摘要
BACKGROUND: A total of 50 missense mutations of presenilin-1 (PS-1) have been found thus far in early-onset familial Alzheimer disease (EOFAD), PS-1 gene might be a causative gene for Chinese EOFAD. OBJECTIVE: To investigate mutation of PS-1 gene in the blood of Chinese patients with familial Alzheimer disease (FAD). DESIGN: A design with randomized control and repeated sequencing. SETTING: Department of Neurology, the Second People’s Hospital of Wuxi. PARTICIPANTS: The experiment was carried out in Huaihua Hospital Affiliated to Nanhua University in September 1993. Eight FAD patients were graded as FAD group. There were 6 males and 2 females with the mean age of (36±16) years. The control group was composed of 42 persons, including 8 hospitalized SAD patients diagnosed according to the criteria of Practical Neuralgia and conformed to the revised fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DCM-Ⅳ-TR), 11 dementia patients caused by multiple cerebral infarction, 13 normal persons in the FAD family mentioned above family, and 10 normal healthy adults provided by the health examination section of our hospital. METHODS: GeneAmp PCR System 2400 (Applied Biosystems, USA), DNA-Sequencer Model 310 (Perkin Elmer, USA), Taq DNA Polymerase (Fermentas, Canada). All reagents used for DNA extraction were prepared with analytical reagents manufactured in China. The samples were stratified carefully, collected the leukocytic cream from the interface, added STMT to each sample and vortexed to suspend evenly. Then the samples were centrifugated. The nuclear pellet was resuspended in digestion solution with proteinase K and incubated under appropriate condition. Genomic DNA was extract with phenol/chloroform, precipitated with dehydrated ethanol, and washed with 70% sterilized ethanol. Finally, genomic DNA was dissolved in ultra pure water and stored for later use. The sequences were 5’-ACT AAC AAT GGA TGA CCT GGT GAA ATC-3’ and 3’-ACG GTC TGA CCT AAG TGA ATA GTA GAG -5’ to flank the exon 2 of PS-1 gene, and 5’-CGT TAC CTT GAT TCT GCT GAG AAT CTG-3’ and 3’-CAG TAG TAG TGG AAC TTC CGG AGA CGT-5’ to flank the exon 3 and 4 of PS-1 gene. Samples dealt with PCR were put on type 310 DNA sequencing to measure exon 2, 3 and 4 of PS-1 gene of 1 in FAD group and all subjects in control group. MAIN OUTCOME MEASURES: Observation and evaluation of mutation positions in PS-1 gene. RESULTS: A heterozygous point T to A mutation at 6 519 nt on exon 4 of PS-1 gene was found by DNA sequencing in the member of FAD family, resulting in a change of Leu50Ile in the encoded protein. But no mutation was detected in all other patients and normal control. CONCLUSION: The results suggest that the T to A mutation on exon 4 of PS-1 gene may be associated with Chinese EOPAD.
BACKGROUND： A total of 50 missense mutations of presenilin-1 （PS-1） have been found thus far in early-onset familial Alzheimer disease （EOFAD）, PS-1 gene might be a causative gene for Chinese EOFAD.
OBJECTIVE： To investigate mutation of PS-1 gene in the blood of Chinese patients with familial Alzheimer disease （FAD）.
DESIGN ： A design with randomized control and repeated sequencing
SETTING： Department of Neurology, the Second People＇s Hospital of Wuxi
PARTICIPANTS： The experiment was carried out in Huaihua Hospital Affiliated to Nanhua University in September 1993. Eight FAD patients were graded as FAD group. There were 6 males and 2 females with the mean age of （36±16） years. The control group was composed of 42 persons, including 8 hospitalized SAD patients diagnosed according to the criteria of Practical Neuralgia and conformed to the revised fourth edition of the Diagnostic and Statistical Manual of Mental Disorders （DCM-Ⅳ-TR）, 11 dementia patients caused by multipie cerebral infarction, 13 normal persons in the FAD family mentioned above family, and 10 normal healthy adults provided by the health examination section of our hospital.
METHODS： GeneAmp PCR System 2400 （Applied Biosystems, USA）, DNA-Sequencer Model 310 （Perkin Elmer, USA）, Taq DNA Polymerase （Fermentas, Canada）. All reagents used for DNA extraction were prepared with analytical reagents manufactured in China. The samples were stratified carefully, collected the leukocytic cream from the interface, added STMT to each sample and vortexed to suspend evenly. Then the samples were centrifugated. The nuclear pellet was resuspended in digestion solution with proteinase K and incubated under appropriate condition. Genomic DNA was extract with phenol/chloroform, precipitated with dehydrated ethanol, and washed with 70% sterilized ethanol. Finally, genomic DNA was dissolved in ultra pure water and stored for later use. The sequences were 5＇-ACT AAC AAT GGA TGA CCT GGT GAA ATC-3＇ and 3＇-ACG GTC TGA CCT AAG TGA ATA GTA GAG -5＇ to flank the exon 2 of PS-1 gene, and 5＇-CGT TAC CTT GAT TCT GCT GAG AAT CTG-3＇ and 3＇-CAG TAG TAG TGG AAC TTC CGG AGA CGT-5＇ to flank the exon 3 and 4 of PS-1 gene. Samples dealt with PCR were put on type 310 DNA sequencing to measure exon 2, 3 and 4 of PS-1 9ene of I in FAD group and all subjects in control group.
MAIN OUTCOME MEASURES： Observation and evaluation of mutation positions in PS-1 gene
RESULTS ： A heterozygous point T to A mutation at 6 519 nt on exon 4 of PS-1 gene was found by DNA sequencing in the member of FAD family, resulting in a change of Leu5011e in the encoded protein. But no mutation was detected in all other patients and normal control.
CONCLUSION ： The results suggest that the T to A mutation on exon 4 of PS-1 gene may be associated with Chinese EOPAD.
