A novel splice-altering frameshift variant in the COL1A1 gene underlies osteogenesis imperfecta type I: molecular characterization of a four-generation Chinese pedigree and literature review.

Backgroud: Osteogenesis imperfecta (OI) is a phenotypically and genetically heterogeneous group of inherited connective tissue disorder. This investigation aims to elucidate the molecular etiology underlying a four-generation Chinese family affected by OI.

Methods: Whole-exome sequencing was employed to identify pathogenic variants in the proband, with subsequent Sanger sequencing performed for familial co-segregation analysis. A minigene assay was conducted to investigate the effect of variant on splicing patterns. The pathogenic potential of variant was evaluated through protein structural modeling and HEK293 cell-based functional studies. COL1A1 splicing variants were further collated to analyze its occurrence frequency across geographically diverse OI cohorts, intronic distribution patterns and potential hotpots for mild versus severe subtypes.

Results: Multiple affected members within an non-consanguineous Chinese pedigree exhibited clinical manifestations fitting OI-associated phenotypic spectrum. A novel heterozygous COL1A1 splicing variant (c.370-2A > C) inherited from the mother was identified in the proband. The splicing variant altered the canonical acceptor site (AG) at the intron 4-exon 5 junction, activating a adjacent cryptic splicing site in exon 5. This abberrant splicing event introduced a frameshift variant (c.370_379delGGACCCGCAG), and generated a premature termination codon that truncates the COL1A1 protein (p.Gly124Alafs*138). AlphaFold3-based protein structural modeling revealed the loss of the triple-helical domain in this truncated protein. In vitro functional assays showed that mRNA and protein expression levels of mutant COL1A1 were significantly increased than wild-type COL1A1 (p < 0.05). Comprehensive literature analysis indicated that COL1A1 splicing variants account for < 10% of variants in OI cohorts from the vast majority of regions. The acceptor site of intron 9 and the donor sites of intron 35 are hotspots for COL1A1 splicing variant occurrence. Moreover, the majority of COL1A1 splicing variants, expecially those proximal to the 5' and 3' terminal regions, result in mild manifestations of OI type I, whereas variants at donor sites of introns 14, 20, and 46, may be candidate hotspots for lethal OI type II.

Conclusions: Our study revealed the pathogenic mechanism of a novel COL1A1 splicing variant in a four-generation Chinese family with OI, and provided updated data on COL1A1 splicing variants and its potential hotpots for mild versus severe OI subtypes.