SCCM Resource Library-02-Sur1 Pathway Genetic Variability May Impact MMP9 Secretion, Ca2+ Signaling, ICP, and Outcome in TBI

Video Transcription

Hello everyone, thank you so much for being here today. My name is Ben Zussman, I'm a fourth year medical student at the University of Pittsburgh School of Medicine.  Today, I'm going to be discussing a study that we carried out looking at genetic variants within the SIR-1 pathway, how those variants are associated to outcome and which biological  pathways might be suggested by the variants that are significant. Before I begin, I have no personal conflicts of interest to disclose, but my mentor and  the PI of this study, Dr. Ruchira Jha, is a paid consultant for Biogen, who are currently sponsoring a trial in the role and efficacy of SIR-1 inhibition in TBI and acute ischemic  stroke. To give some background information, the SIR-1 trypin-4 nonspecific monovalent cation channel  is a heteromeric ATP sensitive ion channel that likely mediates various secondary injuries  after TBI, including cerebral edema formation and traumatic intracerebral hemorrhage progression.  As I just mentioned, there's currently a phase two trial of the SIR-1 inhibitor glybaride ongoing in adult contusional TBI.  In previous studies, we used a candidate gene approach to demonstrate the genetic variants in the genes encoding SIR-1 and trypin-4 are associated with intracranial hypertension,  hemorrhagic contusion expansion, and functional outcome. However, the SIR-1 trypin-4 channel is only one part of a much more complex molecular  pathway and the components of this pathway, much less the significance of genetic variation within it, remain largely undetermined.  Thus, the goal of this study was to use a bioinformatics driven approach to first identify putative components of the molecular pathway associated with SIR-1 and trypin-4, test for  associations of genetic variants within the genes identified with outcome and secondary injuries, and finally, to query bioinformatics knowledge bases to identify molecules and  pathways that may merit further investigation. We began by using Ingenuity Pathway Analysis, or IPA, to identify a network of genes that  had previously been associated with SIR-1 and trypin-4 in the literature, not necessarily in the context of TBI.  Then, we queried exomic genetic variation within these genes in a single center cohort of severe TBI patients using an Illumina multiplex platform.  The identified polymorphisms were then tested for associations with hemorrhagic contusion  expansion, intracranial pressure, and Glasgow outcome scale score using backwards elimination multivariable regression models.  Finally, we reintroduced the list of genes that had polymorphisms associated with outcome to different bioinformatic tools in order to assess for prominent molecular functions,  cellular pathways, and possible regulators. For this final part of the analysis, we used three different bioinformatics platforms.  The first was Gene Ontology Knowledge Base, which allows us to identify biological processes and molecular functions associated with a gene list.  We subsequently entered the list of significant genes back into IPA in order to identify predicted  gene processes and functions and possible upstream regulators of the group of genes that we had found to be significant.  Finally, we used STRING, a platform which generates networks of protein-protein interactions in order to assess for functional enrichment of molecular functions.  The diagram shown here demonstrates the results of our initial IPA query. The genes encoding SIR1 and TRIPM4 can be seen in the center in blue, with the literature-defined  interactors filling out the remainder of the network. As you can see, a wide variety of both protein gene products and non-protein biological molecules  have been found to interact with SIR1 in the literature. All told, the network defined by IPA was comprised of 125 genes.  Of these, our platform queried for exomic variation in 113 of these genes for a total of 1,450 SNPs queried.  However, over half of these SNPs either had no variability in our sample set or insufficient variability to be successfully introduced into our regression model, and so 520 SNPs  across 97 genes were ultimately tested for an association with one of our three defined outcomes.  After using the BUI method for control of the false discovery rate, we found that 47 SNPs across 22 genes within the pathway, not including ABCC8 and TRIPM4 themselves, were  associated with one of our three outcomes. Interestingly, NFAT, one of the key regulators of the immune response and a target of the  drug cyclosporine, which has garnered significant interest itself in both preclinical models  and clinical trials of TBI, was the only gene other than ABCC8 and TRIPM4 to be associated with each of the three outcome classifications tested.  When the gene list was introduced to the gene ontology knowledge base, the top biological process activated was regulation of matrix metallopeptidase secretion with a greater  than 100-fold enrichment of this process. Of note, the GAMES-RP trial in acute ischemic stroke found that SIR1 inhibition with the  sulfonylurea glyburide significantly reduced plasma MMP9 levels. When the list was separated by outcome classification, there were no processes or functions significantly  enriched in genes associated with contusion expansion. Among the other two outcome classifications, both top processes and functions were largely  involved with activation of the immune response. When the gene list was reintroduced to IPA, perhaps unsurprisingly, the canonical pathway  most associated with the list was neuroinflammation, with the top disease being neurological disease and top molecular function of cell death and survival.  Of note, however, the top upstream regulator identified was SP1, which has previously been suggested to regulate ABCC8 or SIR1 expression in preclinical models of TBI.  On visually inspecting the network generated by IPA, which is shown to the right, many of the elements ultimately converge on BCL2, potentially signifying the importance of this  pathway to apoptosis and calcium regulation, two of the critical functions of the BCL2 protein.  Many of the other central nodes of the network, such as PARP1 and TLR4, have been previously studied in our laboratory as key regulators of TBI.  Finally, when the gene list was entered into STRING, it appeared to identify two separate protein interaction networks, one largely centered on NF-kappa B, another key regulator  of the immune response, and a second centered on SLC6A1, a sodium- and chloride-dependent GABA transporter.  Other central nodes include PARP1, one of the key mediators of necroptosis, TLR4, one of the most widely studied pattern recognition receptors, and a TNF receptor, another key  immune regulator. Looking at the top gene ontology terms identified by STRING, they were dominated primarily by cell surface receptors and ion transporters.  In this study, we identified several genes and genetic variants and biological processes that may impact outcome after TBI.  Our finding that matrix metallopeptidase regulation was the top biological process among significant  genes is consistent with the GAMES-RP trial, one of the most promising trials of SIR1 inhibition  in acute ischemic stroke, which found that SIR1 inhibition caused a reduction in plasma MMP9 levels and improvement in spedema-related outcomes.  SP1, the top upstream regulator identified by IPA, has previously been identified as  a possible regulator of SIR1 and of apoptotic mediators after TBI and could be worth investigating as a key molecular driver of secondary injury in this patient population.  Although cyclosporin has had inconsistent benefits in preclinical and clinical studies of TBI, the fact that NFAT was the only gene associated with all three outcome classifications  suggests that the putum molecular mechanism may still be of interest. And finally, many of the other nodes in the IPA and STRING analyses, including PARP1,  BCL2, TNF receptors, and TLR4, have been suggested to be key mediators of TBI and were performed both in our laboratory and others.  Ultimately, this study was meant to be primarily hypothesis generating and has a number of limitations.  We were limited by exomic data from a single center of largely white patients with a sample relatively small by genetic standards.  Our results should be replicated in a larger multicenter study. Our network itself was further limited by the literature searched by IPA, which is constantly changing.  And beyond this, almost one quarter of the genes identified in the pathway did not have sufficient variability in our sample to test for association with outcome.  And this is not including the multiple non-protein molecules that are identified as part of the pathway.  And finally, even if our identified genotype outcome associations are proven to be non-spurious,  the association between a genetic variant and outcome may be entirely unrelated to the expression or function of the gene product encoded by the gene within which the variant  is located. In conclusion, in this study, we identified a number of protein encoding genes and pathways that may play an important role in the development of TBI pathology.  NFAD and SP1 may play a key role in regulating secondary injury cascades after TBI and merit further study.  Regulation of ion transporters, including SIR1, ATP2A3, and SLC6A1, and enzymes requiring  divalent cations for activation such as calcium-calmodulin-dependent kinases and matrix metallopeptidases may be therapeutically actionable mediators of secondary injury.  Finally, our results cumulatively highlight that there are likely important differences  between the regulation of SIR1 and its associated pathway after TBI and the regulation of SIR1 and its pathway in the context of metabolic regulation.  And again, these findings should certainly be replicated in a large-scale multicenter study. I'd like to thank you for listening to my presentation, and I would like to thank my  mentors and collaborators, some of whom are shown here. I would especially like to thank Dr. Ruchira Jha for her exceptional mentorship over the  last several years, and Drs. David Okonkwo and Eva Puccio, in addition to the staff of the University of Pittsburgh Brain Trauma Research Center for their assistance in collecting  information and data for this study. Thank you again for listening to my presentation, and I hope that you enjoy the rest of this conference. I'm happy to take any questions.

Video Summary

The speaker, Ben Zussman, discusses a study he conducted on genetic variants within the SIR-1 pathway and their association with outcomes in traumatic brain injury (TBI) patients. The study utilized bioinformatics tools to identify genes associated with SIR-1 and TRPM4, and then tested genetic variants within these genes for associations with outcomes such as intracranial pressure and contusion expansion. The study found that certain genes and pathways, including NFAT and SP1, may play a role in secondary injury cascades after TBI. Additionally, regulation of ion transporters and enzymes may be therapeutically actionable mediators of secondary injury. Further research in a larger study is needed to validate these findings.

Asset Subtitle

Neuroscience, 2021

Asset Caption

The Society of Critical Care Medicine's Critical Care Congress features internationally renowned faculty and content sessions highlighting the most up-to-date, evidence-based developments in critical care medicine. This is a presentation from the 2021 Critical Care Congress held virtually from January 31-February 12, 2021.

Benjamin Zusman

Introduction/Hypothesis: The Sur1-Trpm4 channel is a promising therapeutic target for cerebral edema and contusion expansion in traumatic brain injury (TBI), with an ongoing Phase-2 clinical trial of pharmacologic channel inhibition. We employed a bioinformatic approach to identify genetic contributors of Sur1-related pathways to secondary injury and outcome.

Methods: We used Ingenuity Pathway Analysis (IPA) to construct a pathway of genes potentially related to Sur1, and assessed exomic single nucleotide polymorphisms (SNPs)in a prospective cohort of severe TBI patients (N=386) using multiplex platforms. Phenotypes included intracranial pressure (ICP), contusion expansion (CE), and Glasgow Outcome Scale (GOS). We used stepwise regression models, controlling for multiple comparisons using the Benjamini-Yekutieli method. We identified biological processes and molecular functions using the Gene Ontology Knowledgebase.

Results: IPA identified 113 genes putatively contributing to the Sur1 pathway: 97 had analyzable variants, totaling 520 SNPs. 47 SNPs in 23 genes were associated with ICP, CE, or GOS after TBI (p-values: 5.9x10-3-8.0x10-9). Regulation of matrix metallopeptidase (MMP) secretion was the top biological process (Fold enrichment [FE]: >100, FDR = 0.008) and pattern recognition receptor activity was the top molecular function (FE: >100, FDR = 0.008). Among 16 genes associated with ICP, response to abiotic stimulus was the top biological process (FE: 8.88, FDR = 0.014). Catalase activity was the top molecular function (FE: >100, FDR = 0.036). In 6 genes associated with GOS, the top biological process was regulation of response to macrophage colony-stimulating factor (FE: >100, FDR = 0.004) and NAD(P)+ nucleosidase activity was the top molecular function (FE: >100, FDR = 0.013). No associations with CE were identified.

Conclusions: We identified genetic variability in biological processes that may contribute to differences in Sur1 related inflammation/Ca2+ signaling, ICP and outcome in TBI. This may help identify novel Sur1-related therapeutic targets. Regulation of MMP secretion was the top biologic processâ€“ this is consistent with the GAMES-RP trial findings, where Sur1 inhibition in stroke lowered plasma MMP9. Further defining individual variability in the Sur1 pathway may allow for targeted interventions.

Meta Tag

Content Type Presentation

Knowledge Area Neuroscience

Knowledge Level Advanced

Membership Level Select

Tag Traumatic Brain Injury TBI

Year 2021

Keywords

genetic variants

SIR-1 pathway

traumatic brain injury

bioinformatics tools

intracranial pressure

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