Molecular Human Reproduction, Vol. 5, No. 1, 5-9,
January 1999
© 1999 European Society of Human Reproduction and Embryology
No association between the –308 polymorphism in the tumour necrosis factor 
(TNF) promoter region and polycystic ovaries
1 Reproductive Medicine Unit, The University of Adelaide, The Queen Elizabeth Hospital, Woodville 5011, South Australia, Australia
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Abstract |
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The tumour necrosis factor (TNF)2 allele appears to be linked with increased insulin resistance and obesity, conditions often found in overweight patients with polycystic ovary syndrome (PCOS). The significance of TNF
polymorphism in relation to the clinical and biochemical parameters associated with PCOS was investigated in 122 well-characterized patients with polycystic ovaries (PCO). Of these, 84 had an abnormal menstrual cycle and were classified as having PCOS, while the remaining 38 had a normal menstrual cycle and were classified as having PCO. There were a further 28 individuals without PCO (non-PCO) and 108 individuals whose PCO status was undetermined (reference population). The promoter region of the TNF gene was amplified by polymerase chain reaction (PCR), and the presence or absence of the polymorphism at –308 was determined by single-strand conformational polymorphism (SSCP) analysis. The less common TNF allele (TNF2) was found as TNF1/2 or TNF2/2 in 11/38 (29%) of PCO subjects, 25/84 (30%) of PCOS subjects, 7/28 (25%) of non-PCO subjects, and 45/108 (42%) of the reference population. There was no significant difference in the incidence of the TNF2 allele between the groups. The relationship of TNF genotype to clinical and biochemical parameters was examined. In both the PCO group and the PCOS group, the presence of the TNF2 allele was significantly associated with lower glucose values obtained from the glucose tolerance testing (P < 0.05). The TNF genotype was not significantly associated with any clinical or biochemical parameter measured in the PCO, PCOS or non-PCOS groups. Thus, the TNF –308 polymorphism does not appear to strongly influence genetic susceptibility to polycystic ovaries.
polycystic ovary syndrome/polymorphism/tumour necrosis factor
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women presenting for investigation of infertility, hirsutism and obesity. It represents a large proportion of women with anovulation (Franks, 1995
) and may be present in 5–10% of all pre-menopausal women. Family studies have demonstrated the importance of genetic factors in PCOS (Legro, 1995; Norman et al., 1996) but the precise mode of inheritance and the molecular basis for PCOS remain uncertain. The study of PCOS is partly hampered by the lack of a universally accepted definition (Zawadski and Dunaif, 1992; Franks, 1995) as a result of disagreement concerning the relative merits of ovarian sonography, endocrinology and clinical features as well as the recognized genetic heterogeneity of the condition. Various candidate genes have been proposed as important contributors to PCOS (Legro, 1995; Jahanfar and Eden, 1996) but none have yet achieved acceptance as major causes for this important clinical condition.
Recent data have suggested a role for TNF in insulin resistance (Hotamisligil and Spiegelman, 1994), obesity (Kern et al., 1995), and pre-eclampsia (Chen et al., 1996). The gene for TNF lies on the short arm of chromosome 6 (Jäättelä, 1991; Browning et al., 1993). The TNF locus lies in a region of the class III region of the major histocompatibility complex (MHC) (Trowsdale, 1993). TNF expression is regulated at both the transcriptional and post-transcriptional levels (Sariban et al., 1988). Transcription of the TNF gene is regulated by the promoter region which consists of an 1100 base pair stretch of DNA (Wilson et al., 1995a). A biallelic polymorphism which involves the substitution of guanine by adenosine at position –308 in the promoter region (Wilson et al., 1993) produces the less common TNF2 allele, which has been associated with elevated serum concentrations of TNF in certain clinical states (Wilson et al., 1994, 1995b; Danis et al., 1995; Fong et al., 1996; Hajeer et al., 1996; Jacob et al., 1996). The –308 TNF polymorphism has been associated with susceptibility to infection (Cabrera et al., 1995; Mizuki et al., 1995; Wilson et al., 1995c; Xia et al., 1995; Bouma et al., 1996a,b; Nadel et al., 1996; Stuber et al., 1996), autoimmune diseases (Verjans et al., 1992, 1994; Brigitta et al., 1994; Wilson et al., 1994; Danis et al., 1995; Galbraith and Pandey, 1995; He et al., 1995; McGuinness et al., 1995; Zipp et al., 1995; Braun et al., 1996; Fong et al., 1996; Jacob et al., 1996; McManus et al., 1996), and pre-eclampsia (Chen et al., 1996). Association of the TNF2 allele with insulin dependent diabetes has also been reported (Pociot et al., 1993a,b; Monos et al., 1995), but this association may be related to linkage disequilibrium between TNF and HLA haplotypes (Deng et al., 1996). The presence of the TNF2 allele has been linked to both insulin resistance development and increasing adiposity (Fernandez-Real et al., 1997). TNF appears to inhibit follicle stimulating hormone (FSH)-induced oestradiol secretion in small follicles from the human ovary (Rice et al., 1998). These two actions of TNF may link the ovarian steroidogenesis problems with those of insulin resistance.
In view of the strong evidence implicating TNF in adiposity, insulin resistance and anovulation (all of which are features of PCOS), the object of this study was to determine the frequency of the TNF promoter polymorphism (alleles TNF1 and TNF2) in a group of well-characterized subjects. Those subjects who demonstrated distinctive clinical changes but remained with a normal menstrual cycle were classed as PCO patients, while those who additionally had an abnormal menstrual cycle were classed as PCOS patients. A control group known to be free of PCOS, and a reference population of unknown PCOS status were also included. We have used single-strand conformational polymorphism (SSCP) analysis with silver staining, to determine the genotype at the TNF promoter. The second aim of this study was to relate the presence of the TNF2 allele to indices relevant to obesity, insulin resistance and ovulation in PCOS.
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Materials and methods |
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Patient samples
PCOS patients and normal control group
A total of 122 female subjects were recruited from the Reproductive Medicine and Gynaecology Clinics at the Queen Elizabeth Hospital. These patients were defined as those who showed characteristic changes in reproductive hormones, clinical features and distinctive morphological changes to the ovary on ultrasound examination. Of these, 22 subjects were included who showed evidence of PCO by ultrasound criteria only, with no other clinical sequelae, while the remaining 100 patients had clinical manifestations of PCOS. Of the 122 patients, 38 had normal menstrual cycles (22–33 days) and were classified as PCO patients. The remaining 84 patients had abnormal menstrual cycles (
21 or 
34 days) and were classified as PCOS patients. In addition, there were 28 patients who were proven to be non-PCO on the basis of clinical, biochemical and ultrasound evaluation. Further details of the phenotypic evaluation including assay methods and 2 h glucose tolerance testing of the PCO and PCOS patients has been previously published (Norman et al., 1995).
Reference population
108 patients whose PCO status was unknown were recruited from the female blood donor population to determine the population frequency of the polymorphism. No clinical details were available on these subjects.
DNA preparation and determination of the –308 TNF polymorphism
Genomic DNA was prepared from leukocyte-rich EDTA plasma using the Progenome II reagent kit (Progen, Queensland, Australia). Aliquots were stored at –20°C. The TNF promoter polymorphism was typed using a modification of the method described in Wilson et al. (1993). Briefly, a 107 bp fragment was amplified by PCR from genomic DNA using the sense primer sequence 5'-AGGCAATAGGTTTTGAGGGCCAT-3' and the anti-sense primer 5'-TCCTCCCTGCTCCGATTCCG-3'. A PCR reaction mix (50 µl) containing 500 ng of genomic DNA, 10 µmol/l of each primer, 1.5 mmol/l MgCl2, 100 µmol/l each of dATP, dTTP, dGTP and dCTP, 0.5 U AmpliTaq DNA polymerase in a 10 mmol/l reaction buffer of Tris–HCl (pH 6.3) and 50 mmol/l KCl (Perkin Elmer, Norwalk, CT, USA) was placed in a PTC-100 thermal cycler (M J Research, Boston, MA, USA). Amplification conditions were denaturation at 94°C for 3 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min, followed by 35 cycles of 1 min each at 94°C, 60°C and 72°C, followed by 94°C for 1 min, 60°C for 1 min, and 72°C for 5 min. Amplification efficiency was determined by agarose gel electrophoresis.
SSCP analysis
Twenty µl of the PCR product was denatured with 20 µl of deionized formamide containing 0.05% each of bromophenol blue and xylene cyanole FF as markers. This mixture was heated at 100°C for 5 min then immediately cooled and kept on ice. Polyacrylamide gel electrophoresis was performed on the whole 40 µl mixture using 9% polyacrylamide (19:1 polyacrylamide/bis) in 0.5x tris borate EDTA (TBE) buffer. Electrophoresis was performed at a constant 0.5 W/cm for 5 h using a 4°C circulating water bath. DNA was visualized using a silver staining reagent (BioRad, Hercules, CA, USA) and methanol (40%)/ethanol (10%) without acetic acid as a fixative.
Statistical analysis
The 
2-test was used to compare the frequencies of the different genotypes. The significance of association between the clinical parameters and the genotypes was assessed using the two-sided t-test.
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Results |
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Clinical, biochemical and ultrasound parameters were measured in 150 female subjects; 38 with PCO, 84 with PCOS, and 28 normal controls (non-PCO). TNF
genotype was determined in these individuals and a further 108 population controls of unknown PCOS status, where specific clinical information was not available. Following the PCR amplification of the genomic DNA and subsequent partial denaturation, the 258 individuals were categorized for the –308 polymorphism using SSCP analysis and the alleles visualized with silver staining. Grouping into the three genotypes could be accomplished by discriminating between the presence or absence of the two alleles. Figure 1 shows the SSCP analysis of the genotypes TNF1,1 (wild type), TNF1,2 (heterozygous) and the uncommon (homozygous) TNF2,2. The frequency of the TNF genotypes for the PCO group, the PCOS group, the control group and the reference group are shown in Table I. In the PCO group 2/38 (5%) and in the PCOS group 3/84 (2%) were homozygous for the TNF2 allele, compared with 0/28 of the non-PCO control group and 3/108 (3%) of the reference population.
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TNF1,2 heterozygotes were combined with the TNF2,2 homozygotes for statistical comparison between the four clinical groups. The frequency of the occurrence of individuals with at least one copy of the TNF2 allele did not differ significantly between the PCO group (11/38, 29%), the PCOS group (25/84, 30%), the non-PCO (control) group (7/28, 25%), and the population reference group (45/108, 42%).
Tables II and III list relevant clinical and biochemical parameters measured as part of this study according to the TNF genotype groups in the PCO, PCOS and the non-PCO control groups. Within both the PCO and PCOS groups, the mean integrated glucose value during the 2 h oral glucose tolerance test was 770.1 (± 80.9), and 815.0 (± 224.9) mmol/l respectively in TNF1 homozygotes compared with 549.0 (±213.6), 706.0 (±237.7) mmol/l respectively in those with at least one copy of the TNF2 allele (P < 0.05). In the other variables examined, there were no statistically significant differences according to TNF genotype in either the PCO, PCOS or the non-PCO group.
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Discussion |
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TNF is present in the human ovary in follicular fluid (Zolti et al., 1992
), granulosa cells (Roby and Terranova, 1989) and the oocyte (Kondo et al., 1995). A suggestion exists that TNF may induce atresia in the follicle or, alternatively, block follicular growth as occurs in PCO by inhibiting FSH-stimulated aromatase activity as well as stimulating androstenedione production (Terranova et al., 1991). While concentrations of TNF in ovarian cultures do not appear to be different between normal and polycystic ovaries (Jasper and Norman, 1995), TNF is an attractive candidate as a contributor to ovarian changes associated with PCO/PCOS. The current study does not exclude a local role for TNF in both insulin resistance and ovarian hyperandrogenaemia, cardinal features of anovulatory PCOS, but more information on RNA expression in PCO and normal ovaries is required.
In this study the biallelic polymorphism at position –308 of the TNF promoter was genotyped in a total of 258 individuals (Table I
). We modified the genotyping methodology for the –308 TNF polymorphism described by Wilson et al. (1993), using silver staining to visualize the SSCP bands. This was found to produce better resolution of the bands corresponding to the two alleles, and stronger band intensity than ethidium bromide staining. There were no significant differences in the frequency of the less common TNF2 allele between the PCO group, the PCOS group, the non-PCO controls, and the reference population. The frequency of the TNF2 allele in the Australian population in this study was similar to that observed in other Caucasian groups studied (Verjans et al., 1992; Brigitta et al., 1994; Cabrere et al., 1995; Wilson, et al., 1995)
It is noteworthy that the only parameter to show a significant association with the –308 TNF genotype was the integrated glucose value derived from the oral glucose tolerance test in both the PCO and the PCOS groups. Recent studies have implicated TNF in physiological pathways of glucose metabolism (Grunfeld and Feingold, 1991; Stephens and Pekala, 1992). It is perhaps surprising that the glucose response should be diminished in those with at least one TNF2 allele. However, this finding should be verified in a second data set before further comment.
The aetiology of PCOS is unknown but genetic factors are known to be of importance (Legro, 1995). The inherited component of PCOS has been demonstrated in family studies (Norman et al., 1996). Determining the mode of inheritance of PCOS has proved difficult, and it is likely that the genetic component is polygenic, although the existence of a single major gene with minor genetic modifiers is a potential model.
Our results show that there is no strong association of the –308 TNF polymorphism and PCO. The finding of altered response to oral glucose tolerance testing according to TNF genotype in the PCO and PCOS groups merits further investigation. We intend to repeat the phenotypic analysis of these groups as part of an ongoing study in the natural history of PCOS. This will provide an opportunity to reassess the association between altered glucose tolerance and TNF genotype. Studying large pedigrees in which PCOS segregates, or a multicentre sibling-pair study may be other approaches to further improve our limited knowledge of the underlying molecular basis for PCOS.
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Notes |
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2 Current address: Walter and Eliza Hall Institute of Medical Research, Melbourne 3000, Australia
3 To whom correspondence should be addressed
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References |
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Submitted on April 7, 1998; accepted on September 25, 1998.
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