Preliminary Exploration Of The Role Of Susceptibility Genes In The Pathogenesis Of Systemic Sclerosis Associated Interstitial Lung Disease

Background and ObjectiveSystemic sclerosis(SSc)is a common connective tissue disease characterized by localized or diffuse skin thickening and fibrosis,which can affect the visceral,such as heart,lung,digestive tract,kidney and so on.Pulmonary involvement is a common extra-skin feature of SSc and serves as the leading cause of death.Interstitial lung disease(ILD)is the most common and potentially most devastating manifestation of SSc in the lung.Some epidemiological studies,molecular biology researches,and genetic reports may provide evidence in the pathogenesis and prognosis of SSc associated ILD(SSc-ILD),nevertheless,it is not enough to fully explain the mechanism of lung involvement in SSc patients.Moreover,most of the researches at the genetic level are based on the studies of European and American races rather than Chinese people.Therefore,an exploration of the rare mutated genes possibly inducing ILD in Chinese patients with SSc might further verify the existing research results,simultaneously,predicting the risk of ILD in patients in the early course of disease so as to timely initiate appropriate monitoring and treatments.This work aims to explore the potential candidates for SSc-ILD upon the whole exome sequencing(WES).Furthermore,combined with the clinical characteristics of patients,the biological functions of candidate genes and the corresponding changes of downstream products,this research attempts to analyze the possible pathogenesis of SSc-ILD from the perspective of genetic level.MethodsA total of 80 outpatients admitted to Peking Union Medical College Hospital between 2016 and 2019 were enrolled,including 30 patients with SSc without ILD and 50 patients with SSc-ILD.Totally,208 subjects from health examination centers were selected as the control group.Peripheral blood samples were extracted from all patients for whole-exome sequencing and sex hormone concentration.The gene variations found by the sequencing were annotated,filtered by a series of methods including the mutant frequency,deleterious analysis,SKATO analysis and geneotype-phenotype association,to find the potential pathogenic genes of SSc-ILD.Furthermore,in combination of the clinical characteristics of patients,the biological functions of candidate genes and the corresponding changes of downstream products,including testosterone,dehydroepiandrosterone sulfate,estrogen and prolactin,this research attempts to analyze the susceptibility genes of SSc-ILD.Results(1)In comparison of patients with SSc without ILD,patients with SSc-ILD showed older age(P<0.001),longer duration of disease(P=0.009)and higher levels of antibodies(anti-Scl-70 P=0.004)and inflammatory indicators,like ESR(P=0.004),NLR(P<0.001).However,there showed no statistical difference in sex,ACA,hsCRP and glucocorticoid therapy alone(P>0.05).(2)After manual review and prioritization,we identified eight known genetic mutations(DSP c.269A>G p.Q90R,DSP c.2453T>C p.L818S,CTGF c.622G>A p.A208T,MAPT c.256G>A p.G86S,SOX5 c.1102A>C p.S368R,NLRP1 c.1606C>T p.R536W,IRF5 c.179A>G p.D60G and MUC2 c.5627C>T p.P1876L)contributing to the SSc-ILD phenotype and one denovo mutation(SLAIN1 c.219₂20insGG p.A73fs)in the trio family.(3)The SKATO analysis mainly showed that RGPD4 mutation may be related to SSc-ILD disease(P=8.13×10-7,OR=17.25).(4)Combined with the clinical presentation of the patients,patients carrying the truncating mutation of RGPD4 showed the female predominance,higher titer of ANA and anti-Scl-70 antibodies and NSIP demonstration in the radiological findings.Additionally,this mutation might result in loss of function of protein by predicting the secondary structure of the protein in 3D model,which might correlate with the severe clinical manifestation of patient.(5)RGPD4 mainly expressed in the testis in human.RGPD4 mainly involve in the interaction of another four proteins by PPI analysis,including RAN,UBE2I,SUMO1 and XPO1,and three of these four genes modulate the function of androgen receptor(AR).Gene oncology(GO)analysis identified that RAN and SUMO1 together regulate the binding feature of steroid hormone receptor(GO:0035258,FDR=0.0378).Upon the GO and PPI analysis,we supposed that RGPD4 might indirectly affect sex hormone levels by interacting with proteins such as RAN,UBE2I,or SUMO1.(6)Lower testosterone concentration was found in the patients with SSc-ILD than the healthy controls in small sample verification experiment.Further,among patients with SSc-ILD,those carrying mutation of RGPD4 owned lower level of testosterone than those without RGPD4 mutation(P=0.08).Another three sex hormones showed no difference between the patients carrying RGPD4 or not.From point-biserial correlation analysis,the mutated status of RGPD4 showed a negative correlation with the concentration of testosterone(B=-0.509,P=0.037).ConclusionGenes are involved in the development of SSc-ILD,and RGPD4 might be a novel pathogenic gene for SSc and is related to the ILD phenotype.Carrier status of RGPD4 variants might correlate with the levels of testosterone in patients with SSc-ILD and lower levels testosterone might lead to dysfunction in immune system and telomere length.However,prospective studies investigating RGPD4 mutations in Chinsese SSc-ILD patients are necessary to refine risk estimates and to determine the clinical implications of the current findings.