The feasibility of storing ovarian tumor cells on databasing paper: establishing a library of ovarian cancer DNA.

The purpose of this study was to assess the feasibility of establishing a library of ovarian cancer nucleic acids using paper matrix by: 1) confirming the stability of DNA stored on paper matrix over a prolonged period of time, 2) determining the amount of genetic material required for storage, and 3) establishing the stability of RNA. Tumor tissue from 66 patients with ovarian cancer was collected intraoperatively, frozen, and dissociated with collagenase and trypsin. A cell suspension was then prepared and spotted onto the paper. The numbers of cells that were stored on the paper were counted using a hemocytometer. The cell suspension was serially diluted and spotted on the paper matrix until the minimum cell number that can be stored and produce a PCR product was determined. PCR, STR genotyping and direct sequencing were performed on tissue stored on the paper matrix. FTA paper was used as RNA template, and RT PCR converted the RNA to cDNA. Ten to 50 mg of ovarian cancer tissue was stored on FTA paper. We stored 7 x 10(4) cells on ISOcode paper and 18 x 10(4) cells on FTA and obtained extractable DNA. PCR analysis on cards with DNA stored 18 months ago enabled us to establish the stability of DNA after storage. RNA was stable for 6 months when stored on FTA cards. Since genetic material is extractable from the paper matrices after passage of time, it could be a suitable medium for the storage of genetic material in cancer tissue banks.

Mutational and polymorphic analysis of the estradiol receptor-alpha gene in men with symptomatic vertebral fractures.

In view of the importance of estrogens in the maintenance of the skeleton in men, we have carried out mutational analysis of all the exons of the estrogen-receptor-alpha (ER-alpha) gene in 64 men (36 patients with symptomatic vertebral crush fractures and 28 control subjects). Initial screening of the ER-alpha gene, carried out by single-strand conformation polymorphism analysis followed by sequencing, showed conservative mutations in exon 4 which resulted in a single base substitutions producing GGG-->GGC transition in codon 274. We also carried out polymorphic analysis of the ER-alpha gene at the PvuII restriction site in 82 men with a range of bone density measurements (53 with symptomatic vertebral fractures and 29 controls). The frequencies of PP, Pp, and pp genotypes were 20.7%, 48.8%, and 30.5%, respectively. The distribution of the alleles was similar in the patients with symptomatic vertebral crush fractures and male control subjects. There was no association between ER-alpha genotypes and bone mineral density or arthropometric parameters. This relatively small study suggests that mutations in the ER-alpha gene are unlikely to be a common cause of osteoporosis in men with vertebral fractures. Furthermore, polymorphic variation of the ER-alpha gene appears to have little effect on the pathogenesis of osteoporosis in men.

Vitamin D binding protein gene in male osteoporosis: association of plasma DBP and bone mineral density with (TAAA)(n)-Alu polymorphism in DBP.

Vitamin D binding protein (DBP) is a major carrier protein for the vitamin D metabolites, but may also play an important role in osteoclast differentiation. Polymorphisms of the DBP gene have been reported, including (TAAA)(n)-Alu repeat polymorphisms downstream of intron 8. We have examined the relationship between polymorphisms of the DBP gene and bone mineral density (BMD) and vertebral fractures in a group of 26 men with vertebral fractures but no underlying secondary cause of osteoporosis (median age 64, ages 27-72 years) and 21 male control subjects (median age 65, ages 40-77 years). There was no apparent effect of DBP phenotype on BMD, but there was a relationship between certain genotypes of (TAAA)(n)-Alu repeats and reduced BMD and vertebral fracture. Lumbar spine and femoral neck BMD were significantly lower in men with 10/8 genotype than 10/10 genotype (P < 0.05). Furthermore, the predominant genotype in men with vertebral fractures was 10/8, whereas the most common genotype in control subjects was 10/10 (odds ratio 56; 95% confidence interval 7-445). Plasma DBP was higher in men with 10/8 genotype than those with 10/10 genotype (P < 0.05), and patients with vertebral fractures were found to have higher levels than control subjects (P < 0.0005). Although our study is small because of the relative rarity of idiopathic osteoporosis in men, the results suggest that (TAAA)(n)-Alu polymorphism may have an important effect on plasma levels of DBP, bone density and fracture risk in men.