NOVEL SPLICING MUTATION OF COL1A1 GENE CAUSING OSTEOGENESIS IMPERFECTA TYPE I IN CHINESE PEDIGREE

Objective To detect the peculiar mutation in a Chinese family with osteogenesis imperfecta, COL1A1 and COL1A2 being analysed. Methods A genome screen was undertaken covering COL1A1 at 17q21- 22 and COLIA2 at 7q22.1. The Linkage （ Version 5. 1 ） was used for 2-point analysis. DNA sequencing was used to screen and identify the mutation. Results A linkage to the markers on chromosome 17q21-22 was observed. Se- quence analysis of COLIA1 revealed a splicing mutation （IVSS-2A 〉 G） that converted the 3＇ end of intron 8 from AG to GG. Conclusion This mutation （ IVS 8-2A 〉 G） is novel, and has not yet been registered in the Human Type I and Type Ⅲ Collagen Mutations Database.

The collagen type Ⅰ alpha 1 chain gene is an alternative safe harbor locus in the porcine genome

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.Safe harbor loci in the animal genome enable consistent overexpression of foreign genes,without side effects.However,relatively few safe harbor loci are available in pigs,a fact which has impeded the development of multi-transgenic pig research.We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain(COL1A1)gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.After the knock-in of a 2A peptide-green fluorescence protein(2A-GFP)transgene in the last codon of COL1A1 in multiple porcine cells,including porcine kidney epithelial(PK15),porcine embryonic fibroblast(PEF)and porcine intestinal epithelial(IPI-2I)cells,quantitative PCR(qPCR),Western blotting,RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus.The qPCR results showed that the GFP knock-in had no effect(P=0.29,P=0.66 and P=0.20 for PK15,PEF and IPI-2I cells,respectively)on the mRNA expression of COL1A1 gene.Similarly,no significant differences(P=0.64,P=0.48 and P=0.80 for PK15,PEF and IPI-2I cells,respectively)were found between the GFP knock-in and wild type cells by Western blotting.RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation(P<2.2e–16)with that of the wild type cells,indicating that the GFP knock-in did not alter the global expression of endogenous genes.Furthermore,the CCK8 assay showed that the GFP knock-in events had no adverse effects(P_(24)h=0.31,P_(48)h=0.96,P_(72)h=0.24,P_(96)h=0.17,and P_(120)h=0.38)on cell proliferation of PK15 cells.These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment.

一罕见成骨不全Ⅳ型的基因诊断

目的对一疑似成骨不全Ⅳ型或其他类型的患儿实施基因诊断,以揭示患儿发病及频繁骨折的内在原因,为今后实施对症治疗和产前基因诊断创造必要的前提条件。方法对经症状、体征观察和X线检查初诊为成骨不全Ⅳ型或其他类型的患儿,在抽取外周血制备DNA模板后,采用PCR、DNA直接测序法,对患儿的COL1A1基因进行突变检测,然后对所发现的突变进行分析和鉴定。结果在COL1A1基因的编码区内发现一典型的错义突变(c.823G>C1/p.G275R),经查HGMD数据库证实为成骨不全Ⅳ型的致病性突变。结论先证者为一罕见的成骨不全Ⅳ型患儿,所发现的p.G275R突变为中国人群首次报道的病理性突变。