ESPE Abstracts

Background and Objective: The spliceosome-associated protein CWC27 (CWC27) is the main component of the spliceosome and plays an important role in the post-transcriptional modification of mRNA. Retinitis pigmentosa with or without skeletal abnormalities (RPSKA) is an autosomal recessive syndrome caused by variation in the CWC27 gene. The main clinical manifestations of RPSKA include short stature, retinitis pigmentosa, craniofacial deformity, and intellectual impairment. Patients with RPSKA consistently presented with short stature but how CWC27 variants lead to short stature has not been studied.

Methods: We collected the clinical data of a patient with short stature and performed the genetic testing. The construction of the exon-trapping plasmid and the minigene splicing assay were used to verify the abnormal splicing caused by the splicing site variant of CWC27 gene. Furthermore, we knocked down the CWC27 gene in chondrosarcoma cell line (SW1353) by shRNA. Full-length transcriptome sequencing was performed to screen for chondrogenesis-associated differentially expressed genes and aberrant transcript fusion.

Results: The patient is an eight-year-old girl with short stature (-2.28 SDS), retinitis pigmentosa, special face (triangular face, oblique left eye, low ear position), and severe intellectual disability (by WISC-III NL Wechsler intelligence scale for children). Whole exome sequencing indicated that the patient carried a homozygous splice site variant of CWC27 (NM_005869.4) c.397-1G>A. The splicing variant leaded to three kinds of abnormal transcripts (r.614del, p.Val133Leufs14*; r.614_620del, p.Val133Glyfs12*; r.614_712del, p.Val133_Glu165del), and the CWC27 protein stability of all transcripts decreased obviously. In cellular models, CWC27 knock-down SW1353 cells showed significant differences in extracellular matrix, extracellular structure, growth factor binding, and transforming growth factor beta binding pathways compared to wild type. Among them, the expression of cartilage extracellular matrix-related genes (CHI3L1, CTSD, and FN1) was decreased. In addition, two abnormal transcription fusions related to cartilage development, ADAMTS6 - NMI and FGF2-PSMD11, have also been found.

Conclusion: In this study, we verified the pathogenicity of the CWC27 variant in vitro functional assay. Moreover, loss of CWC27 protein function caused abnormal gene expression and gene fusions.

Keywords: RPSKA, CWC27 gene, full-length transcriptome sequencing, extracellular matrix.