A germline TaqI restriction fragment length polymorphism in the progesterone receptor gene in ovarian carcinoma

Nj Mckenna,Dg Kieback,Dr Headon,Dn Carney,J Mclinden,M Fanning

doi:10.1038/bjc.1995.92

Abstract

Clinical outcome in ovarian carcinoma is predicted by progesterone receptor status, indicating an endocrine aspect to this disease. Peripheral leucocyte genomic DNAs were obtained from 41 patients with primary ovarian carcinoma and 83 controls from Ireland, as well as from 26 primary ovarian carcinoma patients and 101 controls in Germany. Southern analysis using a human progesterone receptor (hPR) cDNA probe identified a germline TaqI restriction fragment length polymorphism (RFLP) defined by two alleles: T1, represented by a 2.7 kb fragment; and T2, represented by a 1.9 kb fragment and characterised by an additional TaqI restriction site with respect to T1. An over-representation of T2 in ovarian cancer patients compared with controls in the pooled Irish/German population (P < 0.025) was observed. A difference (P < 0.02) in the distribution of the RFLP genotypes between Irish and German control populations was also observed. The allele distributions could not be shown to differ significantly from Hardy-Weinberg distribution in any subgroup. Using hPR cDNA region-specific probes, the extra TaqI restriction site was mapped to intron G of the hPR gene.

Highlights

S_.manr Clinical outcome in ovarian carcinoma is predicted by progesterone receptor status, indicating an endocrine aspect to this disease
Peripheral leucocyte genomic DNAs were obtained from 41 patients with primary ovarian carcinoma and 83 controls from Ireland, as well as from 26 primary ovarian carcinoma patients and 101 controls in Germany
Using human progesterone receptor (hPR) cDNA region-specific probes, the extra TaqI restriction site was mapped to intron G of the hPR gene

Summary

Material and methods

The hPR-2 cDNA probe (Figure 1c) was generated by PCR using sense primer no. HPR-3 probe (Figure Id) was generated by PCR amplification using the sense primer no. 3 (5'-TCGACTTAAGAGAAAAAATCIl-lAC-3') (hPR cDNA sequence 3133-3156 bp, numbered from ATG1) (Kastner et al, 1990), and primer no. After centrifugation at 5000 r.p.m. for 10 min at 4°C, the pellet was resuspended in 10mM Tris-HCI pH 8.0-5 mM EDTA-l% sodium dodecyl sulphate (SDS)-0.4% Proteinase K (Sigma, St Louis, MO, USA), incubated at 37TC overnight, and treated as described previously (Miller et al, 1988). Low-melt agarose gel-purified probe was 32P-labelled uing Prime-A-Gene (Promega, Madison, WI, USA) to a specific activity of 2 x 108 c.p.m. iLg-' Prehybridisation was carried out in 6 x SSC, 5 x Denhardt's solution [0.02% bovine serum albumin, 0.02% polyvinylpyrollidone, 0.02% Ficoll (Pharmacia, Uppsala, Sweden)], 0.5% SDS and. The membranes were exposed to Kodak X-OMAT XAR-5 autoradiographic film (Sigma, St Louis, MO, USA) at - 70°C with intensifying screens for 48 -72 h

Statistical analysis

Characterisation of TaqI polymorphism

Allelic distribution andfrequency in ovarian cancer patients and controls

German Cases Controls'

Ovarian carcinomas express several forms of the progesterone

Analysis of the PvuII restriction fragment length polymorphism