/D) antigen-negative RBCs to hospitals. Details vary but include providing results on the unit labeling intended for clinical use without retyping or providing results on packing documents or via computer query requiring confirmation. Recent regulatory changes allow labeling with historical minor antigen results, defined as previously performed by the donor center on two different donations with results linked to the donor and confirmed concordant. Here we investigate causes of discrepancies and identify critical process steps. STUDY DESIGN AND METHODS: Nine years (2009-2017) of data were reviewed for number, antigen system, and root cause of discrepancies flagged by the computer when retyping donors prior to labeling (internal discrepancies) or reported by the hospital when retested (external discrepancies). Licensed automated (CcEeK) and tube methods were used. RESULTS: Among 300,000 samples phenotyped for CcEe, K, Fya/b , Jka/b , Ss (>3 million antigens), ∼1,389,960 were repeated on 2nd donation with 397 (1/3501) discordant; 205 Fy, 118 Rh, and 74 others. Of ∼682,691 antigen-negative phenotypes provided on unit labeling, ∼37.5% (256,118) were retyped by hospitals with 29 discrepancies (1/8832), primarily Rh variants. CONCLUSION: When repeating minor antigen types by serology, discrepancies are primarily associated with weak Fyb , among Caucasian donors, and weak/partial Rh antigens in donors of African ancestry. DNA-based testing avoids these. To label with historical results, accuracy is increased by automated testing with direct computer interface. Testing on two donations with results confirmed to be concordant is not inferior to testing on the current donation.

Multiple GYPB gene deletions associated with the U- phenotype in those of African ancestry.

BACKGROUND: The MNS blood group system is defined by three homologous genes: GYPA, GYPB, and GYPE. GYPB encodes for glycophorin B (GPB) carrying S/s and the "universal" antigen U. RBCs of approximately 1% of individuals of African ancestry are U- due to absence of GPB. The U- phenotype has long been attributed to a deletion encompassing GYPB exons 2 to 5 and GYPE exon 1 (GYPB*01N). STUDY DESIGN AND METHODS: Samples from two U-individuals underwent Illumina short read whole genome sequencing (WGS) and Nanopore long read WGS. In addition, two existing WGS datasets, MedSeq (n = 110) and 1000 Genomes (1000G, n = 2535), were analyzed for GYPB deletions. Deletions were confirmed by Sanger sequencing. Twenty known U- donor samples were tested by a PCR assay to determine the specific deletion alleles present in African Americans. RESULTS: Two large GYPB deletions in U- samples of African ancestry were identified: a 110 kb deletion extending left of GYPB (DEL_B_LEFT) and a 103 kb deletion extending right (DEL_B_RIGHT). DEL_B_LEFT and DEL_B_RIGHT were the most common GYPB deletions in the 1000 Genomes Project 669 African genomes (allele frequencies 0.04 and 0.02). Seven additional deletions involving GYPB were seen in African, Admixed American, and South Asian samples. No samples analyzed had GYPB*01N. CONCLUSIONS: The U- phenotype in those of African ancestry is primarily associated with two different complete deletions of GYPB (with intact GYPE). Seven additional less common GYPB deletion backgrounds were found. GYPB*01N, long assumed to be the allele commonly encoding U- phenotypes, appears to be rare.

Overcoming the challenges of interpreting complex and uncommon RH alleles from whole genomes.

BACKGROUND AND OBJECTIVES: Rh is one of the most diverse and complex blood group systems. Recently, next generation sequencing (NGS) has proven to be a viable option for RH genotyping. We have developed automated software (bloodTyper) for determining alleles encoding RBC antigens from NGS-based whole genome sequencing (WGS). The bloodTyper algorithm has not yet been optimized and evaluated for complex and uncommon RH alleles. MATERIALS AND METHODS: Twenty-two samples with previous polymerase chain reaction (PCR) and Sanger sequencing-based RH genotyping underwent WGS. bloodTyper was used to detect RH alleles including those defined by structural variation (SV) using a combination of three independent strategies: sequence read depth of coverage, split reads and paired reads. RESULTS: bloodTyper was programmed to identify D negative and positive phenotypes as well as the presence of alleles encoding weak D, partial D and variant RHCE. Sequence read depth of coverage calculation accurately determined RHD zygosity and detected the presence of RHD/RHCE hybrids. RHCE*C was determined by sequence read depth of coverage and by split read methods. RHD hybrid alleles and RHCE*C were confirmed by using a paired read approach. Small SVs present in RHCE*CeRN and RHCE*ceHAR were detected by a combined read depth of coverage and paired read approach. CONCLUSIONS: The combination of several different interpretive approaches allowed for automated software based-RH genotyping of WGS data including RHD zygosity and complex compound RHD and RHCE heterozygotes. The scalable nature of this automated analysis will enable RH genotyping in large genomic sequencing projects.

Automated typing of red blood cell and platelet antigens from whole exome sequences.

BACKGROUND: Genotyping has expanded the number red blood cell (RBC) and platelet (PLT) antigens that can readily be typed, but often represents an additional testing cost. The analysis of existing genomic data offers a cost-effective approach. We recently developed automated software (bloodTyper) for determination of RBC and PLT antigens from whole genome sequencing. Here we extend the algorithm to whole exome sequencing (WES). STUDY DESIGN AND METHODS: Whole exome sequencing was performed on samples from 75 individuals. WES-based bloodTyper RBC and PLT typing was compared to conventional polymerase chain reaction (PCR) RHD zygosity testing and serologic and single-nucleotide polymorphism (SNP) typing for 38 RBC antigens in 12 systems (17 serologic and 35 SNPs) and 22 PLT antigens (22 SNPs). Samples from the first 20 individuals were used to modify bloodTyper to interpret WES followed by blinded typing of 55 samples. RESULTS: Over the first 20 samples, discordances were noted for C, M, and N antigens, which were due to WES-specific biases. After modification, bloodTyper was 100% accurate on blinded evaluation of the last 55 samples and outperformed both serologic (99.67% accurate) and SNP typing (99.97% accurate) reflected by two Fyb and one N serologic typing errors and one undetected SNP encoding a Jknull phenotype. RHD zygosity testing by bloodTyper was 100% concordant with a combination of hybrid Rhesus box PCR and PCR-restriction fragment length polymorphism for all samples. CONCLUSION: The automated bloodTyper software was modified for WES biases to allow for accurate RBC and PLT antigen typing. Such analysis could become a routing part of future WES efforts.

A whole genome approach for discovering the genetic basis of blood group antigens: independent confirmation for P1 and Xga.

BACKGROUND: Although P1 and Xga are known to be associated with the A4GALT and XG genes, respectively, the genetic basis of antigen expression has been elusive. Recent reports link both P1 and Xga expression with nucleotide changes in the promotor regions and with antigen-negative phenotypes due to disruption of transcription factor binding. STUDY DESIGN AND METHODS: Whole genome sequencing was performed on 113 individuals as part of the MedSeq Project with serologic RBC antigen typing for P1 (n = 77) and Xga (n = 15). Genomic data were analyzed by two approaches, nucleotide frequency correlation and serologic correlation, to find A4GALT and XG changes associated with P1 and Xga expression. RESULTS: For P1, the frequency approach identified 29 possible associated nucleotide changes, and the serologic approach revealed four among them correlating with the P1+/P1- phenotype: chr22:43,115,523_43,115,520AAAG/delAAAG (rs66781836); chr 22:43,114,551C/T (rs8138197); chr22:43,114,020 T/G (rs2143918); and chr22:43,113,793G/T (rs5751348). For Xga , the frequency approach identified 82 possible associated nucleotide changes, and among these the serologic approach revealed one correlating with the Xg(a+)/Xg(a-) phenotype: chrX:2,666,384G/C (rs311103). CONCLUSION: A bioinformatics analysis pipeline was created to identify genetic changes responsible for RBC antigen expression. This study, in progress before the recently published reports, independently confirms the basis for P1 and Xga . Although this enabled molecular typing of these antigens, the Y chromosome PAR1 region interfered with Xga typing in males. This approach could be used to identify and confirm the genetic basis of antigens, potentially replacing the historical approach using family pedigrees as genomic sequencing becomes commonplace.

Novel mutations in KLF1 encoding the In(Lu) phenotype reflect a diversity of clinical presentations.

BACKGROUND: Mutation in the KLF1 gene is the cause of the In(Lu) (Inhibitor of Lutheran) Lu(a-b-) phenotype and more than 60 alleles have been associated with this phenotype. Here we describe findings from investigation of seven cases: six presenting with a Lu(a-b-) phenotype including the historical index case and one referred from a patient with chronic anemia. STUDY DESIGN AND METHODS: Serologic testing was by standard methods. DNA testing included amplification and sequencing of KLF1 and LU coding regions. A StuI polymerase chain reaction-restriction fragment length polymorphism was designed to target c.304T>C in KLF1. RESULTS: Five different KLF1 alleles were identified. Three are new: KLF1*90A (p.Trp30Ter), KLF*911A (p.Thr304Lys), and KLF1*304C,318G (p. Ser102Pro, Tyr106Ter) present in two unrelated individuals. Two, including the index case, had c.954dupG (p.Arg319Glufs*34), that is, KLF1*BGM06. The child with unexplained anemia had c.973G>A (p.Glu325Lys), associated with congenital dyserythropoietic anemia. The common c.304T>C was found in two of the seven samples investigated and in 60 of 100 blood donors. CONCLUSION: Mutations in KLF1 are pleiotropic and although most are benign, others are associated with hematologic abnormalities. We report three new KLF1 alleles associated with benign In(Lu) and document both the molecular basis of the original In(Lu) phenotype using a frozen sample stored for more than 50 years and the cause of unexplained anemia in a child. We also confirm previous observations that c.304C (p.102Pro) is not, by itself, associated with an In(Lu) phenotype in donors self-identified as U.S. minorities.