Blood Donor Genotyping

Transfusion of blood is one of the oldest and most widely used clinical interventions. In 2020 the World Health Organisation reported that globally 118.5 million blood donations had been collected worldwide. This blood will be used to provide life-saving transfusion support for millions of individuals with a wide range of medical conditions. To ensure the safety of each blood transfusion it is common policy to identify and ensure compatibility between the ABO and RhD antigens of both donor and recipient. Although this policy prevents the majority of adverse haemolytic transfusion reaction’s (HTR), approximately 3% of recipients become sensitised following an immune reaction to a non-self blood group antigen after a single transfusion episode. This proportion can rise dramatically in patients requiring frequent transfusion support, with immunisation rates as high as 60% being reported for some haemoglobinopathy patients. Sensitisation to non-self red blood cell (RBC) antigens confers a lifetime risk of HTRs, which from 2013 through 2017 were responsible for 17% (32 of 185) and 6% (7 of 110) of transfusion-related deaths reported to the US Food and Drug Administration and Serious Hazards of Transfusion UK, respectively. Furthermore, sensitisation can render transfusion-dependent patients non-transfusable and cause haemolytic disease in pregnancy which is potentially life-threatening to the fetus. A more precise blood matching policy will reduce sensitisation rates, however, adoption of this is resisted because of perceived logistical challenges, donor typing costs, and the lack of evidence from large scale clinical trials that reducing sensitisation rates results in health gains. Antibody-based haemagglutination tests are the current gold standard for RBC antigen typing; however, reliable reagents and high-throughput techniques are not available for all clinically relevant antigens. DNA-based tests have been used to overcome these limitations, and a range of in-house and commercial assays have been developed for donor genotyping. However, due to the limited number of antigens typed for and the low throughput capacity of these assays, they have not been widely applied to blood donor typing. Advances in the technologies used for genome-wide genotyping and sequencing have substantially reduced the cost of generating genetic variation data at population scale. Multiple studies have demonstrated that it is possible to extract antigen typing information from the data produced by these technologies, indicating that they could be used to deliver genomics-based precision transfusion medicine to the patient bedside. However, the same studies also highlight a series of challenges that would have to be overcome before these technologies could be safely integrated into the clinical laboratory. In this thesis, I will present the work that has been done to overcome some of the issues surrounding the interpretation of blood cell antigen typing from genomic data and the development of a universal donor genotyping platform which can be used by blood supply organisations worldwide to implement of a policy of precision transfusion medicine

Old Drugs To Treat Resistant Bugs: Methicillin-Resistant Staphylococcus aureus Isolates with mecC Are Susceptible to a Combination of Penicillin and Clavulanic Acid.

β-Lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by the expression of an alternative penicillin-binding protein 2a (PBP2a) (encoded by mecA) with a low affinity for β-lactam antibiotics. Recently, a novel variant of mecA, known as mecC, was identified in MRSA isolates from both humans and animals. In this study, we demonstrate that mecC-encoded PBP2c does not mediate resistance to penicillin. Rather, broad-spectrum β-lactam resistance in MRSA strains carrying mecC (mecC-MRSA strains) is mediated by a combination of both PBP2c and the distinct β-lactamase encoded by the blaZ gene of strain LGA251 (blaZLGA251), which is part of mecC-encoding staphylococcal cassette chromosome mec (SCCmec) type XI. We further demonstrate that mecC-MRSA strains are susceptible to the combination of penicillin and the β-lactam inhibitor clavulanic acid in vitro and that the same combination is effective in vivo for the treatment of experimental mecC-MRSA infection in wax moth larvae. Thus, we demonstrate how the distinct biological differences between mecA- and mecC-encoded PBP2a and PBP2c have the potential to be exploited as a novel approach for the treatment of mecC-MRSA infections.This work was supported by a Medical Research Council (MRC) Partnership Grant (G1001787/1) held between the Department of Veterinary Medicine, University of Cambridge (M. A. H.), the School of Clinical Medicine, University of Cambridge (S. J. P.), the Moredun Research Institute (R. N. Z.) and the Wellcome Trust Sanger Institute (J. P. and S. J. P.).This is the author accepted manuscript. The final version is available from American Society for Microbiology via http://dx.doi.org/10.1128/AAC.01469-1

Contrasting patterns of longitudinal population dynamics and antimicrobial resistance mechanisms in two priority bacterial pathogens over 7 years in a single center

Abstract: Background: Two of the most important pathogens contributing to the global rise in antimicrobial resistance (AMR) are Klebsiella pneumoniae and Enterobacter cloacae. Despite this, most of our knowledge about the changing patterns of disease caused by these two pathogens is based on studies with limited timeframes that provide few insights into their population dynamics or the dynamics in AMR elements that they can carry. Results: We investigate the population dynamics of two priority AMR pathogens over 7 years between 2007 and 2012 in a major UK hospital, spanning changes made to UK national antimicrobial prescribing policy in 2007. Between 2006 and 2012, K. pneumoniae showed epidemiological cycles of multi-drug-resistant (MDR) lineages being replaced approximately every 2 years. This contrasted E. cloacae where there was no temporally changing pattern, but a continuous presence of the mixed population. Conclusions: The differing patterns of clonal replacement and acquisition of mobile elements shows that the flux in the K. pneumoniae population was linked to the introduction of globally recognized MDR clones carrying drug resistance markers on mobile elements. However, E. cloacae carries a chromosomally encoded ampC conferring resistance to front-line treatments and shows that MDR plasmid acquisition in E. cloacae was not indicative of success in the hospital. This led to markedly different dynamics in the AMR populations of these two pathogens and shows that the mechanism of the resistance and its location in the genome or mobile elements is crucial to predict population dynamics of opportunistic pathogens in clinical settings

Nuclear-mitochondrial DNA segments resemble paternally inherited mitochondrial DNA in humans.

Several strands of evidence question the dogma that human mitochondrial DNA (mtDNA) is inherited exclusively down the maternal line, most recently in three families where several individuals harbored a 'heteroplasmic haplotype' consistent with biparental transmission. Here we report a similar genetic signature in 7 of 11,035 trios, with allelic fractions of 5-25%, implying biparental inheritance of mtDNA in 0.06% of offspring. However, analysing the nuclear whole genome sequence, we observe likely large rare or unique nuclear-mitochondrial DNA segments (mega-NUMTs) transmitted from the father in all 7 families. Independently detecting mega-NUMTs in 0.13% of fathers, we see autosomal transmission of the haplotype. Finally, we show the haplotype allele fraction can be explained by complex concatenated mtDNA-derived sequences rearranged within the nuclear genome. We conclude that rare cryptic mega-NUMTs can resemble paternally mtDNA heteroplasmy, but find no evidence of paternal transmission of mtDNA in humans

Genomic identification of cryptic susceptibility to penicillins and β-lactamase inhibitors in methicillin-resistant Staphylococcus aureus.

Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all β-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with β-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin-binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models shows that penicillin/β-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for 'susceptible' MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin but the absence of clavulanic acid, which suggests that penicillin/β-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections.MRC - G1001787/1 MRC - MR/N002660/1 WT098600 HICF-T5-342 MR/S00291X/1 201344/Z/16/Z MR/P007201/

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans

Author Correction: Nuclear-mitochondrial DNA segments resemble paternally inherited mitochondrial DNA in humans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus.

Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component

Author Correction: Nuclear-mitochondrial DNA segments resemble paternally inherited mitochondrial DNA in humans

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Nuclear-mitochondrial DNA segments resemble paternally inherited mitochondrial DNA in humans

Abstract: Several strands of evidence question the dogma that human mitochondrial DNA (mtDNA) is inherited exclusively down the maternal line, most recently in three families where several individuals harbored a ‘heteroplasmic haplotype’ consistent with biparental transmission. Here we report a similar genetic signature in 7 of 11,035 trios, with allelic fractions of 5–25%, implying biparental inheritance of mtDNA in 0.06% of offspring. However, analysing the nuclear whole genome sequence, we observe likely large rare or unique nuclear-mitochondrial DNA segments (mega-NUMTs) transmitted from the father in all 7 families. Independently detecting mega-NUMTs in 0.13% of fathers, we see autosomal transmission of the haplotype. Finally, we show the haplotype allele fraction can be explained by complex concatenated mtDNA-derived sequences rearranged within the nuclear genome. We conclude that rare cryptic mega-NUMTs can resemble paternally mtDNA heteroplasmy, but find no evidence of paternal transmission of mtDNA in humans