Identifying and Narrowing Down Potential Causative Candidate Genes Associated

Identifying and Narrowing Down Potential Causative Candidate Genes Associated with Mast Cell Hypersensitivity in Hypermobility Ehlers-Danlos Syndrome Introduction Mast cells are a type of white blood cell found throughout the human body in connective tissue and are important in inflammatory response. Mast Cell Hypersensitivity (MCH) is a result of Mast Cell Activation Disorders (MCADs) that occur when mast cells degranulate or release mediators like cytokines, tryptase, histamine, and others through exocytosis at increased or abnormal rates.1 The physical manifestations of MCH is presented through profusely sweating or developing redness/irritation on the skin.2 Prior research has shown that many patients with the connective tissue disorder Hypermobile Ehlers-Danlos Syndrome (hEDS) present signs of MCH and are associated with MCADs.1-6 It is speculated that due to hEDS affecting elastin and collagen components of the Extracellular Matrix (ECM) that makes up connective tissue, this causes mast cells to be altered in stiffness and results in easier activation for degranulation. 7,8 However, this relationship is only strongly suggested, and MCH is not considered in the official hEDS diagnostic criteria which revolves around the Beighton Score and family history instead. 9,10,11 Thus, the purpose of this study is to clarify possible genes and pathways that could be responsible for causing MCH in patients with hEDS, strengthen this relationship, and pose potential candidate genes enabling genetic testing which currently does not exist for diagnosing hEDS. Q: Why do we swell after getting stung by a bee? A: Mast Cells! Results These images demonstrates joint hypermobility and skin hyperextensibility, key symptoms of hEDS Methodology Compiling a list of Possible Candidate Genes: A systematic literature review was carried out through PubMed, Google Scholar, and ScienceDirect to gather a list of genes linked to MCH and hEDS. Case studies involving a target audience and literature on the key pathways of either mast cell activation (MCA) or the effects of hEDS was reviewed. The following bullet points summarize each candidate gene and their pathway. ● TNXB — Encodes for Tenascin X which is an ECM protein organizing bridges formed between collagen fibrils.12 We propose that a TNXB mutation changes the collagen structure in connective tissue, consistent with the effects of hEDS, impacting mast cells and potentially showing an association with MCH. Numerous studies have also demonstrated that point mutations and TNXB haploinsufficiency have been identified in people with hEDS, further supporting this speculation.12 ● TPSAB1 — Encodes for alpha I tryptase or beta I tryptase, key proteins that are secreted when mast cells degranulate and can be used as biomarkers of some MCADs. 11,13 A previous study has found a subset of patients that had elevated tryptase levels (due to duplication and triplications of this gene) to be positive for hypermobility (Beighton Score > 4). 13 ● FMR1 — The Fragile X Mental Retardation Gene codes for fragile X mental retardation protein (FMRP) which lacks in patients diagnosed with fragile X premutation. 14 This lack of FMRP has also been shown to be associated with connective tissue disorders like hEDS as the level of FMRP regulates translation of proteins related to the connective tissue. 14 The study also found that 2 out of a sample size of 5 hEDS patients reported mast cell-like symptoms and had CGG repeat mutations in this gene.14 ● ADGRE2 — Encoding for the ADGRE2 adhesion G protein-coupled receptor protein in mast cells, a study found that a missense mutation in the gene most likely weakens the α and β subunit bond of the protein, causing mast cells to be more susceptible to degranulation caused by vibration/physical touch breaking the subunits’ bond easily.15 This phenomenon could explain why hEDS patients, who have weakened mast cell structures caused by altered ECM, present signs of flushing due to MCH. ● VIM — Encodes for vimentin filaments, which are the intermediate structural filaments that root granules in mast cells. We propose that mast cell stiffness is decreased due to irregular collagen network and softness in ECM caused by hEDS.8 A change in vimentin filament organization could lower the mast cell’s ability to resist compression and increase granule mobility and degranulation instead.8 ● FN1, ITGA5, ITGB1 — These encode for fibronectin, integrin alpha 5, and integrin beta 1 respectively. Integrin alpha 5 and beta 1 are subunits that come together to form integrin α5β1. This integrin anchors mast cells to the fibronectin glycoproteins of the ECM and are mechanotransducers participating in cellular communication.8,12,16 These are the results of the genetic analysis in the 20 patients WGS. Gray boxes means there was no presence of a variant on that gene in our sample size. Discussion & Conclusion: Genetic Analysis: Negative results for finding variations in FMR1, VIM, FN1, ITGA5, and ITGB1 indicate that those genes and their pathways are not responsible for causing MCH in patients with hEDS. However, there was a FN1 gene variant present in an hEDS patient without MCH, indicating its potential role in hEDS only. The presence of variants in TNXB, TPSAB1, and ADGRE2 point to their pathways and genes as potentially being causative of MCH in hEDS. More specifically, the presence of TNXB variations only corroborates the speculation by multiple previous studies for being a possible causative gene.17,18 The TPSAB1 mutation reinforces a case study of elevated tryptase levels being present in those with joint hypermobility and duplications of TPSAB1, pointing to the gene’s usage as a possible marker of hEDS and MCH.13 Finally, presence of an ADGRE2 mutation indicates that it is a viable candidate gene and pathway, as supported by a study which proved that the magnitude of strain on mice mast cells affected degranulation.19 In all, finding variants in these three genes is consistent with prior research speculating each of their respective impacts in causing MCH in hEDS patients. Significance: The overarching purpose of this study is to find potential causative genes for MCH in patients with hEDS and increase awareness about the disorder. The reason for this is because infants with hEDS can present multiple fractures due to bone fragility. However, because of limited awareness and a lack of genetic testing, pediatricians suspect the parents with Child Abuse & Neglect (CAN) and take away their children. Furthermore, this research study is one of the first to specialize in genetic analysis of MCH in patients with hEDS. At the time of writing this, there are no other studies focusing on the genetic basis behind the cause of MCH in hEDS, though some literature reviews have proposed causative pathways. In the future, these genetic variants should be analyzed in a larger population to confirm results, as many of these variants have not been reported or documented as significant before in ClinVar. Although the results are not fully conclusive about which specific genes are responsible due to the limited sample size, it demonstrates that MCH is a symptom that should be considered in the official criteria for diagnosing hEDS and aids a new field of research involving MCH in hEDS. Gathering Samples from Patients for Whole Genome Sequencing: Parents filled out a questionnaire to notify of hEDS symptoms and medical history for their child. A physical examination followed to verify signs of hEDS, prior to collecting DNA from buccal swabs for whole genome sequencing (WGS) of 20 patients (16 with hEDS and 4 without). Carrying Out WGS: WGS was used to detect genetic variations in DNA samples. Following sample quality control, Swift 2S protocol to minimize handling was used in performing PCR-free library preparatory process to obtain optimal variant detection.7 Then, HiSeq 2000 carried out WGS prior to running Fast Quality Control on the data and mapping the reads using BWA-MEM to chimerically align the sequences with a reference genome.7 The data was then uploaded to Illumina BaseSpace where genetic filters set to include prevalence even below 3% were used to detect genetic variations of the target genes.7 Genetic filters also utilized exact chromosome location, allele frequency, pathogenicity, and prevalence of variation. This graphic visualizes how mast cells may be impacted by altered ECM due to hEDS.8 These three images depict MCH physical manifestations in hEDS patients.