Mutation Analysis Of The LAMA3 And LAMB3 Gene In Junctional Epidermolysis Bullosa

Objective:To detect the LAMA3 gene and the LAMB3 gene in six patients with Laryngo-onycho-cutaneous(LOC)syndrome and one patient with lethal Herlitz-type Junctional Epidermolysis Bullosa(JEB-H).Methods:1.Clinical data of the seven patients were consummated,and genomic DAN was extracted from peripheral blood leucocytes of these patients and their relatives.2.Amplifying coding exons and intron sequences of the LAMA3 gene and the LAMB3 gene by PCR and following the DNA sequencing.3.Analyzing comprehensively of the sequencing results and related sites already reportted in the literature.4.Designed experiments in vitro,and detected the effects of LAMA3 truncated protein caused by LAMA3 nonsense mutation to the wood healing of the two cases,who had been reported in the literature,throuth the methods of Realtime-PCR and Western Blot.5.Application of minigene assay to testify how the splice site mutation of the LAMB3 gene of the patient with JEB-H influence his m RNA splicing.Results:1.One of the six patients suffered from LOC syndrome with compound heterozygous mutations as IVS39-1G>A and p.R793*of the LAMA3.The patient suffers from JEB-H with compound heterozygous mutations as IVS11-22G>A and p.C325* of the LAMB3 gene.The mutations above had not been reported in the literature,and it is not detected in 300 normal control individuals.2.We collected two nonsense mutations p.E292*and p.Q511*of the patients with LOC syndrome had reported in the literature.3.The results of Realtime-PCR and Western Blot show that both two LAMA3 truncated proteins promote the expression of type?collagen and type ? collagen comparing with the normal control.4.RT-PCR identified the vector control produced a 248 bp amplicon,whereas the wild-type and the as IVS11-22G>A showed a 404 bp amplicon and a 424 bp amplicon respectively.Conclusion:1.The LAMA3 truncated protein promotes the secretion of type?collagen and type ?collagen by fibroblasts,which is likely to be the main cause that affecting the wound healing in the patient.2.The splice site mutation as IVS11-22G>A in the patient with JEB-H influences his m RNA splicing,mergering with the nonsense mutation p.C325* is likely to be the main cause to develop the disease.