Genetics of Type 1 Diabetes
The Australian Childhood Diabetes DNA Repository
Sarah Lilley (Network Coordinator); Dr Jemma Berry
The Australian Childhood Diabetes DNA Repository ("Repository") is a NHMRC funded project. The aim of the national Repository is to facilitate the identification of genes affecting the risk of developing Type 1 and child-onset Type 2 diabetes. To do so, we invite trio families (mother, father and affected child) to donate a DNA sample. We will archive this DNA and make it available to all qualified Australian researchers, enhancing their ability to identify causes of diabetes. Having DNA available from the parents of children with diabetes increases the ability to define diabetes susceptibility genes.
Dr Jemma Berry is currently supervising the extraction, collation and characterisation of the samples with Sarah Lilley. Sarah has been responsible for coordinating recruitment of families from around Australia. As of the end of January 2008, the Repository contains 3100 samples from over 1110 families. For 1000 of these families, DNA is available from both parents as well as the child with diabetes.
Jemma has inventoried and re-stocked the DNA from affected sib-pair and trio families brought over from WEHI by Prof Morahan. There are currently 119 sib-pair families and 449 trio families in this collection. These are now being processed for whole genome amplification to replenish the stocks prior to being added to the Repository archive, and further genetic analysis.
The Repository samples are being typed for a polymorphism (change in genetic layout) and are in the process of being replicated to increase the stock for distribution to other research laboratories. For further information, please see the ACDDR website.
Type 1 Diabetes Genetics Consortium
Dr Jemma Berry
Dr Jemma Berry is responsible for maintenance and organisation of our T1DGC DNA samples. Over 4,500 DNA samples have arrived from all over the world and these have been sorted into families and plated out ready for analysis. These samples are being tested for DNA variations in a region on human chromosome 19 that we previously identified. Our aim is to refine the linkage peak in this region and to identify candidate genes.
Jemma has also examined potential involvement of the "Toll-like receptor 5" (TLR5) gene in diabetes. This gene participates in the immune response by assisting the inflammatory reaction to foreign (microbial) agents. TLR5 has been examined in some of our T1DGC samples, identifying some new variants in this gene, however, they did not show a significant association with T1D.
Jemma is exploring a new technique to identify DNA polymorphisms. High resolution melting can sense the small temperature changes that occur with changes in the genomic DNA sequence. The process is being developed to analyse a common promoter polymorphism (IL12B) and, once established, will save time and money in processing samples. The technique is also being applied to other diseases to scan for polymorphisms in candidate genes identified via the systems genetics approach.
The major aim of the T1DGC is to use the huge DNA bank it has assembled to identify T1D genes. To do so, it is undertaking some of the largest genetic studies ever conducted. Prof Morahan is helping to plan and coordinate these studies as part of his role on the Steering Committee of the Consortium.
Programs in Systems Genetics
Funded separately from the Centre for Diabetes Research, we have established a special Program in Systems Genetics with the support of the University of Western Australia. Systems Genetics is the name coined by Prof Morahan for "third generation" genetic research, which aims to identify networks of interacting genes. This world-leading research will be applied to tissues relevant to diabetes, such as the pancreatic islets; and tissues involving the immune system. As described above, this work has already allowed us to make initial plans to begin Systems Genetics studies in human T1D.
Systems Genetics is a newly emerging science which incorporates data from many different levels of an organism, and integrates these data with the underlying genetic data. We are performing Systems Genetics analysis of the genetically well defined mouse strains (BXD) that descended from C57BL/6 and DBA/2 parental strains.
In this study, defined organs or tissues such as the thymus, spleen and pancreatic islets are being studied using microarray technology. As T1D is an autoimmune disease, these tissues and the genetic interactions within are of particular interest to us. Microarray technology allows us to analyse the gene expression patterns of over 48,000 transcripts per sample. The results are being accumulated in a curated, public database named WebQTL. The microarray data enable the online investigation of quantitative trait loci, allowing us to identify genetic networks and genes functionally upstream or downstream of a gene of interest. The data will be freely available to all researchers enabling them to define complex genetic interactions in animal models. These data will significantly improve our understanding of the complex genetic interactions that occur in tissues important to the development and progression of diabetes.
Genetic networks within brain. Each of the 20 mouse chromosomes is represented on one edge of a 20-sided polygon; each interior line connects a gene whose expression is significantly (P<10-4) correlated with the genotype of another gene.
Identifying "Molecular Signatures" of Type 1 Diabetes
Dr James Jooste
Type 1 diabetes (T1D) is caused by an immune response that goes wrong. We do not know what causes this to happen, but we do know that children who get T1D have certain genes which make them susceptible. We predict that immune system cells found in the blood (white blood cells) will have differences in expression of these genes and also in other genes which are influenced by these T1D "master genes". We now have the ability to measure the exact amount of expression that each of our 30,000 genes can produce at any one time. This is possible due to microarray technology.
In another arm of this research program, we will also test for the presence of the very immune system cells we think cause T1D. These are a special type of white blood cell (called T cells) that make an important immune system mediator (interferon gamma) when they encounter insulin. Scientists now think that these interferon-gamma producing T cells are the culprits which kick off the immune response leading to T1D. Prof Morahan has found that different versions of another immune system modifier gene, IL12B, could lead to either more interferon being made by these T cells (in which case T1D was more likely) or less interferon (in which case, people were less likely to get T1D.) The different patterns in gene expression that we see can also lead to protein changes in the blood plasma, so we will test for these changes as well. The different patterns of gene expression or the make up of plasma protein will be markers (molecular signatures) of the disease process.
As a result of this research program, we hope to define such signatures, and therefore be able to define patterns of either genes or proteins that increase the risk of developing T1D. This will lead to better diagnostic tests for people at risk, as well as identifying targets for prevention treatments.
Can this work? We think it can, because other researchers have found that people with another autoimmune disease do show altered gene expression patterns in their white blood cells. What we will be doing, for the first time in the world, is to see if we can identify molecular signatures of T1D in children who have different diabetes susceptibility genes. That is, we will not only focus on molecular signatures of T1D, but also molecular signatures of different genetic subtypes of T1D. This work is in an exciting new field of medical research that Prof Morahan is pioneering, and which he has termed "systems genetics". Systems genetics involves going beyond the idea that many genes influence one trait, which most other researchers work with. Instead we are looking at how diabetes susceptibility genes can have an effect on all genes and on the entire body. Research has shown that T1D involves both the immune system and the endocrine system, so we think that this systems genetics approach will be able to provide important new findings. Dr James Jooste will be working on this project in collaboration with Professor Tim Jones and his colleagues in the Department of Endocrinology and Diabetes at Princess Margaret Hospital. One hundred children presenting to the hospital with newly diagnosed T1D will be asked to participate in the study, providing blood samples soon after diagnosis and once again three months later.
James aims to identify T1D "molecular signatures" in three ways, by measuring:
- white blood cell gene expression using the microarray technology to measure the exact amount of expression of every one of our 30,000 genes
- the make up of blood protein composition using a process called mass spectrometry
- production of interferon gamma by T cells
All of these data will then be compared to the genotype of each child with diabetes. We expect to see the different patterns of gene expression, plasma proteins, or interferon production, will relate to the type of diabetes susceptibility genes that the children have. This systems genetics approach to analysis has never been applied to human disease before. A similar approach will be run in parallel using congenic mouse lines from Melbourne and the USA. We expect these results will show new insights into how T1D arises, how we may better identify people who could already be on the road to T1D, and how we may prevent it from occurring in those most at risk.
Mouse models of Type 1 Diabetes
Dr Lois Balmer; Emma Jamieson
We are continuing our experiments to define T1D susceptibility genes in mouse models. Mouse models allow us to look at genes that are relevant in humans. This work involves monitoring large groups of mice maintained in nearly germ-free conditions over periods of up to 300 days. Dr Lois Balmer and Emma Jamieson coordinate the genotyping and breeding of 17 different mouse lineage (strains). The project aims to create special inbred (congenic) mouse strains to help us find regions associated with T1D (candidate regions). We have produced new mouse strains which have narrowed the regions showing diabetes susceptibility genes and now need to test these strains to determine their diabetes status.
We have begun The Gene Mine breeding program, which will produce 1000 congenic mouse strains. We will use these in the search for a better mouse model for T1D. Lois has been responsible for genotyping important indicators (markers) within these strains and 10 have been identified for further study. As part of this project, she is developing a fluorescence-based experiment that will allow more rapid and inexpensive characterisation of relevant mouse gene variants.