The Research On Novel Mutation APPK724M Causing Chinese Early-onset Familial Alzheimer’s Disease

Objects:Alzheimer’s disease (AD) is a common neurodegenerative disorder in the elderly. Although there are the largest aged and dementia population in the world, familial AD (FAD) mutations were scarcely reported in China. Recently, we found a novel APP mutation (APPK724M) in an early-onset familial AD (EOFAD) pedigree. In order to prove APP K724M is a pathogenic mutation for this Chinese EOFAD pedigree and find the underlying mechanism, we screened the AD causal genes and the risk factor (APOE) in this family and investigated the role played by APPK724M in this case of EOFAD.Methods:Peripheral blood DNA samples were collected from the members of EOFAD pedigree and control group, and PCR was conducted using primers designed to amplify encoding exons of PSEN-1and PSEN-2, exons16and17of APP, and exons of APOE gene, respectively. Gene mutations were identified by sequencing the PCR products. HEK293cells were transfected with the plasmids carrying wild or mutant APP gene. The intracellular levels of APP protein were analyzed by Western Blot and the levels of β Amyloid-beta peptide (Aβ), products of APP metabolism, were analyzed by ELISA.Results:The APPK724M mutation was found in each of the tested dementia patients in this pedigree and no coding mutations existed in either PSEN-1or PSEN-2gene. Also no such mutation was observed in the samples from healthy individuals in this family and control group. The gene type of APOE3/3was carried by all the members of this family with the exception of APOE3/4carried by the proband. The similar expression levels of APP protein between the mutant and wild APP transfected HEK293cells were observed by Western Blot. However, the Aβ42level and the ratio of AP42/40were elevated significantly in APP mutant groups compared with APP wild group by ELISA.Conclusion:The APPK724M gene mutation may be the pathogenic factor of EOFAD in the Chinese pedigree and the elevated level of Aβ42from APPK724M metabolism may be the underlying mechanism of the AD patients in this pedigree.