Background/Purpose:

Interstitial lung disease (ILD) is a major complication and leading cause of mortality in scleroderma (SSc, systemic sclerosis). The morbidity and mortality rates in African American scleroderma patients are higher when compared with SSc patients of other races. We previously demonstrated that hepatocyte growth factor (HGF) is reduced in BALF and plasma from African American SSc-ILD patients compared with white SSc-ILD patients. Moreover, in African American SSc fibroblasts the anti-fibrotic effects of HGF are compromised due to a deficiency in phosphorylation of the HGF receptor (cellular mesenchymal-epithelial transition factor, c-MET). The present study was undertaken to identify potential inhibitory mutations in c-MET gene extracted from lung fibroblasts with impaired phosphorylation of the HGF receptor.

Methods:

Lung tissue was collected postmortem from SSc patients who fulfilled the ACR criteria for SSc and had evidence of lung involvement. SSc-ILD was confirmed by histological examination of postmortem lung tissue. Lung fibroblasts were isolated using standard procedures. RNA samples from lung fibroblasts were prepared by QIAGEN RNeasy^® kit; c-MET gene was extracted by QIAGEN OneStep RT-PCR Kit. The coding exons of c-MET were PCR amplified and sequenced on both strands on ABA Prism 377 Genetic Analyzer using taq dye terminator chemistry at our university’s DNA Sequencing Core. The sequence data were analyzed by BLASTN 2.2.18 using CCDS 47689. D1398G c-Met mutant was created using the QuickChange Site-Directed Mutagenesis XL kit from Stratagene; c-Met wild type and D1398G adenoviruses were generated by AdEasy Vector System, Quantum Biotechnology. HGF receptor phosphorylation, connective tissue growth factor (CTGF) and collagen content were studied by immunoblotting.

Results: We have identified six variants of c-Met unique for lung fibroblasts with non-functional HGF receptor. All of them are located in the C-terminus of the c-Met gene. We found that 100% of cell lines with non-functional HGF receptor carry the D1398G variant of c-Met gene; approximately 20% of all tested SSc lung fibroblasts contain C1379W and V1380G variants of c-Met gene; approximately 10% of cells with reduced HGF receptor function contain H1366P, L1351W, or E1350D variants of c-Met gene. None of these mutations was found in the c-Met gene isolated from SSc lung fibroblasts containing functional c-Met receptor. Lung fibroblasts isolated from African American SSc-ILD patients with D1398G variant of c-Met gene and normal lung fibroblasts transfected with D1398G c-Met mutant were characterized by limited effects of HGF on CTGF and collagen and by reduced HGF receptor phosphorylation on tyrosine 1238, 1252, and 1253.

Conclusion: D1398G variant of c-Met gene results in defective HGF signaling in lung fibroblasts. The occurrence of D1398G in African American SSc-ILD patients may explain in part a pathophysiologic link between African American race and poor pulmonary outcomes in SSc patients.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/d1398g-variant-of-hepatocyte-growth-factor-receptor-a-potential-biomarker-of-severe-interstitial-lung-disease-in-african-american-scleroderma-patients/