Generating full-length Killer-cell Immunoglobulin-like Receptor (KIR) gene sequences using Third Generation long-amplicon sequencing to assess the impact of KIR polymorphism on Haematopoietic Cell Transplantation outcomes

Killer-cell Immunoglobulin-like Receptor (KIR) polymorphism is extensive in both allelic and copy number variation. Although multiple assays have been designed to assess the latter form of polymorphism, KIR allelic diversity is less well understood owing to the homologous nature of different KIR genes and, until recently, limitations in sequencing technology. To better understand KIR allelic diversity in the UK, and its impact on haematopoietic cell transplant (HCT) outcomes, I have designed and validated a whole-gene, fully-phased allele sequencing strategy that encompasses third generation sequencing technology to deliver unambiguous genotypes for several different KIR genes. Subsequently, this strategy was applied to a novel, largely T cell deplete UK cohort of patients receiving HCT to treat acute myeloid leukaemia, and their respective donors. This assay utilises a semi-generic targeted polymerase chain-reaction amplification prior to multiplexed library preparation and sequencing, providing a relatively high-throughput methodology amenable to clinical laboratories. Initially, to assess the relevance of presence/absence KIR polymorphism on HCT outcomes, I utilised existing genotyping methods to establish baseline characteristics. In contrast to previous publications, relapse was largely unaffected by KIR polymorphism. However, striking differences related to preparative conditioning regimen were observed in KIR-mediated effects in other HCT outcomes. Presence of donor-encoded centromeric (Cen)-B motifs relayed increased risk of detrimental non-relapse mortality following myeloablative conditioning, whereas the opposite appeared to be true of reduced-intensity conditioning transplants. When the impact of allelic diversity at the KIR2DL1, KIR2DL2/3 and KIR3DL1/S1 loci on HCT outcomes was assessed in my cohort, allelic differences within the Cen-A, Cen-B and telomeric A haplotype motifs provided additional insight into the possible mechanisms of KIR-mediated influence on HCT outcomes. By estimating frequencies of different KIR alleles within a UK population, I have demonstrated that donor selection algorithms could feasibly incorporate allelic KIR genotypes that may improve quality of life after HCT

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate.

Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes

HLA Typing for the Next Generation

<div><p>Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.</p></div

Extensions and corrections to known HLA alleles identified in PacBio results.

<p>Extensions and corrections to known HLA alleles identified in PacBio results.</p

Anomalies observed in the PacBio SMRT consensus sequences as compared to the expected allele.

<p>Anomalies observed in the PacBio SMRT consensus sequences as compared to the expected allele.</p

Basic stages of the Single Molecule Real-Time (SMRT) DNA sequencing method.

<p>SMRTbell adaptors are ligated onto the ends of a blunt-ended PCR amplicon to facilitate continuous sequencing of both strands of the amplicon. The entire sequence generated may include multiple copies of the sense and anti-sense strands of the PCR amplicon in a single read known as the Continuous Long Read (CLR). The post-sequencing bioinformatic post-processes are able to break down the CLR into shorter sub-reads, which encompass the sequence of one strand of the amplicon. These sub-reads can then be compared and used to create a consensus sequence.</p

Homopolymer count in 38 PacBio sequences (total length: 130117 bp).

<p>Homopolymer count in 38 PacBio sequences (total length: 130117 bp).</p