Screening Of Pathogenic Gene Of Congenital Cataract And Apoptosis Of HLE Cells Induced By Mutation Of CRYAB Gene

Congenital cataract refers to a kind of cataract accompanied with symptom of lens of metabolic disturbances or decline in transparency, the patients who suffer from this disease usually occurs mainly at birth or seldom after birth. The morbidity of congenital cataract in our country is 5‰ accounting for major causes for children blindness. Therefore from the perspective of prenatal and postnatal care, we are supposed to adopt urgent precautions against congenital cataract.Inherited genetic factors are the main causes for the formation of the congenital cataract, by which autosomal dominant inheritance (AD) is commonly seen in addition to the autosomal recessive inheritance (AR) and X-linked inheritance (XL). So far the identified and validated disease-caused genes contain:crystallin gene, membrane protein gene, cytoskeletal protein gene, transcriptional regulation factor gene, ferritin light chain gene, etc.My study contains three chapters:In the chapter 1, we screened candidate genes from three autosomal dominant congenital cataract family and one autosomal recessive congenital cataract family by PCR amplification and DNA sequencing. We individually discovered 5,6,7,9 SNP in four families and T deletion (g.8545) in the non-coding region of CRYGB from the proband of CA-010 family, in addition the C> G (c.337) in the CRYGB from the proband of CA-016 family was spotted, thereby causing mutation of histidine into aspartic acid (H113D). Considering the rest of the CA-016 family all bearing such mutation, we speculate that H113D is not the decisive cause for congenital cataract. In view of no critical disease-caused gene being identified and validated, it is mandatory to further study the critical disease-caused gene for congenital cataractgeneisis.In the chapter 2, we constructed the CRYAB wild-type and P20R eukaryotic expression vector and transfected into human lens epithelial cell (HLE). After the transfection, we evaluated the apoptosis rate by flow cytometre and TUNEL. Based on the resulting data, we discussed the effect of CRYAB gene mutation (P20R) on anti-apoptosis function. The results showed that both wild-type and mutate aB-crystallin were abundantly expressed in HLE. The wild-type aB-crystallin could apparently inhibit H2O2-induced apoptosis contrasted with sharply lowering of the mutate type (P< 0.05).In the chapter 3, we explored the expression of aB-crystallin in clinical samples from another commonly seen ophthalmolic disease-pterygia. We applied HE staining and immunochemistry to evaluate and localize the aB-crystallin in pterygia sample. The results showed that aB-crystallin were abundantly expressed both in the normal conjunctival tissue and pterygia sample, among which stratum basale of the epithelial cells was the most enriched. There is no significant difference in aB-crystallin in both tissues (P>0.05), the clinical significance of which in pterygia is worthy of further study.