Functional Analysis Of Transgenic Mice With Novel Mutation In TRPV4 Gene

Objective:This study was based on the discovery of a novel heterozygous mutation（c.2479C>G p.P827A）carrying transient receptor potential vanilloid receptor 4（TRPV 4）gene in a child.The child suffered from spinal epiphyseal dysplasia-Kozlowski type（SMD-K）.In this study,transgenic mice were constructed by gene editing.And the pathogenicity of the mutation was further verified by measuring physiological indexes,applying histopathological techniques and Micro-CT in the animal model.At the same time,the pathogenesis of SMD-K caused by the mutation was explored,providing new ideas and methods for the diagnosis and treatment of the disease.Methods:1.Using CRISPR/Cas9 technology,the heterozygous mutation of TRPV 4 gene（c.2479 C>G p.P827A）was introduced to establish the heterozygous mouse model of TRPV 4 gene.The birth rate of wild type mice,heterozygous mutant mice and homozygous mutant mice were calculated by using the breeding strategy of heterozygous and heterozygous mating.2.This study observed and compared the appearance of wild-type mice,heterozygous mutant mice and homozygous mutant mice at postnatal day 1（P1）/3months/6 months/9 months.3.From one month to six months,the weight and length of mice were measured every other month.The weight and length of wild-type mice,heterozygous mutant mice and homozygous mutant mice were compared.4.Alcian Blue staining was used to observe and compare the skeleton and femoral calcified bone length of wild-type P1 mice,heterozygous mutant P1 mice and homozygous mutant P1 mice.HE staining and Safranin O staining were used to observe and compare the histological structure of the knee and spine of wild-type mice,heterozygous mutant mice and homozygous mutant mice.5.The spinal appearance and bone parameters of wild-type mic,heterozygous mutant mic and homozygous mutant mice were observed and compared by Micro-CT scanning reconstruction and analysis.Results:1.Homozygous mutant mice died at birth.The birth rate（3.7%）is far lower than the theoretical birth rate（25%）.2.The skeletons of homozygous mutant P1 mice were smaller than those of wild type P1 mice and heterozygous mutant P1 mice.And skeletal deformity was not found,showing slow growth and development.3.Comparing the weight and length of heterozygous mutant mice with those of wild-type mice,only the weight of 3-month-old heterozygous mutant male mice（23.99±1.22g）was greater than that of wild-type male mice（22.75±1.47g）.The difference was statistically significant（P<0.05）.4.Compared with the wild type mice,the histological structure of knee joint and spine in the 3-month-old,6-month-old and 9-month-old heterozygous mutant mice were not abnormal.5.Compared with the wild type mice,the spinal appearance and bone parameters of the thirteenth thoracic vertebra in the 3-month-old,6-month-old and 9-month-old heterozygous mutant mice were not abnormal.Conclusion:1.TRPV 4^P827Ahomozygous mutant mice were lethal.Their skeletal system showed growth retardation,suggesting the pathogenicity of the mutation.2.Compared with wild-type mice,TRPV 4^P827Aheterozygous mutant mice have no obvious difference in the growth and development of skeletal system.