SNV Regulation of Diabetic Wound Re-epithelialization Mechanisms: T2D vs T1D

About This Grant

Patients with diabetes mellitus (type 1 or 2) have a total lifetime risk of a diabetic foot ulcer (DFU) complication as high as 25%; 14-24% of them suffer from amputation. People with T1D develop DFU at a younger age and are at a much greater risk of amputation and hospitalization secondary to a DFU compared to T2D. The difference between pathophysiology and outcomes for individuals with T1D versus T2D is poorly understood and understudied. Family and twin-based studies have identified significant genetic components especially single nucleotide variations (SNV) in T1D as compared to T2D subjects. However, systematic patient-based genetic studies of T1D DFU are scanty, and the proposed work is aimed at seeding a novel paradigm in wound healing research. The originality and strength of our study stems from the genome-wide genotyping feasibility studies on robust quality controlled and parametrically qualified genotyped data of 149 chronic wound patients with diabetes status. This study identified 20576 SNV significantly associated with human chronic wounds (p- value<0.01, CR>97%, MAF>0.01). Majority (>60%) of these SNP were predicted to be causative for truncated or nonfunctional proteins using Variant Effector Prediction analysis were identified. To investigate the clinical significance of wound associated SNV, a meta-analysis against the phenotypes annotated in GWAS catalog was conducted as reported. These SNVs were intersected with manually curated >270,000 GWAS SNPs annotated with ~900 GWAS phenotypes collected from ~2500 studies. Enrichment analysis of the above intersected SNVs was performed against these GWAS phenotypes and respective odds ratio, and level of significance were calculated using Fisher’s exact test. These analyses identified “obesity” as the most significantly enriched GWAS-phenotype (log2 odds ratio = 4.06, p-value= 4.94E-12) for wound associated SNPs predominantly present in fat mass and obesity-associated (FTO) gene. This proposal is responsive to RFA-DK-26-009 for the New Investigator Gateway Award for collaborative type 1 diabetes (T1D) Research through Diabetic Foot Consortium (DFC). The objective of the proposed work is to determine SNV T1D and T2D that contribute to diabetic wound closure. This study will investigate the wound tissue already collected from patients with open DFU (N=50 with T1D and n=100 T2D) enrolled in the DFC Master Protocol. The following specific aims are proposed: 1.0 Aim 1. Identify SNV uniquely associated with T1D non-healing phenotype. T1D vs T2D will identify T1D-specific SNV (SNVT1D). Healing vs non-healing will identify SNVT1D-NH. SNVT1D-NH will be shortlisted to obtain candidate SNVT1D- NH (cSNVT1D-NH) based on overlap with obesity-associated SNP. 2.0 Aim 2. Test the functional significance of cSNVT1D-NH in wound healing mechanisms in vitro. Gene editing to specifically induce risk to non-risk alleles of specific cSNVT1D-NH using CRISPR/Cas9 genome editing improves: 2.1 epidermal keratinocyte migration in an in vitro scratch model; 2.2 formation of well-perfused and non-leaky vessels by microvascular endothelial cells using 3D-angiogenesis assay; and 2.3 augmentation of collagen deposition and maturation by dermal fibroblasts.