Eleni Tukuafu and John S.K. Kauwe, Department of Biology
Introduction
Rheumatic heart disease (RHD) is the most common form of acquired cardiovascular disease among children worldwide and results in 233,000 deaths annually. Although genetic and environmental factors are known to contribute to RHD, not much is known about genes that increase susceptibility. RHD results from untreated rheumatic fever (RF). RF develops when a Streptococcus pyogenes infection, such as streptococcal pharyngitis, goes untreated. About 34% of children with an untreated Streptococcus pyogenes infection develop rheumatic fever. Only 1/3 of those children acquire RHD1. This suggests that there is a genetic basis for why some children are prone to the development of RF/RHD, while others are not. Understanding the genetic architecture of RHD will allow for better prevention and awareness of this disease.
Previous studies have identified possible candidate genes, involved in both innate and adaptive immune responses that exhibit predisposing risk factors. My project focuses on identifying singlenucleotide polymorphisms (SNPs) in the toll like receptor 5 gene (TLR5). TLR5 is a pattern recognition receptor that is important for activating innate immunity. Polymorphisms in this gene may affect human defense against RHD.
The rates of RHD in Samoa are elevated and even within Samoa there are small geographic regions with statistically higher rates than others, again suggesting a genetic predisposition. An analysis of prevalence data from screening tests show an excess of RHD in two schools, Faletitu and Lapea. 15% of students from Faletitu and 12% from Lapea screened positive for RHD. The parents of these students were contacted to obtain pedigree identification. When multiple affected individuals were identified within a family, DNA was collected via saliva samples from all available family members. In this study, I will analyze the DNA samples from one of these families and determine the effect of gene variation on susceptibility. Understanding these variations will help us determine the extent to which MBL contributes to the development of RHD.
Methodology
I have access to 14 samples of DNA that were collected using DNA Genotek. The DNA was extracted using DNA genotek protocol using prep IT L2P. The primers used to locate our target DNA fragment, the TLR5 gene, were created using Primer3. I then performed polymerase chain reaction (PCR) using Roche PCR master mix to amplify our gene of interest. The PCR was cleaned using PrepEase PCR Purification 96 well plate kit from USB and then a sequencing reaction was run. The DNA was sequenced using BigDye v3.1 and the product of these reactions were cleaned using Sephadex columns. The clean samples were sent to the sequencing center at BYU to be Sanger sequenced on the ABI3730xl DNA Analyzer. We analyzed the TLR5 gene variants using Sequencher 4.9, a DNA sequence assembly and analysis software.
Results
Our preliminary findings led us to resample a sample pool that was 10x larger than what we initially planned. We have since found the SNP we were looking for through PCR and sequencing. We are continuing to do statistical analysis to find if there is a link between the SNP and RHD.
Discussion/Conclusion
Although environmental factors are known to contribute to the development of RHD in Samoa, our study suggests genetics may also play an important role. Further analysis of this SNP through statistical analysis could potentially reveal the association of the TLR5 gene and RHD. This information could aid in providing unique variant targets and effective prevention for areas of high risk.
1Guilherme L, Ramasawmy R, Kalil J. 2007. Rheumatic Fever and Rheumatic Heart Disease: Genetics and Pathogenesis. Scandinavian Journal of Immunology 66: 99207.