Rhiannon McBean

Approaches to Determination of a Full Profile of Blood Group Genotypes: Single Nucleotide Variant Mapping and Massively Parallel Sequencing

The number of blood group systems, currently 35, has increased in the recent years as genetic variations defining red cell antigens continue to be discovered. At present, 44 genes and 1568 alleles have been defined as encoding antigens within the 35 blood group systems. This paper provides a brief overview of two genetic technologies: single nucleotide variant (SNV) mapping by DNA microarray and massively parallel sequencing, with respect to blood group genotyping. The most frequent genetic change associated with blood group antigens are SNVs. To predict blood group antigen phenotypes, SNV mapping which involves highly multiplexed genotyping, can be performed on commercial microarray platforms. Microarrays detect only known SNVs, therefore, to type rare or novel alleles not represented in the array, further Sanger sequencing of the region is often required to resolve genotype. An example discussed in this article is the identification of rare and novel RHD alleles in the Australian population. Massively parallel sequencing, also known as next generation sequencing, has a high-throughput capacity and maps all points of variation from a reference sequence, allowing for identification of novel SNVs. Examples of the application of this technology to resolve the genetic basis of orphan blood group antigens are presented here. Overall, the determination of a full profile of blood group SNVs, in addition to serological phenotyping, provides a basis for provision of compatible blood thus offering improved transfusion safety.

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.

SARA: a “new” low-frequency MNS antigen (MNS47) provides further evidence of the extreme diversity of the MNS blood group system

Until recently, SARAH (SARA) was a low‐frequency antigen within the 700 series (700.052). SARA was discovered in Australia and subsequently described in Canada where anti‐SARA was implicated in severe hemolytic disease of the fetus and newborn (HDFN). This study investigated whether SARA could be recategorized into an existing, or novel, blood group system.

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.

Blood group genotype analysis of Australian reagent red blood cell donors across three genotyping platforms: consistent detection of 7·0% phenotype genotype nonconcordance

Standards mandate reagent red blood cells (RBCs) must be homozygous for certain antigens. Genotyping provides an enhanced approach to characterizing RBCs as mutations encoding weakened antigen expression may be detected. This study aimed to genotype reagent RBC donors and compare outcomes with serological phenotype. Genotyping was undertaken using three commercially available platforms.

Radiological appearance of coal mine dust lung diseases in Australian workers

Coal Mine Dust Lung Disease (CMDLD) encompasses a spectrum of lung diseases caused by prolonged exposure to coal mine dust. This review presents high‐resolution computed tomography (HRCT) images from men diagnosed with a CMDLD since the resurgence of these diseases in Queensland in 2015.

Novel MRI of mediastinal masses: internal differentiation of a thymoma and lymphoma with T1 and T2 mapping

Routine imaging for mediastinal malignancies includes chest X-ray, CT or MRI. T1 and T2 mapping are novel MRI techniques which may have a role in expanding the assessment of internal tumour characteristics. This case report details two middle-aged women who had similar clinical presentations of mediastinal masses of comparable size and appearance when assessed with routine imaging. T1 and T2 maps were acquired on MRI to investigate whether these tumours could be further differentiated prior to surgery. T1 and T2 mapping supported suspicion for which tumour components were solid and cystic, as subsequently confirmed histologically. Furthermore, comparison between the two tumours showed native T1 values differed within the solid components by 37%, correlating to differences in proteinaceous material within the tumour types. This radiological–pathological correlation provides evidence that T1 and T2 mapping has clinical utility in the assessment and differentiation of mediastinal masses.

Innocent PET avidity in patient with metastatic melanoma

A 53-year-old man presented for a surveillance 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT following recent resection of a melanoma of his right leg and metastasis to his right inguinal region. The scan was acquired approximately 60 min after intravenous injection of FDG. The FDG-PET/CT scan demonstrated no FDG uptake in the right inguinal region. However, intense FDG uptake was observed in skeletal muscles, specifically in the left foot (figure 1A) and around the shoulder, bicep and pectoral regions bilaterally (figure 1B,C). Figure 1 18F-fluorodeoxyglucose positron emission tomography showed intense uptake within the left foot (A) and around the shoulder (B), bicep and pectoral regions bilaterally (C). FDG-PET/CT is an integral part of modern-day practice in oncology imaging …

Prostate artery Embolisation Assessment of Safety and feasibilitY (P-EASY): a potential alternative to long-term medical therapy for benign prostate hyperplasia

To assess the safety, short‐term efficacy and early functional results of prostate artery embolisation (PAE), an emerging minimally invasive treatment for symptomatic benign prostate hyperplasia (BPH).

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−.