For the diplotype frequencies, the previous model can be reformulated as:

The UCP1 A-3826G polymorphism was genotyped by genomic PCR and BclI-RFLP analysis as previously described (Oppert et al., 1994). To genotype the UCP1 C-3737A polymorphism 3–5 μl of the amplified DNA as above was digested by the restriction enzyme DdeI (3 U) for 3 h at 37 °C as recommended by the manufacturer. The fragments were separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide.

where the effect of the diplotype h /h on survival is represented by the i j slope parameter β : if it is significantly different from 0 the frequency ij1 of the carriers of the diplotype h /h goes up if β N0, or down if β b0. i j ij1 ij1

i j ij1 ij1 The binomial logistic regression model has been applied to estimate the effect of each of the three reconstructed haplotypes (A-C, G-A and G-C) and of the relevant diplotypes on survival.

Table 2

Diplotype frequency distributions in the analyzed sample categorized according to the defined age classes.

Diplotype Females Males Age class 1 Age class 2 Age class 3 Age class 1 Age class 2 Age class 3 N=200 N=83 N=113 N=104 N=128 N=87 (40–72 years) (73–91 years) (N91 years) (40–65 years) (66–88 years) (N88 years) A-C/A-C 108 (54.0%) 99 (63.1%) 69 (58.5%) 63 (54.3%) 119 (55.3%) 64 (61.5%) A-C/G-A 27 (13.5%) 17 (10.8%) 14 (11.9%) 21 (18.1%) 45 (20.9%) 9 (8.7%) A-C/G-C 53 (26.5%) 30 (19.1%) 29 (26.4%) 22 (19.0%) 43 (20.0%) 25 (24.0%) G-A/G-A 2 (1.0%) 2 (1.3%) 2 (1.7%) 3 (2.6%) 0 (0.0%) 1 (1.0%) G-A/G-C 4 (2.0%) 8 (5.1%) 1 (0.8%) 4 (3.4%) 2 (0.9%) 4 (3.8%) G-C/G-C 6 (3.0%) 1 (0.6%) 3 (2.5%) 3 (2.6%) 6 (2.8%) 1 (1.0%)

orientation and to confirm that the sequences matched the original genomic sequences with no PCR-generated errors. Cloned vectors were then used for subsequent experiments.

2.3. Bioinformatic analyses

A 463 bp fragment (corresponding to −3967 to −3504) of the 5′ region of the human UCP1 gene including both the A-3826G and C- 3737A polymorphisms was extracted from the National Center for BiotechnologyInformationdatabase(http://www.ncbi.nlm.nih.gov/). The search for putative regulatory elements in this sequence was performed using the MatInspector software (http://www.genomatix. de/matins).

2.5. Transfection assays

MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 4.5 g/l glucose and 2 mM L-glutamine supplemented with 5% fetal bovine serum (Invitrogen) and 1% gentamycin (Invitrogen). T47D cells were cultured in RPMI 1640 medium (Sigma) supplemented with 4.5 g/l glucose (Sigma), 1 mM Sodium pyruvate (Sigma), 10 mM HEPES buffer (Sigma), 0.2 U/ml Insulin (Sigma), 10% fetal bovine serum (Invitrogen), 2 mM L- glutamine (Invitrogen) and 1% gentamycin (Invitrogen). The cells were cultured in a water-humidified incubator at 37 °C in 5% CO2/95% air. Transfections were performed with the Fugene6 reagent as recommended by the manufacturer (Roche Molecular Biochemicals). Briefly, MCF-7 (5×104) and T47D cells (8×104) were transferred into 24-well plates with 1 ml of regular growth medium/well the day before transfection. The growth medium was then discarded and replaced with fresh medium supplemented by a mixture containing Fugene6, 1 μg of each reporter plasmid, and 1 ng of pRL-CMV (Promega), a plasmid containing the Renilla luciferase gene under the cytomegalovirus promoter utilized as an internal control to normalize the effects of transfection efficiencies. After 10 h, the medium was discarded and cells were incubated in fresh medium containing 17β-estradiol 1 μM or Progesterone 30 nM or 9-cis Retinoic Acid 1 μM for 48, 36 and 24 h, respectively. Then, untreated and treated cells were lysed by applying 100 μl of Passive Lysis Buffer (Promega). 20 μl of cell lysate was used for luciferase reporter assay according to the manufacturer's protocol for the Dual Luciferase reporter Assay Kit (Promega). Light intensity was quantified in a Lumat LB9507 luminometer (EG&G Berthold). The luciferase activities of the reporter plasmids were normalized to the Renilla luciferase

2.4. Construction of reporter gene plasmids

The 463-bp region containing the different haplotypes was amplified by PCR from human genomic DNA. Primers were designed to include the restriction sites KpnI and SacI for cloning in the product (Forward: 5′caggtaccTAACAGGGTATTTCCCAG; Reverse: 5′cagactCT- GAGAGGTCACAGAAGTT). PCR was carried out in 25 μl containing 1× buffer, 0.2 mM dNTPs, 0.32 μM primers, 2.5 mM MgCl , and 1.5 U of 2 Go Taq FlexiDNA Polymerase (Promega). The PCR reaction consisted of 35 cycles (95 °C for 60 s for the denaturation step, 55 °C for 45 s for the annealing step, and 72 °C for 60 s for the extension step). A denaturation step of 2 min at 95 °C preceded the first cycle while the last was followed by an extension step of 5 min at 72 °C. The resulting fragments were purified by agarose gel electrophoresis (Wizard SV Gel; Promega) and digested with KpnI and SacI enzymes (Promega) as recommended by the manufacturer. After the enzymatic digestions, the fragments were purified by phenol extraction and then inserted by using T4 DNA ligase (Promega) into the KpnI–SacI site upstream of the firefly luciferase reporter gene in the pGL3-Basic vector containing an SV40 promoter (Promega). DNA constructs were transformed into Top10 Escherichia coli cells by electroporation according to standard protocols. pGL3/UCP1 haplotype constructs were prepared by using the QIAprep Spin Miniprep Kit (Qiagen). The constructs were sequenced with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) to check the correct insert

0.350.30Haplotype frequency (G-A)  —e— Fitted (Females) Fitted (Males)Observed (Females)Observed (Males)      

Table 3

Maximum likelihood estimations of the parameters of the fitted binomial logistic regression models in the analyzed sample.