Molecular Genetics Of A Chinese Family With Avellino Corneal Dystrophy

Purpose: To characterize the clinical features of a Chinese family with Avellino corneal dystrophies(ACD)from Hubei Province, to screen the causative gene for the disease and to explore the molecular mechanisms underlying the disease. To analyze phenotype-genotype correlations on the ACD from the molecular genetic point of view, and provide the basis for prevention and treatment of the disease in the future.Methods: 1. Eighteen people from a Chinese family of three generations were included in this study. All members underwent complete ophthalmic and systemic clinical examination, to rule out the possibility of systemic disease and syndrome, to determine the clinical phenotype.2. Molecular genetic analysis was performed on all subjects included in the study. Genomic DNA was extracted from peripheral blood collected from all affected and unaffected participants. All exons of human TGFβI gene were amplified by polymerase chain reaction(PCR), sequenced and compared with reference sequence.3. Evolutional conservation of both mutation sites were analyzed by homologous sequence alignment among ten species. The potential damage of the missense mutation on the encoded protein was predicted by Poly Phen-2 tools.4. Recombined wild-type and mutants of TGFβI were cloned. The sequences of all constructs were verified by direct sequencing.Results: 1. Five members in the family were diagnosed as Avellino corneal dystrophy, and the rest were asymptomatic. The clinical phenotype of female patients was more severe. All patients showed ocular symptoms only No other physical abnormality was observed.2. A heterozygote missense mutation c.371G>A(p.R124H)in exon 4 of TGFβI gene was identified in this family. Only individuals with the mutation developed ACD, and family members who had no the mutation didn’t show any abnormal phenotype. This mutation caused an amino acid substitution of arginine to histidine at position 124(p.R124H) of the TGFβIp protein. In addition, we also found that the five patients had two synonymous single nucleotide polymorphisms(SNP): c.981G>A(V327V) and c.1626 T>C(F542F).3. The site was highly conserved for TGFβI based on analysis of orthologs from six different species and was predicted to “damage” the structure of the encoded protein.4. Wild type and mutant TGFβI were cloned into an expression vector, and then to be transfected into normal corneal epithelial cells. In the transfected group of the recombinant plasmid TGFβI was expressed. While TGFβI was not expressed in the negative control group and the empty plasmid transfection group. All these provided the basis for further studies on mechanisms underlying the pathogenesis of the disease.5. Within a certain range, estrogen stimulates the expression of endogenous TGFβI gene in human corneal epithelial cells.Conclusions: 1. A heterozygote missense mutation of TGFβI was identified in a Chinese family with ACD(c. 371G>A in exon 4).2. The site of c. 371G>A mutation was highly conserved, and were probably essential for the structure and function of TGFβI.3. Molecular genetic study of the family showed that corneal dystrophies were a group of heterogeneous inherited disorders; the same genetic mutation could have different phenotypes of the cornea.4. The expression of TGFβI gene in human corneal epithelium cells was not detected because of the low expression or no expression of TGFβI gene.5. The phenotype of the female patients is likely to be related to estrogen.