MUC5B Promoter Polymorphism and Survival in Indian Patients With Idiopathic Pulmonary Fibrosis

Methods We performed this prospective, observational study between September 2018 and October 2021 at our Chest Clinic after ethical approval (Institutional Ethics Committee, NK/3773/MD/921). We included consecutive, consenting subjects with IPF who had been diagnosed with the use of standard criteria, as described previously.7-9 We excluded subjects with a family history of interstitial lung disease. The control subjects were recruited from the hospital staff and patients' unrelated accompaniments after exclusion of chronic respiratory illness. We recorded the demographic details, smoking status, date of onset of respiratory symptoms, comorbid illness, baseline FVC that was measured by spirometry and the calculated percentage-predicted FVC, percentage-predicted diffusion lung capacity for carbon monoxide, and findings on CT scan of the chest of the subjects with IPF. We followed the subjects longitudinally and recorded the date of death, when applicable. We finally recorded the vital status of all subjects in October 2021 by contacting them or their next of kin telephonically. We extracted genomic DNA from peripheral blood cells of the IPF and control subjects using a commercial DNA extraction kit (QIAamp DNA Blood Mini Kit; Qiagen). We then amplified the DNA by polymerase chain reaction using primers for the promoter region of the MUC5B gene, purified the DNA (QIAquick; Qiagen) and performed sequencing using the Big Dye terminator sequencing kit version 3.1 on ABI 3500 series genetic analyzer (Life Technologies, Thermo Fisher Scientific).10 The sequencing profiles were analyzed for the genotypes of rs35705950 on Finch TV (Perkin Elmer, Geospiza). We calculated the proportion of subjects with the wild -type (GG), heterozygous (GT), and homozygous (TT) genotypes in the IPF and control cohorts. We explored the difference in the minor allele frequency (MAF) of the MUC5B promoter SNP rs35705950 in the IPF and control cohorts (primary outcome). We also explored the association of this SNP with survival in the IPF cohort (secondary outcome). We analyzed data using the statistical package SPSS (version 23.0, IBM Inc) and assumed statistical significance at a probability value of < .05. To test whether the variant was associated with IPF disease risk, we tested conformity to Hardy-Weinberg equilibrium (HWE) using the chi-square test in the cases and control subjects. We tested conformity to HWE using the chi-square test. We assumed an allele frequency of 12% in the control population.11 We selected the number of control cases as 1.5 times the number of cases. We calculated the sample size (90% power; alpha error of 5%) to detect an association between the SNP of interest and IPF with an OR of at least 3, which is well below the published pooled OR of 4.85.2 We compared the ratio of heterozygous (GT) subjects to those with a wild type (GG) genotype between the cases and control subjects using the chi-square test and presented the OR. We performed a similar analysis for individuals homozygous (TT) for the risk allele. The Kaplan-Meier method was used to estimate cumulative survival from the time of onset of respiratory symptoms in the two genotype groups: wild-type (GG) and variant (GT or TT). Univariate and multivariate Cox proportional-hazards regression analyses were used to evaluate the effect of the genotype and other relevant covariates on survival from the time of onset of respiratory symptoms; the HRs with 95% CIs were calculated.

Automated summary

This study is a prospective observational study that aimed to investigate the relationship between the MUC5B promoter SNP rs35705950 and IPF, as well as its impact on the prognosis of IPF patients. IPF patients who met the standard diagnostic criteria were included as the case group, and control subjects were recruited from hospital staff and patients' companions. By performing DNA sequencing and statistical analysis on the subjects, the study provides evidence for the difference in the genotype proportion of MUC5B promoter SNP rs35705950 between IPF and control groups, and a possible association of this SNP with the survival rate of IPF patients.