Abstract Rationale Aging predisposes to multiple lung diseases including idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and cancer. Somatic mutations are a hallmark of aging. Yet the consequences of somatic mutations for lung diseases such as IPF are unclear. Recent results suggested an association between somatic mutations and IPF; however, this conclusion relied on variant allele frequencies (VAFs) from mRNA, which are distorted by gene expression levels and error-prone RNA polymerases. Moreover, short-read sequencing error rates (1-10%) are much higher than the expected rate of somatic mutations (0.01-1%) in non-cancerous tissues. Thus, the prevalence of somatic mutations in IPF, and the genes affected, still remain uncertain. Methods Duplex sequencing can detect variants at frequencies of 10-7 in as little as 0.5µg of DNA. We prepared libraries from 8 control and 27 IPF lung tissues using a custom paired-end barcoding and hybrid capture kit from TwinStrand Biosciences (Seattle, WA). Tissue diagnoses were validated by a multidisciplinary committee of pulmonologists, pathologists, and radiologists from our University of Colorado lung tissue biobank (IRB #15-1147). Our custom library covering 29 cancer-associated genes across 48.5kb of the genome was sequenced on an Illumina NovaSeqX in the University of Colorado Genomics Core. We identified variants from paired-end FASTQ files using “deepUMIcaller”, converted VCF files into MAF files using “vcf2maf”, and analyzed differences in variant abundance the “maftools” package and generalized linear models in R. Results Somatic variants were distinguished using a VAF0.4 and an allele depth of at least 1000 reads per position. We detected over 17.6x106 variants from 29 cancer-associated genes . Overall, there was a non-significant trend toward higher variant frequencies in IPF specimens. Protein-altering somatic mutations were significantly increased in SMAD4, a regulator of TGFβ family signaling, as well as TP53, a critical regulator of the DNA damage response and cell cycle progression, in IPF samples. Accordingly, we found enrichment for base substitution signatures associated with defective DNA mismatch repair in IPF (SBS6). Conclusions Given the relevance of TGFβ family signaling in IPF pathogenesis the increased variant burden in SMAD4 may suggest a clonal escape mechanism or causative mutations. Moreover, the increased mutation burden in TP53 could be a cause of defective mismatch repair suggested by mutation signature analysis. Further gain-of-function and loss-of-function studies may help sort out the relevance of these variants in IPF. This abstract is funded by: University of Colorado Clinical and Translational Sciences Institute, UM1-TR004399
Kurche et al. (Fri,) studied this question.