Equipment purchase with funding contribution from Open Scheme



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Open Scheme Report: James Traherne



Equipment purchase with funding contribution from Open Scheme

Applicants: James Traherne and John Trowsdale, University of Cambridge.



Amount awarded: £5,000 towards funding of a Caliper Twister II robotics system.
Our research group is investigating the role of the MHC and the leucocyte receptor complex (LRC) in the pathogenesis of human disease. We have published extensively on the genomic structure of these complex chromosomal regions and are now undertaking association studies to further delineate their role in human disease1. The genes that we have investigated most recently are called KIR and encode receptors that control the cytolytic activity of natural killer cells and act as costimulatory receptors on subpopulations of T cells2. These genes have been implicated in the pathogenesis of HIV, HCV, cancer, autoimmune disease and have been shown to have a role in pregnancy-related pathologies and transplantation. They are encoded within LRC on chromosome 19. This is an area of high genetic complexity reflecting recent evolution, with evidence of extensive gene duplication, deletion, conversion and hybrid formation. Consequently, loci within this region are characterised by variable gene copy number and extreme allelic diversity. Each locus shows high sequence similarity with other members of this gene family, in some cases up to 99% homology. Furthermore, their MHC related ligands (alleles of HLA-A, B, C and G) can be variably present and are altered in the context of viral infection. Analysis of this region is therefore difficult but unravelling the genetic complexity of these receptors will provide insights into disease mechanisms, which ultimately may be beneficial to improve human health.
The development of a high throughput assay to type for gene copy number of KIR has the potential to make the biggest long-term contribution to the field. Genotyping to date has been focussed on identifying only the presence or absence of these genes, using cumbersome and expensive methods. Consequently disease association studies have been limited in sample size. Employing state-of-the-art technology, we have developed a high throughput assay capable of determining copy number and are applying this to large cohorts of well phenotyped patients with complex polygenic disease. This is a real-time PCR-based method using multiplexed reactions and fluorescent DNA probes. It requires less than half a microgram of DNA per individual to fully type these genes in quadruplicate together with their known MHC ligands. Briefly, an array of multiplexed PCR reactions are carried out in quadruplicate on DNA samples in 384 well plates using a Roche Lightcycler480 quantitative PCR instrument. Fluorescence from labelled DNA probes is recorded over time, permitting the calculation of a cycle threshold value. Using a reference gene, the copy numbers of the genes of interest are then calculated. This method has been extensively optimised.
In order to meet our project goals and new demand from interested researchers internationally we needed to scale up our experiments. Our request to the Pathological Society was for a contribution towards a Caliper Twister II robotics system and refrigeration unit that integrates with the Lightcycler to allow automated microplate loading. The Twister allows continuous running of the Lightcycler day and night, significantly increasing throughput and making full use of the machine. Our unique assay provides, for the first time, a ‘gold standard’ for rapid KIR typing that is accessible, reproducible, highly informative and cost effective. With the attached robotic system the method is now fully automated and allows 1000 samples to be typed per week. Establishment of this typing resource will be of strategic value to Cambridge, strengthening our worldwide leadership in the field.
In the first instance we have used our technique to genotype the parents and children of over 700 families from the UK and US. This allowed us to properly categorise the haplotype diversity of the KIR complex. So far we have identified over 75 different gene configurations. This level of variation is unprecedented in the human genome. The study was recently presented at the 2011 KIR workshop in Sweden and is described in a forthcoming research article3.
Exploiting our novel method, we are approaching LRC variation and disease with a network of local and international collaborators who have collected large patient cohorts, each in excess of 1000 disease samples. In an early study through collaboration with Profs Bill Cookson and Miriam Moffett from Imperial College London, we used our technique to genotype large family based cohorts of children with asthma and atopic dermatitis. We have found some interesting trends that we are following up in further replication cohorts. Our current model is that these associated loci influence the development of childhood asthma by modifying the adaptive and innate responses to respiratory viral infections. This is supported by good genetic and functional evidence for a disease-modifying role in acute and chronic viral infections including HIV, HCV and Influenza A. Application of our technique with collaborators in Cambridge is broad ranging and equally exciting as illustrated by the following three examples; (i) we are assisting Prof Ashley Moffett at the Centre for Trophoblast Research to dissect how receptor gene variability impacts on reproductive success, (ii) we are facilitating Dr Peter Goon’s project in the Department of Pathology to identify which NK cells are effective against HPV-infection and associated carcinoma, (iii) in collaboration with Dr Steve Sawcer in the Department of Clinical Neurosciences and Prof John Todd in the Department of Medical Genetics we are investigating the role of KIR genes and their interactions in conferring genetic susceptibility to autoimmune conditions, multiple sclerosis and type 1 diabetes.
Beyond disease studies, our assay has application in tissue typing where, for example, it is estimated that a 7% reduction of relapse after transplantation for acute myeloid leukemia (AML) can be achieved by typing donors. In this regard, we are at the initial stages of setting up a clinical diagnostic service for KIR typing with the Molecular Genetics department at Addenbrooke’s Hospital.
Our working hypothesis is that immunity related genes within the LRC together with their cognate MHC ligands contribute to the genetic susceptibility to numerous conditions and their severity. We believe this to be an important question which may identify further disease pathways for investigation or indeed assist in clinical phenotyping. We are excited and optimistic about the advancements that are now feasible and would like to thank the Pathological Society greatly for providing financial support which has helped make this achievable.
1 Kulkarni et al. (2008). “The Yin and Yang of HLA and KIR in human disease.” Semin Immunol 20(6): 343-52.

2 Wei et al. (2011). “Novel KIR haplotypes revealed by typing for copy number variation.” in preparation.
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