Application Of Animal Model In The Study Of Congenital Microtia And Genetic Analysis Of Families With Microtia

Objectiveto investigate the application of Prkra little ear(lear)mouse animal model in the study of congenital microtia and to explore its application as an animal model in the study of microtia.to apply Bmp5 short ear mouse model to the study of congenital microtia and to explore its application as an animal model in the study of microtia.Human microtia is a congenital ear malformation,which can be affected by genetic and environmental factors.at present,the pathogenesis of microtia is not clear.We hope to sequence and analyze the genomic information of patients with microtia,To further understand the relationship between mutations in known pathogenic genes and microtia.Methods1.Ten pregnant mouse were bred in Prkra little ear(lear)mouse,the tail of fetal mouse was sequenced by Sanger method at the age of 14.5.the bone of 40-day-old mouse with auricle deformity and normal auricle was compared with that of 40-day-old mouse with normal auricle for bone Alisin blue staining.2.Sanger sequencing of Bmp5 short ear(se)mice and Allison blue staining of bone of Bmp5 short ear(se)mice were carried out.3.In this study,the genomes of a pair of 2-year-old twin sisters with right microtia were sequenced using Whole genome sequencing(WGS).4.In this study,whole exome sequencing(WES)technique was used to analyze a Chinese genetic family with congenital microtia,and Sanger sequencing technique was used to verify the results.Results1.The general characteristics of Prkra little ear(lear)mouse were that the auricle of homozygous mouse was small.The auricle of Prkra little ear(lear)homozygous mouse was significantly smaller than that of sibling normal mouse.The mutation site is G to A on chromosome 2(76,643,218 bp GRCm38),which is located after exon 3 and may destroy the RNA splicing of pre-mRNA.The whole bone development of stained mice was small.2.The general characteristics of Bmp5 short ear(se)were that the auricle of homozygous mouse was small.The auricle of bmp5 short ear(se)homozygous mouse was significantly smaller than that of sibling normal mouse.Spontaneous mutation:the transition from C to T produces a termination codon at amino acid 208.this mutation is a nonsense mutation that inactivates the BMP5 protein,the product of the Bmp5 mutant gene.The bone development of stained mice was abnormal.3.we identified a previously unknown mutation in the twin sister BMP5 gene(exon4:c.833-4C>G),and also a new mutation(exon2:c.332G>T:p.S111I)in the BMP2 gene,both of which were confirmed by PCR amplification of the corresponding genome regions followed by first generation sequencing.4.1t was found that there were 10 new heterozygous mutations(exonl:c.C169T)at the same site in FGF3 gene in this family,we believe that FGF3 gene is the pathogenic gene mutation site of congenital microtia in this family.The newly discovered mutation in exon 1 of FGF3 gene belongs to heterozygous nonsense mutation(exonl:c.169C>T),which forms a termination codon(p.Q57X)at position 57.All the mutation sites of FGF3 gene in this family were heterozygote,in the family members with FGF3 gene mutation,the other members of the family whose ears were not affected were not completely explicit.Conclusion1.The mouse model of homozygous mutation in Prkra little ear(lear)mouse can be completely suitable for the study of human microtia.Prkra little ear(lear)mouse mutation model is a suitable animal model to study the development mechanism of congenital microtia.2.The mouse model of homozygous mutation of Bmp5 short ear(se)mouse can be completely suitable for the study of human congenital microtia.Bmp5 short ear(se)mouse mutation model is a high quality animal model to study the development mechanism of congenital microtia.3.Our study therefore further demonstrates the power of WGS to discover novel mutations associated with microtia on the whole genome scale,and extends the mutation spectrum of the BMP5 gene.In addition,our data suggest that BMP2 may be another pathogenic gene associated with microtia.4.FGF3 gene is the only mutation in the family that conforms to the basic law of Mendelian inheritance.We believe that the novel heterozygous FGF3 gene mutation site can lead to simple microtia.This may be the first time that the heterozygous mutation of FGF3 gene has been found to be related to human microtia,and it is helpful for us to understand the key regulatory role of FGF3 gene in the development of external ear during embryonic development.