Brett Wilson

Molecular typing for the Indian blood group associated 252G>C single nucleotide polymorphism in a selected cohort of Australian blood donors.

BACKGROUND The Indian blood group antigens, In(a) and In(b), are clinically significant in transfusion medicine. However, antisera to type these antigens are difficult to obtain. The In(b) antigen is a high frequency antigen present in all populations, while the frequency of the antithetical In(a) ranges from 0.1% in Caucasians up to 11% in Middle Eastern groups. This antigen polymorphism is encoded by the single nucleotide polymorphism (SNP) 252G>C in CD44. The aim of this study was to establish and compare two genotyping methods to measure the frequency of the IN*A and IN*B alleles in a blood donor cohort. MATERIALS AND METHODS Donor blood samples (n=151) were genotyped by a novel real-time polymerase chain reaction (PCR) high-resolution meltcurve (HRM) analysis and a custom matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) assay. Samples with the rare IN*A allele were further investigated by nucleotide sequencing, red cell agglutination, and flow cytometry techniques. RESULTS In this study group, 149 IN*B homozygous and 2 IN*A/B heterozygous samples were detected with 100% concordance between HRM and MALDI-TOF MS methods. For PCR HRM, amplicon melting alone did not differentiate IN*A and IN*B alleles (class 3 SNP), however, the introduction of an unlabelled probe (UP) increased the resolution of the assay. Sequencing confirmed that the two non-homozygous samples were IN*A/B heterozygous and phenotyping by red cell agglutination, and flow cytometry confirmed both In(a) and In(b) antigens were present as predicted. DISCUSSION Genotyping permits conservation of rare antisera to predict blood group antigen phenotype. In PCR UP-HRM the IN*A and IN*B alleles were discriminated on the basis of their melting properties. The In(a) frequency in this selected donor population was 1.3%. Application of genotyping methods such as these assists in identifying donors with rare blood group phenotypes of potential clinical significance.

Targeted exome sequencing defines novel and rare variants in complex blood group serology cases for a red blood cell reference laboratory setting

We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single‐nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP‐typing cases.

Duffy blood group phenotype–genotype correlations using high‐resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms

Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fya/Fyb polymorphism, for weak Fyb antigen, and for the red cell null Fy(a−b−) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing.

A novel FY*A allele with the 265T and 298A SNPs formerly associated exclusively with the FY*B allele and weak Fyb antigen expression: implication for genotyping interpretative algorithms

An Australian Caucasian blood donor consistently presented a serology profile for the Duffy blood group as Fy(a+b+) with Fya antigen expression weaker than other examples of Fy(a+b+) red cells. Molecular typing studies were performed to investigate the reason for the observed serology profile.

An alloantibody in a homozygous GYP*Mur individual defines JENU (MNS49), a new high-frequency antigen on glycophorin B.

R ed blood cell (RBC) antigens present in more than 90% of a population are classified as highfrequency antigens. Nine of these are in the MNS blood group system with two antigens, ‘N’ (MNS30) and U (MNS5), on glycophorin B (GPB). Hybrid glycophorins such as GYP(B-A-B) are formed by gene conversion events between homologous genes GYPA and GYPB. Gene conversion resulting in GYPA Exon 3 insertion into the 30 end of GYPB Pseudoexon 3 forms a composite exon with a repaired splice donor site (that is inactive in GYPB) and, depending on the crossover sites, encodes various GP(B-A-B) hybrid glycophorins: GP.HF, GP.Mur, GP.Bun, GP.Hop, and GP.Kip. A patient with thalassemia of Thai ethnicity was transfused with 2 RBC units to correct anemia. Posttransfusion, the patient developed multiple alloantibodies including a pan-reactive alloantibody of unknown specificity. A total of 3600 E–c–Jk–S– RBC units were screened and 2 compatible units were found. We present typing data for the patient and these compatible donors as well as epitope mapping defining this pan-reactive antibody.

Genotyping confirms inheritance of the rare At(a−) type in a case of haemolytic disease of the newborn

The Ata blood group antigen (now AUG2 in the Augustine system) is a high‐frequency antigen with negative phenotype At(a−) found only in individuals of African ancestry. In a twin pregnancy, the fifth pregnancy in a woman of African origin, serological investigations confirmed that the mother was At(a−) and anti‐Ata was detected. DNA samples were exome sequenced and alignment was performed to allow variant calling. It was confirmed that the single nucleotide polymorphism, rs45458701, within the SLC29A1 gene encoding the ENT1 protein, recently reported to be a basis of the At(a−) phenotype was also the basis of the phenotype in this family. The reagents for serological analysis required to identify the rare blood type present in this mother are held in only a few reference laboratories worldwide. This case highlights the utility of genetic methods in resolving complex investigations involving blood grouping and demonstrates that genotyping of variants associated with blood types present in specific ethnic groups may be the fastest method available for identification of the basis of fetomaternal incompatibilities.

A proposed new low-frequency antigen in the Augustine blood group system associated with a severe case of hemolytic disease of the fetus and newborn: Proposed new low-frequency antigen in the Aug Blood Group System

A male infant born at 29 weeks presented with a severe case of hemolytic disease of the fetus and newborn (HDFN) (cord blood direct antiglobulin test 41, hemoglobin 45 g/L with cardiac failure, pleural effusion, and generalized edema). Two exchange and four top-up transfusions were required. The maternal antibody was reactive with paternal red blood cells (RBCs). Later testing revealed that the antibody reacted with RBCs from four additional members of the paternal family. Extensive testing excluded clinically relevant RBC antibodies but failed to reveal a specificity for this antibody. To guide further investigation, specimens from the family (n 5 10) were submitted for blood group genetic studies.

Quantitation of Lan antigen in Lan+, Lan+(w) and Lan- phenotypes.

The Lan antigen (LAN1) is a clinically significant, high-frequency red blood cell (RBC) antigen. Lan was originally described in 1961 and assigned to the LAN blood group system in 20121,2. Lan is currently the only antigen within the LAN blood group system and Lan+, Lan−, Lan+w and Lan+w/− phenotypes have been defined1–4. The Lan− phenotype is very rare worldwide with a frequency of less than 1% in all populations tested to date2,5,6. For example, a Japanese screening study identified 14 Lan− individuals among 713,384 blood donors, giving a frequency of 0.002%2. Lan− individuals are usually identified due to the detection of anti-Lan during investigations into haemolytic disease of the foetus and newborn7,8 or when serological testing is performed to find compatible blood units for a patient5,9. Transfusion support for Lan− individuals is highly challenging due to the scarcity of both compatible blood and suitable anti-Lan reagents for screening for compatible blood. The first monoclonal anti-Lan antibody (OSK43) was produced in 2012 by Helias et al. and this antibody has proven to be of huge benefit as a reliable reagent for screening for Lan− individuals2. The carrier of the Lan antigen is the ABCB6 protein encoded by the ABCB6 gene at chromosome 2q36, containing 19 exons2. Numerous genetic variants have been identified as encoding for the Lan− phenotype2,4,10–12. The initial sequencing study identified ten novel alleles, including frameshift, nonsense and splice-site mutations, within 12 unrelated Lan− individuals and found that each individual was heterozygous for two ABCB6 null alleles2. Subsequently, Saison et al. characterised yet another ABCB6 null allele, a single nucleotide variant missense mutation (c.574C>T)10. To date this single nucleotide variant is the most common mutation causing the Lan− phenotype4,10. It has been suggested that the frequency of ABCB6 null alleles differs between populations as sequencing of 27 Japanese Lan− individuals identified a further ten novel alleles and revealed that none of the donors carried the c.574C>T variant12. The Lan+w phenotype has been described in individuals heterozygous for an ABCB6 null allele and a wild-type allele, and these individuals typically express 50% of the normal level of Lan antigen4,10. The fourth Lan phenotype is referred to as Lan+w/− as cells serologically type as either Lan+w or Lan− depending on the anti-Lan utilised3. A comprehensive study involving serological and molecular characterisation of Lan phenotypes was recently performed by Reid et al. and for the first time alleles encoding Lan+w/− phenotypes were defined4. Lan+w/− individuals are heterozygous for an ABCB6 null allele and a variant allele encoding for weakened Lan expression. Antigens can be quantified by flow cytometry by converting the fluorescent intensity of staining into an antibody-binding capacity (ABC), relating to the number of monoclonal antibody molecules bound to a cell. This is performed utilising populations of calibrated microspheres coated with defined amounts of a capture antibody, to create a calibration curve used for quantitation of ABC. The usefulness of determining ABC values has been well established, particularly for lymphocyte antigens13. In previous studies, calibrated microsphere-based assays have been validated to aid in the diagnosis, prognosis, and treatment monitoring of diseases including chronic lymphocytic leukaemia14,15 and human immunodeficiency virus infection16. In the context of RBCs, glycophorin A and RhD antigen expression has been investigated utilising traditional flow cytometric approaches17,18 and, more recently, calibrated microspheres19,20. The variability of Lan antigen expression has never been quantitatively investigated. In this study, we investigated the expression of Lan antigen by developing a novel indirect staining protocol capable of quantitating the number of Lan sites per RBC in samples reported as Lan+, Lan+w, and Lan−.