RNA Sequencing of Urine-Derived Cells for the Characterisation and Diagnosis of Osteogenesis Imperfecta.

Abstract

DNA sequencing is a reliable tool for identifying genetic variants in osteogenesis imperfecta (OI), but cannot always establish pathogenicity, particularly in variants altering splicing. RNA sequencing can provide functional evidence of the effect of a variant on the transcript but requires cells expressing the relevant genes. Here we used urine-derived cells (UDC) to characterize genetic variants in patients with suspected or confirmed OI and provide evidence on the pathogenicity of variants of uncertain significance (VUS). Urine samples were obtained from 45 children and adolescents; UDS culture was successful in 40 of these participants (age range 4 to 20 years 21 females), including 18 participants with OI or suspected OI who had a candidate variant or VUS on DNA sequencing. RNA was extracted from UDC and sequenced on an Illumina NextSeq550 device. Principal component analysis showed that the gene expression profiles of UDC and fibroblasts (based on GTEx consortium data) clustered close together, and had less variability than those of whole blood cells. Transcript abundance was sufficient for analysis by RNA sequencing (defined as a median gene expression level of ≥ 10 transcripts per million) for 25 of the 32 bone fragility genes (78%) that were included in our diagnostic DNA sequencing panel. These results were similar to GTEx data for fibroblasts. Abnormal splicing was identified in 7 of the 8 participants with pathogenic or likely pathogenic variants in the splice region or deeper within the intron. Abnormal splicing was also observed in 2 VUS (COL1A1 c.2829+5G>A and COL1A2 c.693+6T>G), but no splice abnormality was seen in 3 other VUS. Abnormal deletions and duplications could also be observed in UDC transcripts. In conclusion, UDC are suitable for RNA transcript analysis in patients with suspected OI and can provide functional evidence for pathogenicity, in particular of variants affecting splicing. This article is protected by copyright. All rights reserved.