BIOLOGICAL SCIENCES

MOLECULAR GENETICS

Research in the genetics unit focuses primarily on the genetics of asthma and allergy, but also includes other respiratory diseases such as emphysema and chronic bronchitis. As one of the Australian National Cooperative Research Centres for Asthma and Airways (CRCAA) our work is focused on improving the health of asthmatics through providing new and improved treatments and advanced diagnostic and monitoring tools. More specifically, we aim to identify changes in the DNA of candidate genes and identify whether they contribute towards the clinical features, onset and severity of these diseases.

The basis of our experimental approach is through association studies linking the incidence of a particular disease with changes in the DNA of genes previously shown to be involved in inflammation and tissue repair. When an association is found, functional studies are conducted to determine whether the change has a direct effect the on the gene and its mode of action with respect to disease. In all cases our studies rely on volunteer recruitment and the use of human material from both test cases and healthy controls.

Current Research Activities

The majority of respiratory diseases are mainly as a result of either lung hyper-inflammation or tissue remodelling. It is now widely accepted that airway inflammation is the key factor underlying the pathogenesis of asthma. Inhaled corticosteroids remain the most important anti-inflammatory treatment for asthma. However, they are rather non-specific in their actions and their use raises concerns over side effects and compliance issues, particularly in children and adolescents. Moreover, a significant sub-group of asthmatic patients responds poorly or not at all to high-dose inhaled or systemic steroid treatment. Therefore, much effort is being made to develop novel more specific and safer therapy for asthma.

Abnormal remodelling of the airway wall is a characteristic feature of chronic asthma and is a dynamic process involving extracellular matrix (ECM) production, its degradation and altered structure. In this regard, the matrix metalloproteinases (MMPs), a family of proteases that degrade components of the ECM, and their specific inhibitors known as tissue inhibitors of metalloproteinases (TIMPs), have both been shown to be particularly important in this process.

With this background in mind our genetic studies focus on genes associated with the arachidonic acid pathway investigating the regulation of recognised inflammatory related genes and the MMP and TIMP family of genes association with ECM remodelling in asthma phenotypes.

Association Studies

Several ongoing studies in the lab investigate the presence of single nucleotide polymorphisms (SNPs) identified within specific inflammatory and remodelling family of genes and their link with asthma and/or asthma severity. With the assistance of adult asthmatic volunteers we are building a resource which enables us to screen specific gene SNPs across the different asthma phenotypes and identify whether there are association patterns between them.

MMP-9 and TIMP-1 SNP analysis

Several SNPs have been described in both the MMP-9 and TIMP-1 genes. For the purpose of our initial investigation into the association between SNPs in these genes and asthma phenotypes, we selected four of the five coding region polymorphisms reported by NCBI (www.ncbi.nlm.nih.gov) in the TIMP-1 gene as these may alter interactions with MMP-9, and four polymorphisms in the MMP-9 gene including one in the coding region and three promoter polymorphisms, the latter of which may be involved in altered gene expression.

In summary, we investigated the association between a number of polymorphisms in both MMP-9 and TIMP-1 genes and asthma, asthma severity and atopy using a large, carefully phenotyped white Australian population. Results from this study show that four MMP-9 and TIMP-1 polymorphisms were not associated with asthma, but a novel polymorphism in the TIMP-1 gene (536C.T (Ile158Ile)) was significantly associated with mild asthma in women and TIMP-1 haplotypes were also associated with asthma. The functional significance of the 536C.T polymorphism has still to be determined, but its association with asthma severity highlights the potential importance of proteases and antiproteases in this socioeconomically important disease. (Ref: Thorax. 2005 Aug; 60(8):623-8.)

We are continuing to investigate other SNPs within the tissue remodelling MMP family of genes in our asthma population in an effort to find candidates for improved drug therapy.

Alternative Splicing

In addition to the SNP association approach towards investigating genetic influences in respiratory disease, we have chosen several candidate genes to study the influence of aberrant spliced gene products in relation to asthma and airway disease. This recent advance is in its early stages, however we are hopeful results will provide additional opportunities for the design of new therapies for respiratory disease.

Current Contract Research

N/A

Potential PhD Project Areas

o Pharmacogenetics of the leukotriene pathway in asthma
o Genotype associations and asthma severity
o Genetics of proteases and anti-proteases in chronic obstructive pulmonary disease (COPD)
o Genetics of airway remodelling in respiratory diseases
o Genetics of lung cancer

Contact person (s): Dr Carolyn Williams

Potential Honours Projects

A. Genetic variants of PGE2 receptor genes in asthma.

1. Genotyping of previously identified single nucleotide polymorphisms (SNPs) in a defined asthma cohort to determine whether an association exists between the SNP and disease;
2. Use a variety of cell-based assays to determine the functional significance of SNPs found to be associated with disease; and
3. Perform real-time PCR analysis of alternative splice variants of the PGE2 receptor genes in asthmatic and non-asthmatic individuals.

Techniques:
• Literature search / project design
• Primer design
• PCR
• Population screening
• SNP association and statistical analysis
• Construct design and Cloning
• Luciferase assay

Contact person (s): Dr Estri Arthaningtyas

Potential Undergraduate Cadetship Projects

A. Aspirin-Intolerant-Asthma Project

1. Validate previous association found in the first study. Screen 2 SNPs we have screened previously and confirm original finding, that there is an association between the AIA and Non-AIA cohorts; and
2. New analysis of 2 newly screened SNPs in the entire asthmatic cohort. The student is to decide which 2 SNPs to screen based on scientific merit. Ie create or destroys a transcription binding site etc.

Techniques:
• PCR
• Primer design
• Population screening
• SNP Association statistical analysis

B. Bradykinin B1 Receptor Promoter Functional Studies

1. Site-directed mutagenesis of the wild type BDKR B1 weak promoter to create a construct containing previously validated SNPs; and
2. Functional assays to determine the differences in transcriptional activity between the wildtype BDKR B1 promoter construct and the SNP(s)-containing promoter construct in 2 different cell lines (HeLa, and cultured epithelial cells-16HBE) under different stimulation conditions.

Techniques:
• PCR
• Sequencing
• Site directed mutatgeneiss and cloning
• Cell culture and stimluation
• Luciferase assays
• Statistical analysis

C. iNOS Splice Variants and Asthma

1. Establish techniques for splice variant detection and isolation in normal tissues/cells; and
2. Investigate differences in levels of expression between asthmatic and non-asthmatic samples

Techniques:
• Literature search to identify splice variants and identify best cell line
• Sequence analysis
• Primer design
• PCR
• Sequencing
• Cell culture
• RT PCR
• Real time PCR

Contact person (s): Bradley Shelton

Other Useful Information

Location and Contact Details
WAIMR, First Floor, B Block, SCHG
+ 61 8 9346 7948

carolynw@liwa.uwa.edu.au