ABSTRACT
Objective@#To analysis the pathogenic mutation and the clinical characteristics of a three generation family with congenital pulverulent cataract.@*Methods@#A congenital cataract family was chosen from the First Affiliated Hospital of AnHui Medical University, 5 ml peripheral blood was obtained from each family member to extract genomic DNA.Next generation sequencing was used to detect the mutation in proband (Ⅱ5), Ⅱ6 and Ⅲ8, and Sanger sequencing was applied to verify pathogenic mutation in the whole family members.The mutation site was compared with the gene sequence of 10 000 normal Chinese.PolyPhen-2 and SIFT were applied to analysis the alteration on the protein structure and function and its possible pathogenesis.This study followed the Declaration of Helsinki and was approved by the Ethics Committee of AnHui Medical University (NO.PJ2017-5-17). All patients signed informed consent.@*Results@#The pedigree consisted of 19 members of three generations, including 10 patients and 9 normal family members.Heterozygous mutation of GJA3 gene c. 427G>A (p.G143R) was detected in all patients of the pedigree, but was not found in normal members of the pedigree and 10 000 normal Chinese.The score calculated from SIFT and PolyPhen-2 indicated that the mutation probably had malignant effect on normal protein structure, Swiss-model website analysis showed that the mutation likely altered the secondary structure of the protein CX 46 by reducing an α-helix between 107-115 amino acids.Meanwhile, c.1325-1G>T mutation of HSF4 gene were detected in Ⅱ5 and Ⅲ8, which was not found in other family members and 10 000 normal Chinese.HSF and MaxEntScan results showed that the mutation probably had serious effect on the splicing of mRNA.The cataract development rates of Ⅱ5 and Ⅲ8 were faster than that of the same age in the same generation of the pedigree, and the morphology of lens opacity was changed.@*Conclusions@#The heterozygous c. 427G>A mutation in GJA3 gene is responsible for pulverulent cataract in this family, meanwhile the c. 1325-1G>T mutation in HSF4 gene may change the type of phacoscotasmus and accelerate the progress of disease.
ABSTRACT
Objective To analysis the pathogenic mutation and the clinical characteristics of a three generation family with congenital pulverulent cataract. Methods A congenital cataract family was chosen from the First Affiliated Hospital of AnHui Medical University,5 ml peripheral blood was obtained from each family member to extract genomic DNA. Next generation sequencing was used to detect the mutation in proband (Ⅱ5),Ⅱ6 and Ⅲ8, and Sanger sequencing was applied to verify pathogenic mutation in the whole family members. The mutation site was compared with the gene sequence of 10000 normal Chinese. PolyPhen-2 and SIFT were applied to analysis the alteration on the protein structure and function and its possible pathogenesis. This study followed the Declaration of Helsinki and was approved by the Ethics Committee of AnHui Medical University ( NO. PJ2017-5-17 ) . All patients signed informed consent. Results The pedigree consisted of 19 members of three generations,including 10 patients and 9 normal family members. Heterozygous mutation of GJA3 gene c. 427G>A ( p. G143R) was detected in all patients of the pedigree,but was not found in normal members of the pedigree and 10000 normal Chinese. The score calculated from SIFT and PolyPhen-2 indicated that the mutation probably had malignant effect on normal protein structure,Swiss-model website analysis showed that the mutation likely altered the secondary structure of the protein CX 46 by reducing anα-helix between 107-115 amino acids. Meanwhile,c. 1325-1G>T mutation of HSF4 gene were detected in Ⅱ5 and Ⅲ8, which was not found in other family members and 10000 normal Chinese. HSF and MaxEntScan results showed that the mutation probably had serious effect on the splicing of mRNA. The cataract development rates of Ⅱ5 andⅢ8 were faster than that of the same age in the same generation of the pedigree,and the morphology of lens opacity was changed. Conclusions The heterozygous c. 427G>A mutation in GJA3 gene is responsible for pulverulent cataract in this family,meanwhile the c. 1325-1G>T mutation in HSF4 gene may change the type of phacoscotasmus and accelerate the progress of disease.
ABSTRACT
Objective To explore the cultural method of lingual keratinocytes and make preparations for further investigation in using lingual keratinocytes as a new choice of seed cells for the reconstruction of tissue engineered corneal epithelium.Methods Keratinocytes were enzymatically dissociated from lingual tissue specimens using 0.25%disepase and 0.05%trypsin.The biological characteristics of the cells were observed through phase microscope.Meanwhile.1ingual kemtinoeytes were immtmohistochemically labeled with broad spectrum keratin antibody (AE1/AF3).Results The cells revealed the form of "road metal" and could be passed for 3 or 4 generations.Immunohistochemistry indicated that lingual keratinocytes were positive for AE1/AE3 staining.Condusion Lingual kemtinocytes of rabbits can be cultured with KSFM in vitm and magnitude quantity can be attained,laying a favorable foundation for lingual keratinocytes as a new choice of seed cells for corneal epithelium reconstruction with tissue engineering.