AbstractBicuspid Aortic Valve (BAV) disease is the most common congenital heart defect with an overall frequency of 0.5 – 1.2%. The normal aortic valve has three leaflets (tricuspid aortic valve) regulating blood flow between the aorta and the heart. However, during the embryonic development of individuals with BAV, two adjacent leaflets (cusps) fuse to a single large cusp. Only two leaflets instead of the usual three are formed leading to a bicuspid aortic valve. BAV increases the risk of aortic valve stenosis, aortic valve insufficiency, and thoracic aortic aneurysm. Both aortic valve stenosis and aortic valve insufficiency make the heart work harder and less efficiently. Thoracic aortic aneurysm increases the risk of aortic dissection which can lead to aortic rupture and death. DNA samples were collected from consenting patients by the Yale Cardiovascular Genetics Program. Then the samples were processed for Polymerase Chain Reaction (PCR) analysis and underwent PCR. PCR products then underwent gel electrophoresis. This data was then entered into Microsoft Excel. The data was then filter through based on genes of interest, frequency, significance, bioinformatic databases, etc. ANKRD12 and MNT had been identified as a possible genetic mutation associated with Bicuspid Aortic Valve. Additional data will have to be analyzed to further support this result.IntroductionBicuspid Aortic Valve (BAV) disease is the most common congenital heart defect with an overall frequency of 0.5 – 1.2% (Siu et al.). The normal aortic valve has three leaflets (tricuspid aortic valve) regulating blood flow between the aorta and the heart. However, during the embryonic development of individuals with BAV, two adjacent leaflets (cusps) fuse to a single large cusp. Only two leaflets instead of the usual three are formed leading to a bicuspid aortic valve. BAV increases the risk of aortic valve stenosis, aortic valve insufficiency, and thoracic aortic aneurysm. Both aortic valve stenosis and aortic valve insufficiency make the heart work harder and less efficiently. Thoracic aortic aneurysm increases the risk of aortic dissection which can lead to aortic rupture and death. Bicuspid Aortic Valve has been shown to be heritable (Cripe et al.). A study identified chromosomal regions 18q, 5q, and 13q as likely to contain genes whose mutations can cause BAV (Martin et al.). In addition, mutations in chromosome 16p13.1 have been shown to increase the risk for thoracic aortic aneurysms and dissections (TAAD) (Kuang et al.). TAAD and BAV frequently occur together. All these findings indicate that specific gene mutations may result in BAV. Currently, not many gene mutations have been found that are associated with BAV (Prakash et al.). As a result, identifying gene mutations for the most common congenital heart defect is very important. Based on the study completed by Martin et al., the regions most likely to contain genetic mutations linked to BAV are 18q, 5q, and 13q. It is anticipated that some genetic mutations that are found will be located in these regions.MethodsI will receive the de identified patient data from my mentor. The data will be contained in an Excel data sheet. I will be primarily using the filter features of Excel to narrow down my data and identify a genetic marker. I will restrict the maximum population percentage to be less than 1%. This is done to increase the possibility that the genetic mutation is harmful. If a large percentage of the population carries this genetic mutation, then it is more likely that the genetic mutation is benign. The Phylo score is then filtered such that only the gene mutations with a Phylo score of .5 or more are shown. The Phylo score predicts how dangerous a gene mutation is on a scale from -1 to 1. The closer the score is to 1 the more dangerous is can be. Next, intronic and synonymous gene mutations were filtered out. FACTS ABOUT SYNONYMOUS GENE MUTATION. I then focused on analyzing genetic mutations of specific chromosomes. The first genetic mutations that were analyzed were in chromosome 5, 13, and 18. This was based off the study done by Martin et al. Additional chromosomes were analyzed. Once gene mutations were identified and flagged as possibly associated with BAV, genetic databases were used to find where the genes were expressed. GTEx Portal was used to find the regions of the human body that the gene mutation was expressed in. (The GTEx Consortium) The flagged genetic mutations were further validated or invalidated if they were or were not expressed in the heart or artery. Future data sets will be analyzed to validate the results from this patient data set.ResultsANKRD12 and MNT were identified as a possible genetic mutations associated with Bicuspid Aortic Valve. Using the GTEx Portal, they could increase risk for Bicuspid Aortic Valve because it is expressed in the arteries and the heart. No research was found through OMIM that validated or negated the effects of these genetic mutations. ANKRD12 was located at 18p11.22, while MNT was at 17p13.3 (see table 1). This information was in the Excel spread sheet. This was confirmed using OMIM.GeneChrom.CytobandConsequenceRefAltGenotypeSIFTPolyphenMax. PopulationANKRD121818p11.22Missense variantCAHet. 0/10.020.4980.0001MNT1717p13.3Missense variantTCHet. 0/1000.00017Table 1 Potential genetic markers and some of its attributesDiscussionThe aim of this research was to identify gene mutations associated with Bicuspid Aortic Valve Disease. This was achieved through a combination of Excel filtering and the usage of bioinformatic databases. This experiment was able to identify possible novel genetic markers for BAV. The viability of ANKRD12 and MNT as genetic markers for BAV was supported by bioinformatic databases such as GTEx Portal and OMIM. GTEx Portal listed the types of the human tissue that specific genes have been shown to impact. ANKRD12 and MNT were both shown to be expressed in the heart and arteries. This increases the possibility that ANKRD12 and MNT are viable genetic markers for BAV. OMIM was used to find any previous literature that linked a specific genetic variant to BAV. Both ANKRD12 and MNT contained no previous literature that had linked them to BAV. ANKRD12 is further supported by the Martin et al. study. It had stated that 18q, 13q, and 5q were chromosomal regions that were most likely contained genes associated with BAV. ANKRD12 was in the 18 chromosome, but it was located on 18p not 18q (see table 1). There were some interesting gene mutations that are possible genetic markers for BAV. One such questionable gene mutation was ASPN. GTEx Portal had stated the ASPN was expressed in the arteries. The one detractor that made ASPN questionable was that its maximum population frequency was 0. This made ASPN a questionable genetic marker and will have to discussed in the future. One limitation to this study was that the genome of only one subject was analyzed. Addition subjects will have to be analyzed to add to the credibility of ANKRD12 and MNT as genetic markers for BAV. Future work entails analyzing the genome of more subjects to make ANKRD12, MNT, and other potential genetic markers more credible and viable.ReferencesCripe, L., et al. “Bicuspid Aortic Valve is heritable.” Journal of the American College of Cardiology. 2004.Kuang S.Q., Guo D.C., Prakash S.K., et al. “Recurrent chromosome 16p13.1 duplications are a risk factor for aortic dissections.” PLoS Genet. 2011.Martin L.J., Ramachandran V., Cripe L.H., et al. “Evidence in favor of linkage to human chromosomal regions 18q, 5q and 13q for bicuspid aortic valve and associated cardiovascular malformations.” Hum Genet. 2007.Prakash, Siddharth K. et al. “A Roadmap to Investigate the Genetic Basis of Bicuspid Aortic Valve and Its Complications: Insights From the International BAVCon (Bicuspid Aortic Valve Consortium).” Journal of the American College of Cardiology 64.8 (2014): 832–839. PMC. Web. 15 Jan. 2018.Siu, S.C. and Silversides, C.K. “Bicuspid aortic valve disease.” Journal of the American College of Cardiology, Vol. 55, No. 25, 2010.The GTEx Consortium. “The Genotype-Tissue Expression (GTEx) Project.” Nature genetics 45.6 (2013): 580–585. PMC. Web. 28 Jan. 2018.