| Background:Alzheimer’s disease (AD) is a progressive degenerative disorder of the centr al nervous system with progressive loss of memory, impairment of cognitive fun ction, and changes in personality amony its clinical features. The pathology of A D is characterized by the aggregation and deposition of amyloid-β (AP) peptide r esulting in the formation of extracellular neuritic plaques and hyperphosphorylates tau protein resulting in the formation of intracellular neurofibrillary tangles. Mut ations in the amyloid-β precursor protein, presenilin-1(PS-1), and presenilin-2ge nes have been linked to autosomal dominant familial AD (FAD), with the most common mutations occurring in PS-1. However, the genetics of sporadic AD are less clear, with only the apolipoprotein E (ApoE)4allele considered to be a ke y risk factor in its etiology. In addition, some research has found individuals wit h sporadic AD who have PS-1mutations. The prevailing view of the effects of PS-1mutations in AD is that these mutations increase the production of amyloid-P42(AP42), triggering the pathogenic cascade, by causing gain of toxic function. On the other hand, more recent work has shown that many PS-1FAD mutations have no significant effect on the production of AP42. The precise mechanism, h owever, remains unknown. Additional evidence is needed to clarify the pathogeni c mechanism of PS-1mutations in AD.Objective:We analyzed the13exons and the splice junctions of PS-1for111cases of AD unselected for family history or age at onset to find new PS-1gene mutations.A novel heterozygous initiation codon mutation (from ATG to GTG) was found. mRNA expression levels of PS-1and APP gene and the PS-1proteins expression were detected by quantitative real-time PCR and western blot analysis in transfected cells to investigate the influence of the initiation codon mutation on the expression of PS-1.Methods:A total of266volunteers participated in our study, including111individuals with AD unselected for family history or age at onset, five individuals who were relatives of one of the111patients, and150healthy controls. Genomic DNA was extracted from peripheral blood leukocytes and collected from all participants using the phenol-chloroform extraction method. All PS1gene exons and the flanking intronic sequences were amplified by polymerase chain reaction (PCR). PCR products were direct sequenced by the Sangon Biotech Co. Ltd sequencing service.cDNAs encoding full-length wild-type PS-1and with a GTG as the initiation codon were subcloned into pEGFP-N1. HEK293T and N2a cells were transiently transfected with the empty vector (transfection control) or the confirmed vectors containing either wild-type or mutant PS-1cDNA using Attractene Transfection Reagent according to the manufacturer’s instructions. the GFP fusion proteins were detected by western blot analysis.Results:(1) We analyzed the sequences of the13exons and the splice junctions of PS-1for all111AD cases and found a heterozygous A to G mutation in the i nitiation codon in patient M766. The mutation was also detected in three unaffec ted relatives, but not in two other unaffected siblings,110cases or150healthy controls. The proband, a65-year-old male, had the first symptoms of mild memo ry impairment at the age of57years and had a gradual progressive cognitive di sorder that made him unable to recognize family members6years later. While t he proband’s93-year-old mother and63-year-old younger sister carrying the sam e mutation were both healthy. In addition, three previously reported polymorphis ms were found (rs1800839, rs116882898, and rs65932).(2) We analyzed mRNA expression levels of PS-1and APP gene in transf ected cells.The mRNA expression of mutant PS-1gene was significantly lower t han normal PS-1gene and the APP gene was not obviously altered.(3)The mutant PS-1with GTG as the initiation codon could be expressed in HEK293T, but not in N2aConclusion:(1) The unaffected carriers in our study may provide evidence that the mutation of PS-1does not cause AD and it might serve as a risk factor.Since no other mutations or polymorphisms were detected in our patiens, mutations in the coding regions and splice junctions of PS-1are likely to be rare in AD.(2) In our study, the mutation was also observed in three unaffected relatives as well as one individual with AD and all the carriers showed normal neuronal function including the individual’s mother and younger sister at the age above the onset age of the proband. It seemed that the mutation failed to segregate with the disease.(3) We have detected the expression of mutant cDNA carrying GTG as the start codon in HEK293T cells, but not in N2a cells. These data suggest that the GTG initiation codon is inefficient in neuron-like cells.(4) The results of mRNA expression levels of PS-1and APP may suggested that overexpression of PS-1do not affect the expression of APP. |
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