Molecular Human Reproduction, Vol. 6, No. 10, 873-876,
October 2000
© 2000 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
No evidence for mutations of the leptin or leptin receptor genes in women with polycystic ovary syndrome
1 Department of Medicine, University of Helsinki, 2 Department of Obstetrics and Gynecology, University of Helsinki, 3 Department of Public Health, University of Helsinki, and 4 Department of Clinical Chemistry, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland
Abstract
Polycystic ovarian syndrome (PCOS) is often associated with obesity and insulin resistance, both of which are features that are linked to the leptin and leptin receptor (LEPR) genes. Analysis of the leptin gene by sequencing samples from 38 well-characterized patients with PCOS revealed no mutations of the coding exons. In single-stranded conformational polymorphism (SSCP) analysis and subsequent sequencing of the LEPR gene revealed previously identified amino acid variants in exons 2, 4 and 12 as well as the pentanucleotide insertion in the 3'-untranslated region (3'-UTR). The allele frequencies of these polymorphisms did not differ from those in the general population, as assessed in 122 female controls. Compared with non-carriers, serum insulin concentrations tended to be lower in the carriers of the variant LEPR exon 12 allele as well as in the carriers of the variant LEPR 3'-UTR allele, a marker previously suggested to be associated with serum insulin concentrations. In conclusion, PCOS is not commonly a consequence of mutations of the leptin or LEPR genes. However, our data support the hypothesis that variations in the LEPR gene locus have an effect on insulin regulation.
insulin/leptin/leptin receptor/obesity/polycystic ovary syndrome
Introduction
Polycystic ovary syndrome (PCOS) is a common endocrine disorder with an estimated prevalence of 4% in premenopausal women (Knochenhauer et al., 1998
). It is characterized by chronic anovulation and hyperandrogenaemia, and is associated with hirsutism, insulin resistance and obesity. A familial aggregation of PCOS, suggesting a genetic aetiology, has been established (Hague et al., 1988), and both an autosomal dominant and an oligogenic mode of transmission have been proposed (Franks et al., 1997). Genes in several metabolic pathways, including those regulating insulin metabolism and insulin action, have been proposed as contributing to the development of PCOS (Dunaif, 1997).
Leptin is an adipose tissue-secreted protein (Zhang et al., 1994) that acts on the hypothalamus (Caro et al., 1996). The plasma leptin concentration is correlated with body fat content and is usually higher in obese subjects (Maffei et al., 1995; Considine et al., 1996). In addition to signalling nutritional status to the central nervous system, leptin appears to have a role in reproductive physiology. Thus, treatment of mice with leptin accelerates the maturation of the female reproductive tract resulting in an earlier onset of the oestrous cycle and reproductive capacity (Chehab et al., 1997). Furthermore, leptin receptors have been shown to be expressed in ovaries (Cioffi et al., 1996). In normal individuals, chronic hyperinsulinaemia increases both the accumulation of leptin mRNA in adipose tissue and serum concentrations of leptin. Pancreatic ß-cells represent a potential site of insulin–leptin interaction, since these cells express leptin receptors (Kieffer et al., 1996), and possibly mediate leptin-induced inhibition of insulin secretion (Emilsson et al., 1997; Kieffer et al., 1997). A leptin surge in normal adolescents is associated with the onset of puberty (Mantzoros et al., 1997). The exact role of leptin in sexual maturation remains unclear, however leptin and leptin receptor-deficient individuals lack a normal pubertal development (Clément et al., 1998; Strobel et al., 1998). These findings, linking the leptin system to insulin regulation and gonadal function, prompted us to study whether variations of the leptin or leptin receptor (LEPR) genes could play a role in causation of the neuroendocrine and metabolic abnormalities characteristic of PCOS.
Materials and methods
Subjects
A total of 38 women with PCOS recruited from Helsinki University Central Hospital were examined. The diagnosis of PCOS was based on typical ovarian morphology assessed by transvaginal ultrasonography, hyperandrogenism (i.e. elevated total or free testosterone or androstenedione concentrations), and chronic anovulation (Tiitinen et al., 1994). The age range in the study group was 23–45 years (mean ± SD, 34 ± 6 years), and the mean body mass index (BMI) was 30.7 ± 9.5 kg/m2. In all, 26 subjects were obese (as defined by a BMI of >27 kg/m2). One subject had type 2 diabetes, and two had treatment for hypertension. Non-hirsute women (n = 18) with regular menstrual cycles were studied as a control group for serum leptin concentrations. The mean age of the control group was 30 ± 4 years and the mean BMI was 22.2 ± 2.1 kg/m2.
Reference population
Subjects used for determination of leptin receptor variant allele frequencies in the general population consisted of 122 apparently healthy females recruited from the Finnish Red Cross Blood Transfusion Service. They ranged in age from 40–50 years (mean ± SD, 45 ± 3 years). Although they all were Finnish and from the same geographical region of Southern Finland as the PCOS subjects, no data was available on their menstrual status or possible symptoms of PCOS and, as regular blood donors, they may not be fully representative of the population at large. The study protocol was approved by the Ethical Review Committee of the Departments of Obstetrics and Gynecology and the Department of Medicine, University of Helsinki, and all subjects gave their written informed consent.
Mutation screening of the LEPR gene by single-stranded conformational polymorphism (SSCP)
The coding and the adjacent flanking areas of the LEPR gene were screened for mutations and variations using SSCP. The polymerase chain reaction (PCR) primers used to amplify all 18 exons were always designed according to the intronic sequences (Chung et al., 1996) flanking the individual exons, as previously described (Oksanen et al., 1998).
DNA sequencing and genotype assays
The leptin gene was amplified by PCR and sequenced using previously reported oligonucleotide primer pairs (Maffei et al., 1996). The LEPR gene fragments showing an abnormal migration pattern in SSCP were similarly sequenced using an ABI 377 automated sequencer (PE Applied Biosystems, Foster City, CA, USA). The codon 109, 223 and 656 amino acid variants in exons 2, 4 and 12 respectively, and the 3'-untranslated region (3'-UTR) polymorphism were screened as previously described (Gotoda et al., 1997; Oksanen et al., 1998) using PCR and restriction fragment length polymorphism (RFLP) analysis. The PCR primers and restriction endonucleases are shown in Table I.
|
Hormone determinations
Serum leptin concentrations were measured by radioimmunoassay with a commercial kit purchased from Linco (Linco Research Inc., St Charles, MO, USA) with intra- and inter-assay coefficients of variation of
5%. Serum glucose concentrations were measured by a commercial glucose dehydrogenase technique (Banauch et al., 1975). Serum insulin concentrations were measured by a double antibody radioimmunoassay (Phadeseph Insulin RIA; Pharmacia, Uppsala, Sweden) (Desbuquois and Aurbach, 1971). Serum concentrations of LH, FSH, and sex-hormone binding globulin (SHBG) were measured by solid phase, two-site time-resolved immunofluorometric assays (DELFIA; Pharmacia Wallac, Turku, Finland). The intra- and inter-assay coefficients of variation were 5.2 and 9.4% for LH; 4.7 and 4.3% for FSH; and 5.0 and 7.0% for SHBG respectively. Total testosterone was determined using a commercial radioimmunoassay kit (Farmos Diagnostica, Turku, Finland). The intra- and inter-assay coefficients of variation were 13.0 and 15.6% respectively. Androstenedione and dehydroepiandrosterone sulphate (DHEAS) were determined by use of radioimmunoassay kits (Diagnostic Products Corporation, Los Angeles, CA, USA). The intra- and inter-assay coefficients of variation were 8.3 and 9.2% for androstenedione and 4.7 and 4.6% for DHEAS respectively.
Statistical analysis
The 
2-test was used to compare the frequencies of the different genotypes. Equality of the means of the quantitative traits in the different genotypes of the PCOS subjects were tested with non-parametric statistics using Mann–Whitney U-test using PROC NPAR1WAY in SAS (SAS Institute Inc, Cary, NC, USA). Values for serum leptin, insulin and SHBG were adjusted by linear regression on BMI prior to testing; P < 0.05 was considered to be statistically significant.
Results
A total of 38 women with PCOS were examined. Serum leptin concentrations (mean ± SE), adjusted for BMI and age, were similar in patients with PCOS (24.4 ± 2.6 ng/ml) and 18 regularly menstruating controls (28.5 ± 3.6 ng/ml, P = 0.39). Complete sequencing of the coding exons and adjacent flanking areas of the leptin gene revealed no mutations in DNA samples from the PCOS patients. SSCP screening and subsequent sequencing of the exonic and their immediate flanking regions of the LEPR gene revealed three amino acid variants in exon 2 (Lys109Arg), exon 4 (Gln223Arg) and exon 12 (Lys656Asn), intronic variants at position –27 (A
G) of intron 12, upstream of exon 13 and at position +37 (AC) of intron 17, a silent TC mutation at position 1029 (Ser343) of exon 7, a silent AC mutation at position 3057 (Pro1019) of exon 18, and a pentanucleotide insertion (CTTTA) localized in the 3'-UTR of the LEPR gene, 60 nucleotides after the stop codon.
The allele frequencies of the amino acid variants or the 3'-UTR insertion/deletion polymorphism of the LEPR gene did not differ from those in the general population as assessed in 122 population controls (Table II). When the BMI of the PCOS patients were compared according to the LEPR genotypes listed above, no differences were seen in the current BMI (Table III), or in the BMI at the age of 20 years, or the maximal BMI ever attained (data not shown). The prevalence of amenorrhoea was not increased in any of the genotypes either (data not shown).
|
|
Mean serum insulin values, adjusted for BMI, suggested an association with both LEPR exon 12 (P = 0.005) and LEPR 3'-UTR (P = 0.02) genotype (Table III
), with a tendency towards lower insulin concentrations in individuals heterozygous for the rare alleles. There was a weaker association between the 3'-UTR genotype and BMI-adjusted serum leptin concentrations, with lower leptin concentrations in the heterozygous subjects than in the carriers of the common allele (P = 0.05, Table III), and serum testosterone concentrations tended to be higher in the carriers of the variant 3'-UTR insertion allele, compared with the carriers of the common 3'-UTR deletion allele (P = 0.03). However, none of the associations were significant after correction for multiple comparisons. When serum LH, FSH, LH/FSH, SHBG, androstenedione, and DHEAS concentrations or free androgen index (FAI = testosterone/SHBG) were examined, there were no significant associations between any of these variables and the LEPR genotypes in the PCOS patients (data not shown). Discussion
The leptin receptor is expressed in the human ovaries (Cioffi et al., 1996) and leptin has been shown to have direct effects on ovarian function in vitro (Kitawaki et al., 1999). Although serum leptin concentrations do not appear to differ between PCOS patients and control subjects (Chapman et al., 1997), leptin and its receptor are attractive candidates as contributors to altered energy homeostasis, insulin metabolism and ovarian androgen synthesis associated with PCOS. In this study, we tested the hypothesis that part of the metabolic disturbances in PCOS are due to an inherited defect in the leptin or LEPR genes. Our data are in agreement with a previous report which showed that serum leptin concentrations do not differ between women with PCOS and controls (Chapman et al., 1997). Furthermore, we found no genetic variations in the coding area of the leptin gene. This is in agreement with a previous study (Urbanek et al., 1999), in which a marker in close vicinity of the leptin gene failed to associate with PCOS, as tested with a transmission disequilibrium test in 150 nuclear families. In contrast, screening of the LEPR gene revealed one new DNA variant in intron 12. Its location relatively distant from the exon–intron junction suggests that it may not cause an RNA splicing defect, although detailed studies to confirm this were not carried out. Previously identified amino acid variants corresponding to exons 2, 4 and 12 and the 3'-UTR insertion/deletion polymorphism of the LEPR gene were detected with allelic frequencies similar to those found in other Caucasian populations (Echwald et al., 1997; Oksanen et al., 1998).
Serum insulin values were associated with both the variant LEPR exon 12 and the variant LEPR 3'-UTR genotypes, but after correction for multiple comparisons, these results were no longer significant (Table II). However, they are in agreement with our previous findings in morbidly obese subjects, in which the presence of the variant insertion allele in the 3'-untranslated region of the LEPR gene was associated with low serum insulin concentrations in the carriers of the insertion allele, possibly due to an effect on the stability of the LEPR mRNA (Oksanen et al., 1998). In addition, another study (Francke et al., 1997) reported a trend towards lower serum insulin values 30 min after an oral glucose load in insertion/deletion heterozygotes compared with deletion/deletion homozygotes. Thus a plausible biological background as well as several congruent observations speak against a chance association, although the exact underlying mechanism remains to be explored.
In conclusion, we found no evidence for altered serum leptin concentrations in PCOS, nor could we demonstrate a causal role for mutations or variations of the leptin or LEPR genes in the development of the syndrome. However, the present study supports the hypothesis that the LEPR gene plays a role in the regulation of serum insulin concentrations.
Acknowledgments
We thank Susanna Tverin, Tuula Soppela-Loponen and Merja Lindfors for expert technical assistance. We also thank Dr Tom Krusius (Finnish Red Cross Blood Transfusion Service) for providing us with the blood samples of healthy blood donors. This work was supported by grants from The Sigrid Juselius Foundation, The Medical Council of the Finnish Academy (grant #42044), The Emil Aaltonen Foundation, The Finnish Medical Foundation, The Maud Kuistila Foundation, The Finnish Foundation for Cardiovascular Research and The Finnish Cultural Foundation.
Notes
5 To whom correspondence should be addressed at: Department of Medicine, University of Helsinki, FIN-00290 Helsinki, Finland. E-mail: kimmo.kontula{at}hus.fi 
References
Banauch, D., Brümmer, W., Ebeling, W. et al. (1975) Eine Glucose-dehydrogenase für die Glucose-bestimmung in Körperflüssigkeiten. Z. Klin. Chem. Klin. Biochem., 13, 101–107.[Web of Science][Medline]
Caro, J.F., Sinha, M.K., Kolaczynski, J.W. et al. (1996) Leptin: the tale of an obesity gene. Diabetes, 45, 1455–1462.[Web of Science][Medline]
Chapman, I.M., Wittert, G.A., Norman, R.J. (1997) Circulating leptin concentrations in polycystic ovary syndrome: relation to anthropometric and metabolic parameters. Clin. Endocrinol., 46, 175–181.[Medline]
Chehab, F., Mounzih, K., Lu, R. et al. (1997) Early onset of reproductive function in normal female mice treated with leptin. Science, 275, 88–90.
Chung, W.K., Power-Kehoe, L., Chua, M. et al. (1996) Genomic structure of the human OB receptor and identification of two novel intronic microsatellites. Genome Res., 6, 1192–1199.
Cioffi, J.A., Shafer, A.W., Zupancic, T.J. et al. (1996) Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. Nature Med., 2, 585–589.[Web of Science][Medline]
Clément, K., Vaisse, C., Lahlou, N. et al. (1998) A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature, 392, 398–401.[Medline]
Considine, R.V., Sinha, M.K., Heiman, M.L. et al. (1996) Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N. Engl J. Med., 334, 292–295.
Desbuquois, B. and Aurbach, G.D. (1971) Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J. Clin. Endocrinol., 33, 732–738.
Dunaif, A. (1997) Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev., 18, 774–800.
Echwald, S.M., Sørensen, T.D., Sørensen, T.I. et al. (1997) Amino acid variants in the human leptin receptor: lack of association to juvenile onset obesity. Biochem. Biophys. Res. Commun., 233, 248–252.[Web of Science][Medline]
Emilsson, V., Liu, Y.L., Cawthorne, M.A. et al. (1997) Expression of the leptin receptor mRNA in pancreatic islets and direct inhibitory action of leptin on insulin secretion. Diabetes, 46, 313–316.[Abstract]
Francke, S., Clément, K., Dina, C. et al. (1997) Genetic studies of the leptin receptor gene in morbidly obese French Caucasian families. Hum. Genet., 100, 491–496.[Web of Science][Medline]
Franks, S., Gharani, N., Waterworth, D. et al. (1997) The genetic basis of polycystic ovary syndrome. Hum. Reprod., 12, 2641–2648.
Gotoda, T., Manning, B.S., Goldstone, A.P. et al. (1997) Leptin receptor gene variation and obesity: lack of association in a white British male population. Hum. Mol. Genet., 6, 869–876.
Hague, W.M., Adams, J., Reeders, S.T. et al. (1988) Familial polycystic ovaries: a genetic disease? Clin. Endocrinol., 29, 593–605.[Medline]
Kieffer, T.J., Heller, R.S., Haberner, J.F. (1996) Leptin receptors expressed on pancreatic -cells. Biochem. Biophys. Res. Commun., 224, 522527.
Kieffer, T.J., Heller, S.R., Leech, C.A. et al. (1997) Leptin suppression of insulin secretion by activation of ATP-sensitive K+ channels in pancreatic -cells. Diabetes, 46, 10871093.
Kitawaki, J., Kusuki, I., Koshiba, H. et al. (1999) Leptin directly stimulates aromatase activity in human luteinized granulosa cells. Mol. Hum. Reprod., 5, 708–713.
Knochenhauer, E.S., Key, T.J., Kahsar-Miller, M. et al. (1998) Prevalence of the polycystic ovary syndrome in unselected black and white women of the Southeastern United States: a prospective study. J. Clin. Endocrinol. Metab., 83, 3078–3082.
Maffei, M., Halaas, J., Ravussin, E. et al. (1995) Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nature Med., 1, 1155–1161.[Web of Science][Medline]
Maffei, M., Stoffel, M., Barone, M. et al. (1996) Absence of mutations in the human ob gene in obese/diabetic subjects. Diabetes, 45, 679–682.[Abstract]
Mantzoros, C.S., Flier, J.S., Rogol, A.D. (1997) A longitudinal assessment of hormonal and physical alterations during normal puberty in boys. V. Rising leptin levels may signal the onset of puberty. J. Clin. Endocrinol. Metab., 82, 1066–1070.
Oksanen, L., Kaprio, J., Mustajoki, P. et al. (1998) A common pentanucleotide polymorphism of the 3'-untranslated part of the leptin receptor gene generates a putative stem-loop motif in the mRNA and is associated with serum insulin levels in obese individuals. Int. J. Obes., 22, 634–640.[Web of Science]
Strobel, A., Issad, T., Camoin, L. et al. (1998) A leptin missense mutation associated with hypogonadism and morbid obesity. Nature Genet., 18, 213–215.[Web of Science][Medline]
Tiitinen, A., Simberg, N., Stenman, U. et al. (1994) Estrogen replacement does not potentiate gonadotropin-releasing hormone agonist-induced androgen suppression in treatment of hirsutism. J. Clin. Endocrinol. Metab., 79, 447–451.[Abstract]
Urbanek, M., Legro, R.S., Driscoll, D.A. et al. (1999) Thirty-seven candidate genes for polycystic ovary syndrome: Strongest evidence for linkage is with follistatin. Proc. Natl Acad. Sci. USA, 96, 8573–8578.
Zhang, Y., Proenca, R., Maffei, M. et al. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature, 372, 425–432.[Medline]
Submitted on January 20, 2000; accepted on July 4, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Simoni, C.B. Tempfer, B. Destenaves, and B.C.J.M. Fauser Functional genetic polymorphisms and female reproductive disorders: Part I: polycystic ovary syndrome and ovarian response Hum. Reprod. Update, September 1, 2008; 14(5): 459 - 484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Diamanti-Kandarakis and C. Piperi Genetics of polycystic ovary syndrome: searching for the way out of the labyrinth Hum. Reprod. Update, November 1, 2005; 11(6): 631 - 643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. F. Escobar-Morreale, M. Luque-Ramirez, and J. L. San Millan The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome Endocr. Rev., April 1, 2005; 26(2): 251 - 282. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||