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
Open Scheme Report: James Traherne
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
2
Kulkarni et al. (2008). “The Yin and Yang of HLA and KIR in human disease.” Semin Immunol 20(6): 343-52.
Wei et al. (2011). “Novel KIR haplotypes revealed by typing for copy number variation.” in preparation.