The period 2004-2007 saw substantial advances with introduction of new technology resulting in the development of capacity for medium and high throughput genotyping for the first time in the Unit. Initially a Taqman instrument was installed which is suitable for smaller scale projects and subsequently an ABI 3730xl for sequence analysis and medium scale genotyping studies. These instruments together with two new liquid handling robots and new equipment for determining the quality of DNA are used by all programmes. In addition new genetic research projects began on malaria, liver cancer and tuberculosis.

The period also saw organisational changes in genetic research with the introduction of a new stream of work in Pharmacogenetics and the subsequent merging of Pharmacogenetics with Human Genetics into a single Genetics programme with a broader remit under the leadership of Dr Robert Walton. Dr. Giorgio Sirugo was the Head of the Human Genetics laboratory and of the DNA Bank until August 2006.

1. Genetic effects on pharmacokinetics of antimalarial drugs and their effectiveness in the treatment of malaria in children.

Genetics staff: Ramatoullie Janha, Dr Fatou Joof, Mr Archibald Worwui
Internal collaborators: Dr David Conway, Dr Sam Dunyo, Dr Giorgio Sirugo
External collaborators: Professor Munir Pirmohamed, Professor Peter Winstanley Liverpool University. Dr Paul Milligan, London School of Hygiene and Tropical Medicine
Funding: EDCTP PhD Fellowship

The aim of these studies is to investigate the effects of common variants in the cytochrome P450 enzymes that activate the antimalarial biguanides proguanil and chlorproguanil. These drugs have been extensively used worldwide for many years and are components of new therapeutic combinations such as Malarone and Lapdap. Studies in Europeans and Asians suggest that a significant proportion of the population carry alleles that lead to poor activation, however the frequency of these alleles in Africa was previously not known. In addition there may be novel African alleles that affect function which are not present in other populations. These studies seek to explore the genomic variations that may affect activation of these antimalarial compounds and to investigate their effects on pharmacokinetics and clinical efficacy. Initial bioinformatic analysis of the CYP2C19 gene which encodes the cytochrome P450 mainly responsible for the activation of antimalarial biguanides is shown in Figure 1.

Analysis of patterns of linkage disequilibrium in the CYP2C cluster shows two blocks of LD, one containing CYP2C18, CYP2C19 and CYP2C9 and the other CYP2C8 (Walton et al Nature Genetics 2005)

Forty two people who participated in a pharmacokinetic study of Lapdap in Farafenni were contacted and gave consent for a sample of DNA to be taken for pharmacogenetic analysis. Preliminary results correlating known alleles with detailed pharmacokinetic parameters show that the recently described *17 allele has the greatest effect on pharmacokinetics in our population followed by the *2 allele. The frequencies of these gene variants are remarkably similar to those in Europeans and we have found that poor metabolising alleles are much less common than in Asia. Several novel alleles have been found and their effects on drug metabolism are currently being characterised. Typing of 600 children treated for malaria with Lapdap in a randomised controlled trial has recently been completed for the *2 allele. Early results suggest that this allele does not affect the outcome of treatment - typing for the *17 allele is currently underway.

2. Investigating the effects of genetic variants in the innate immune system and disease progression in HIV infection.

Genetics staff: Mr Louis-Marie Yindom, Dr Peter Aka
Internal collaborators: Professor Rowland-Jones, Dr Giorgio Sirugo, Dr Matt Cotten and Mr Clayton Onyango External collaborator: Professor Mary Carrington, National Cancer Institute, Frederick, USA
Funding: EDCTP PhD Fellowship

This project seeks to elucidate the role of inherited variations in receptors on natural killer cells (KIR) in controlling infection with the HIV2 virus. Initial KIR typing has now been completed on 347 people in the Caio cohort, 178 of these are cases and the remainder controls. The presence or absence of 15 KIR genes in all participants has been ascertained using PCR with sequence specific primers. Work is now in progress to identify the alleles present by DNA sequencing on the newly installed 3730xl genetic analyser. Primers have been designed to make the detection of the KIR genes more time efficient using the SNPlex system on the 3130xl. This new method will be used on future samples and should contribute a novel technological advance in this field.

The relationship between KIR alleles, case status and viral load for HIV2 will then be investigated together with an exploratory analysis of the effects of KIR variants on HIV1 and HTLV1, although the number of people with HIV1 in Caio is relatively small. Work has commenced on similar studies in Fajara for HIV1 where 160 of a target 500 HIV negative controls have been recruited so far from those who present for retroviral serology at the GUM clinic. These people will act as controls for the Fajara HIV 1 cohort which has already been established.

SNPlex assays have been designed for fine resolution genetic typing of the TRIM5 gene encoding an antiviral restriction factor thought to be important in controlling retroviral infection. Currently reliant on information from the HapMap database, these assays will be supplemented by including novel polymorphisms of the splice variant TRIM5alpha which have been identified by sequencing cDNA from TRIM5alpha mRNA extracted from peripheral blood lymphocytes from participants in Caio (Professor Rowland-Jones, Dr Matt Cotten and Mr Clayton Onyango). Thus the generic assay will be tailored to the specific study population. This is collaboration with. The resulting SNPlex assay will be used to explore the relationship between genetic variants of TRIM genes in large scale studies in the Fajara and Caio cohorts.

4. Gambian National DNA collection

Genetics staff: Mr Mathurin Diatta, Mr Cyrille Bissaye Mr Archibald Worwui
Funding: MRC Core

The DNA Bank provides a service for extracting quantifying and standardising DNA for studies conducted by all the programmes in the Unit. A photograph of the team is shown in Figure 3.

DNA samples are bar coded and archived using the Biobase computer system, which records routine data related to sample usage such as storage location, when aliquots are taken and the amount of DNA that remains. We have recently added routine information on the purity of each sample, as measured by the protein concentration, to the genetic database. A system has been recently implemented so that principal investigators can view all data on their samples over the MRC intranet using a web browser interface.

In 2006 a low volume genotyping capacity was added to the services which had previously been provided solely for DNA sample curation. The ABI Taqman system using real-time PCR for genotyping is currently being extensively used mainly for genetic studies in the TB case control cohort which should reach completion in early 2007.

The SNPlex system for high throughput typing will be made available for use by other programmes within the Unit in a similar fashion. The two systems are complementary with Taqman being suited for low volume typing where specific SNPs must be evaluated and SNPlex being used for larger studies where a haplotype tagging approach is used. Thus both systems will be maintained to enable maximum flexibility in genotyping as a service to the Unit.