The method used was that described by Green et al.3 Genomic DNA was analyzed. Oligonucleotide primers were those described by Humphries et al.13 Amplification involved 36 cycles of 93°C (60 seconds), 55°C (60 seconds), and 72°C (120 seconds). Subsequently, the PCR product was digested with the restriction enzyme Msp I (Boehringer Mannheim), and the digestion products were separated by electrophoresis on ethid ium bromide-stained 3% agarose gel. The fragment of the 353R allele was 205 bp and that of the 353Q allele, 272 bp.

Centenarians (96 women and 28 men 100 to 109 years old, white) were ambulatory, self-sufficient, and living in their homes. None of them were current smokers; 31 had stopped smoking between 5 and 10 years before blood sampling, whereas the remaining 93 had never smoked or had stopped smoking at least 10 years before. They were selected from the whole group of centenarians identified in the same communities because they were judged to be healthy on the basis of the Senieur protocol1,0 which establishes strict enrollment criteria based on clinical his tory, physical examination, and laboratory data for studies on healthy elderly people. Exclusion criteria were chronic disorders such as dementia, malignancy, infection, diabetes, a history of arterial and venous thrombosis, and current or past cardiovascu lar, renal, and liver disease. Hemocytometry and erythrocyte sedimentation rate and plasma concentrations of glucose, sodium, potassium, albumin, alkaline phosphatase, aminotrans ferases, urea, and creatinine were measured. Centenarians with values outside the 2.5 and 97.5 percentiles of the reference val ues of these measurements were excluded. An electrocardiogram was also obtained. Values of serum triglycerides, a measurement not included in the Senieur protocol, were available in only 101 centenarians and in 60 control subjects.

PAI-1 Polymorphism

The guanine insertion/deletion polymorphism is located at position -675 in the promoter region of PAI-1, where one allele has a sequence of four guanines (4G) and the other has a fifth guanine inserted (5G).14 The polymorphism is associated with plasma PAI-1 levels.614 Individuals homozygous for the 4G allele (frequency =0.50) have 25% to 30% higher PAI-1 levels than individuals with at least one 5G allele.

oligonucleotide melting technique described by Dawson et al,14 with some modifications. An 890-bp region of the PAI-1 pro moter was amplified by PCR using the primers described Dawson.14 Amplification involved 35 cycles of 95°C (60 sec onds), 55°C (60 seconds), and 72°C (60 seconds). Samples (3 5 uL) of each PCR product were spotted onto two sheets Hybond N+ nylon membranes (Amersham). Samples were dena tured in 1.5 mol/L NaOH, 0.5 mol/L NaCl for 5 minutes, neu tralized with 3x SSC, and fixed with ultraviolet cross-linking. Two 14-mer oligonucleotides were designed to cover the poly morphic site of each allele: 5'-CACGTGGGGAGTCA-3' and 5'-ACGTGGGGGAGTCA-3'. T4 polynucleotide kinase (Promega) was used to phosphorylate the 5' ends with [32P]ATP. Membranes were prehybridized individually by immersion for at least 2 hours in a solution containing 5x SSPE buffer, Denhardt's solution (0.1% Ficoll 400, 0.1% polyvinylpyrroli done, 0.1% BSA), 100 ng/mL salmon sperm DNA, and 0.5% SDS. Then the labeled probes were added separately to each solution at a final concentration of 105 cpm/mL for at least 1 hour. Membranes were washed in 5x SSC, 0.1% SDS for minutes to remove background hybridization. Prehybridization, hybridization, and washing were carried out at 55°C for oligonucleotide complementary to the 4G allele and at 58°C the oligonucleotide complementary to the 5G allele. Autoradio- grams were obtained by exposing films to membranes intensifying screens for 16 to 20 hours at -70°C. Samples individuals with known genotypes determined by allele-specific oligonucleotide melting14 and kindly supplied by S. (London, UK) were used as controls.

Young individuals (82 women and 48 men) were all <31 years old (mean age, 25.3±3.7 years; range, 14 to 30 years) and were randomly selected from the same metropolitan communi ties (hospital staff, high-school students). Arterial or venous thrombotic disease was excluded on the basis of clinical history and the results of physical examination. None of them were cur rent or recent smokers.

Blood samples for DNA analysis were drawn directly into vacuum containers containing sodium citrate as anticoagulant. Genomic DNA was prepared from whole blood by a salting-out procedure."

from http://ahajournals.org by on March 13, 2020

Fibrinogen Polymorphism

The biallelic polymorphism is located in the pro moter region of the (3-fibrinogen gene. The allele containing polymorphic cutting site for the restriction enzyme Haelll identified as G-455, and the allele without the cutting site is iden tified as A-455. Carriers of the less frequent allele A"455 (=0.20 in the general population) have higher fibrinogen levels (=20% 30%) than carriers of the G~455 allele.12

The method used was that described by Thomas et with some modifications. Briefly, a portion of the (3-fibrinogen gene, -551 to -109 bp from the start of transcription, was amplified by polymerase chain reaction (PCR) using the primers 5'-ATA- GAATAGGGTATGAATTTG-3' and 5'-GAACCATTTTAT- CATTTAAGC-3' in a 50-uL mixture containing 200 to 500 ng DNA, 0.1 mg/mL BSA, 200 umol/L of each nucleotide triphos phate, 100 pmol of each primer, 2 mmol/L MgCh, 10 Tris, 50 mmol/L KC1 (pH 8.3), and 2.5 U Taq Amplification involved 33 cycles of 93CC (20 seconds), 50°C (10 seconds), and 70°C (45 seconds). Subsequently, the PCR product was digested with the restriction enzyme Haelll (Boehringer Mannheim). Finally, the digestion products were separated by electrophoresis on 3% agarose gel and stained ethidium bromide. The fragments of the G"455 allele contained 346 and 96 bp and the fragment of the A"455 allele, 442 bp.

Statistical Analysis