Hum. Reprod. Advance Access originally published online on January 5, 2006
Human Reproduction 2006 21(4):943-951; doi:10.1093/humrep/dei443
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Specific haplotypes of the CALPAIN-5 gene are associated with polycystic ovary syndrome
1 Centro Avanzado de Fertilidad (CAF), Unidad de Reproducción y Genética Humana, Instituto Médico Serman, 2 Unidad Materno-Infantil, Hospital Virgen de las Montañas, Cádiz and 3 Neocodex, Departamento de Genómica Estructural, Sevilla, Spain
4 To whom correspondence should be addressed at: Departamento Genómica Estructural, Neocodex, Centro de Negocios Charles Darwin, Avda, Charles Darwin, s/n; Isla de la Cartuja, 41092 Sevilla, Spain. E-mail: aruiz{at}neocodex.es
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Abstract |
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BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age. The aim of the present study was to investigate the role of CALPAIN-5 (CAPN5) gene in PCOS susceptibility. METHODS: We analysed four intronic polymorphisms of the CAPN5 gene in 148 well-characterized women with PCOS and 606 unrelated controls. We performed a case-control study and an intracohort analysis of clinical characteristics associated with PCOS. RESULTS: Analysis of haplotypes distribution between PCOS population compared to controls showed a strong deviation (P = 0.00029). The haplotypes GGCA and GGTG were overrepresented in PCOS patients (P = 0.009 and P = 0.001, respectively). In addition, we identified several CAPN5 haplotypes associated with phenotypic differences observed between PCOS patients, such as the presence of obesity (P = 0.02), cardiovascular complications (P = 0.02), familial antecedents of obesity (P = 0.003) and of hypertension (P = 0.007) and type 2 diabetes mellitus aggregation (P = 0.04). CONCLUSIONS: These results suggest a role of CAPN5 gene in PCOS susceptibility in humans. Moreover, novel candidate risk alleles have been identified, within CAPN5 gene, which could be associated with important phenotypic and prognosis differences observed in PCOS patients.
Key words: anovulation/CAPN5 gene/PCOS/SNP/sterility
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflicts of interestAcknowledgementsReferences |
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Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting women of reproductive age (Asteria, 2000
). Specifically in Spain, a prospective study reported an overall 6.5% prevalence of PCOS (Asuncion et al., 2000). It is considered to be a syndrome and not a disease, although the term polycystic ovarian disease is often used synonymously, but incorrectly, to describe the syndrome which is manifested by heterogeneous clinical features. The most common features of PCOS are irregular menstrual cycles (oligomenorrhoea or amenorrhoea), signs of androgen excess (hirsutism, acne and alopoecia) and often obesity. However, only 5–10% of women with PCOS express the typical clinical features of the syndrome (Balen, 1999). Data on the epidemiology of PCOS are variable because of the lack of well-accepted criteria for diagnosis. Defining PCOS has generated controversies between clinicians for many years (Dewailly, 1997). In fact, definition consensus of PCOS, proposed by the National Institute of Health–National Institute of Child Health and Human Development (NIH–NICHHD) conference in 1990 (Zawadzky and Dunaif, 1992), does not include the ovarian morphologic phenotype observed on ultrasound scans, because the ovarian phenotype (polycystic ovaries, PCO) is a common trait observed in a great proportion of women (Legro et al., 1998; Legro and Strauss, 2002). In contrast, a definition consensus definition has recently been proposed, in which ultrasound morphology should be performed in order to confirm diagnosis of PCOS in the presence of clinical symptoms. Nevertheless, normal ovary morphology would not exclude patients with clinical anovulation, menstrual disturbances and hyperandrogenism as far as diagnosis would be ascertained by biochemical examinations (Homburg, 2002).
Despite its prevalence, little is known about the aetiology of PCOS, but there is increasing evidence for an important genetic involvement (Legro et al., 1998; Govind et al., 1999). However, the mode of inheritance of PCOS is still uncertain, and recent studies indicate that this disorder could be a complex trait (Crosignani and Nicolosi, 2001). This means that several genes are interacting with environmental factors (principally dietary) to determinate the typically heterogeneous, clinical and biochemical phenotype (Weiss and Terwilliger, 2000). Biochemical parameters including fasting insulin levels or hyperandrogenaemia seem to be highly heritable parameters, suggesting that some clinical signs, symptoms or biochemical parameters of PCOS could be transmitted as Mendelian autosomal dominant or X-linked traits (Legro and Strauss, 2002).
Present data strongly support an association between PCOS and several long-term disease risks. Conditions that have been linked to PCOS include non-insulin-dependent diabetes mellitus (NIDDM), hypercholesterolaemia, hypertension, cardiovascular disease, gestational diabetes mellitus, pregnancy-induced hypertension and endometrial cancer, and recently associations between PCOS and breast cancer and ovarian cancer have been reported (Solomon, 1999; Lobo and Carmina, 2000). These data emphasize the need for early diagnosis of the syndrome and close follow-up of women with PCOS.
Looking for the molecular basis of PCOS, numerous research teams have begun a systematic search of genetic risk factors involved in PCOS susceptibility and prognosis (Legro and Strauss, 2002). Several genes involved in reproduction, genes affecting the secretion or action of insulin and those involved in obesity and energy regulation have been tested as candidate genes. In particular, attention has been focused on genes coding for steroidogenic enzymes in the androgen biosynthetic pathway (Carey et al., 1994; Gharani et al., 1997; Urbanek et al., 1999) and on those involved in the secretion and the action of insulin (Talbot et al., 1996; McKeigue and Wild, 1997; Waterworth et al., 1997; Eaves et al., 1999; Urbanek et al., 1999).
PCOS is associated with a two- to sevenfold risk of NIDDM. Previous epidemiological and genetic studies have revealed that PCOS and NIDDM could share genetic susceptibility factors. Using this working hypothesis, several studies have suggested that genes related to NIDDM may play a role in PCOS pathogenesis (Ehrmann et al., 2002a,b; Escobar-Morreale et al., 2002; Gonzalez et al., 2002). The first NIDDM susceptibility gene revealed by a genome-wide scan and positional cloning was an ubiquitously expressed member of the calpain-like cysteine protease family CALPAIN-10 (CAPN10) (Horikawa et al., 2000). Association studies using intragenic markers of CAPN10 gene have revealed that different alleles may contribute to genetic predisposition to NIDDM in several populations (Horikawa et al., 2000; Evans et al., 2001; Garant et al., 2002). Recently, we and others have identified novel candidate risk alleles and genotypes, within CAPN10 gene, which could be associated with important phenotypic and prognostic differences observed in PCOS patients (Ehrmann et al., 2002a; Gonzalez et al., 2002, 2003).
CAPN5 is a paralogue of CAPN10 gene (genes that have originated through duplication of an ancestral gene), which, like the latter, is a protease that has been implicated in the regulation of a variety of cellular functions, including intracellular signalling, proliferation, differentiation and apoptosis (Suzuki et al., 1992; Sorimachi et al., 1997; Ono et al., 1998).
We can infer that CAPN5 is expressed in the human ovary since the protein expression is ubiquitous (Waghray et al., 2004). In addition, it would have the function recognized for CAPN5 in the differentiation and maturation of the germline cells of Caenorhabditis elegans. It has been demonstrated that this member of the calpain gene family (tra3, homologue of CAPN5 in humans) plays a crucial role in the development of the germline of the hermaphrodite earthworm by means of the post-transcriptional control of the genes tra2 and phosphatases 1–3. All these proteins seem to control the correct production of germ cells of hermaphrodite sex and also the sexual differentiation of C. elegans (Barnes and Hodking, 1996). Given the role of CAPN5 paralogue in the sexual development of model animals, and its structural and functional relationships with CAPN10 gene, we also hypothetized that CAPN5 could be a good candidate gene for linkage and association studies in PCOS. Furthermore, in humans, OMP (olfactory marker protein) is a intragenic gene located within intron 3 of CAPN5 gene and could be implied in the olfactory dysfunction (anosmia) that is associated with certain types of infertility (Mroueh and Kase, 1968).
The aim of the present study was to investigate the role of CAPN5 gene in PCOS susceptibility. We decided to examine this hypothesis with an association study using a population-based series of patients with PCOS for the frequency of four intronic polymorphisms within CAPN5 gene (rs948976, rs4945140, rs2233546 and rs2233549) compared to normal controls. To further analyse the role of CAPN5 in PCOS, we present a haplotype–phenotype correlation study of CAPN5 gene in 148 women showing ecographically detected PCO combined with one or more clinical features of PCOS.
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Materials and methods |
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Patients
The study population consisted of 148 unrelated women with PCOS. We also included 606 unselected and non-related individuals. The ethnicity of all probands and controls was Caucasian (white Europid). The referral centre for this study is the Centro Avanzado de Fertilidad (CAF, Jerez, Andalucia, Spain). Informed consent was obtained from all patients.
PCOS was defined, according to Homburg (2002), as previously reported by us (Gonzalez et al., 2003). Selected probands were examined by one of the study investigators (A.G.). The clinical profile of the population studied is summarized in Table I. To estimate population frequencies of single-nucleotide polymorphisms (SNPs), haplotypes or genotypes in the Spanish population, 606 unselected controls from the same geographical region were genotyped in an anonymized fashion.
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SNP discovery
The DNA sequence used to carry out this study corresponds to the genomic sequence of the CAPN5 gene. Genomic sequence containing CAPN5 gene (Genomic Contig NT 033927, locus ID 726) was identified using BLAT tool at UCSC Genome Bioinformatics (http://genome.ucsc.edu/index.html) with CAPN5 mRNA probe (GenBank accession number NM004055). The gene spans 59 192 pb comprising 14 exons and 13 introns at 11q.14.
Looking for SNPs, we used germline DNA pooling from 40 unrelated individuals, and we performed the screening of this gene selecting two candidate clusters located at introns 1 and 3. Using bi-directional automated DNA sequencing, we identified four DNA variant sites: (i) two polymorphic markers located in the 5' region, intron 1 of CAPN5 gene: rs948976 and rs4945140 (according to GenBank accession number AY547311 [GenBank] ) and (ii) two polymorphisms located in the coding region of the OMP gene, intron 3 of CAPN5 gene: rs2233546 and rs2233549 (according to GenBank accession number U01212 [GenBank] ). Information concerning the markers detected was compared with the UCSC Genome Bioinformatics and also with Genome Database (GDB) (Figure 1).
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Genotyping
Cluster 1: rs948976 and rs4945140
Parallel analysis of these polymorphisms was performed using automated DNA-sequencing methods. For the PCR, amplification primers covering the entire 5' region of study were designed (Table II). PCR was carried out with a final volume of 10 µl using 10 ng of genomic DNA, 1 mM of each amplification primer, 4.4 mM MgCl2 and 1 µl of LC Faststart DNA Master SYBR green I (Roche Applied Science, Indianapolis, IN, USA). PCR amplification conditions for the Lightcycler were an initial denaturation step of 95°C for 7 min, followed by 40 cycles of 95°C for 0 s, 67°C for 10 s and 72°C for 45 s. PCR products were purified and sequenced directly using Capn5F and Capn5R primers (Table II). Sequencing reactions were performed using CEQ Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Fullerton, CA, USA), according to the manufacturer’s instructions, and analysed on CEQTM 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA).
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Cluster 2: rs2233546 and rs2233549
We designed and synthesized amplification primers and fluorescent-detection probes for the PCRs of the CAPN5 gene using the Web Primer software (genome-www2.stanford.edu/cgi-bin/SGD/web-primer) following the manufacturer’s instructions. The selected primer pairs and detection probes are shown in Table II.
PCR conditions
Real-time PCR was performed in the LightCycler system (Roche) using reaction conditions previously published (Real et al., 2001; Buch et al., 2003; Gonzalez-Gomez et al., 2003). PCR was performed to amplify the segment of the CAPN5 gene that flanks the two polymorphic sites within intron 3. The PCR reactions were carried out in a final volume of 10 µl using 10 ng of genomic DNA, 0.5 mM each amplification primer, 4.4 mM MgCl2, 0.5 mM each detection probe and 1 µl of LC Faststart DNA Master hybridization probes (Roche). We used an initial denaturation step of 95°C for 7 min, followed by 40 cycles of 95°C for 0 s, 68°C for 10 s and 72°C for 40 s.
Melting curves
The conditions to obtain optimal melting curves and spectrofluorimetric genotypes were 95°C for 0 s, 63°C for 25 s, 45°C for 0 s and 80°C for 0 s (with a temperature-transfer speed of 20°C/s in each step, except the last step, in which the speed of temperature transfer was 0.1°C/s). In the last step, a continuous fluorometric register was performed (F3/F1 for rs2233546 and F2/F1 for rs2233549), fixing the gains of the system at 1, 50 and 50 on channels F1, F2 and F3, respectively. Genotype results using real time-PCR are shown in Figure 1. To test the specificity of these assays, selected amplicons of different melting patterns were sequenced using an automated DNA sequencer (Beckman Coulter CEQTM 8000 Genetic Analysis System, data not shown).
Statistical analysis
To compare alleles and genotypes frequencies between patients and unselected controls, conventional Chi-square tests with Yate’s correction were performed using Statcalc and Analysis software (EpiInfo 5.1, Center for Disease control, Atlanta, GA, USA).
For statistical analysis of genotype distribution, test for deviation of Hardy–Weinberg equilibrium or two-point association studies, we employed tests adapted from Sasieni (1997). These calculations were performed in the online resource at the Institute for Human Genetics, Munich, Germany (http://ihg.gsf.de). For genotypes studies, the Bonferroni correction has been employed to adjust for the multiple testing, considering that both polymorphisms in each cluster are in complete linkage disequilibrium (LD), so statistical significance was established as P < 0.025.
LD value (D') between the genetic markers studied, haplotype frequencies and haplotype-based association analysis were calculated using Thesias software (http://genecanvas.org) based on the SEM algorithm (Tregouet et al., 2003, 2004). This method allows one to estimate haplotype frequencies and haplotype effects by comparison to a reference (the intercept) taken here as the most frequent one. Haplotypes effects are expressed as odds ratios (OR). Statistical significance level was defined at 0.05.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflicts of interestAcknowledgementsReferences |
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We have analysed 148 cases of PCOS compared to 606 controls for the frequency of polymorphic alleles at four loci within CAPN5 gene: rs948976, rs4945140, rs2233546 and rs2233549. Allelic frequency in Spanish population was 0.74 for A allele in rs948976, 0.57 for G allele in rs4945140, 0.94 for C allele in rs2233546 and 0.80 for G allele in rs2233549. The difference in allelic frequencies in these genetic markers between PCOS and controls was not significant (data not shown). Genotype frequencies observed during this study are in accordance with the Hardy–Weinberg equilibrium law (P > 0.38). After Bonferroni correction, there were no statistically significant differences in the genotypes distribution between PCOS patients and controls (P > 0.04, data not shown).
To analyse the degree to which these polymorphisms are in LD, we performed standardized pairwise LD (D') using Thesias software. The pairwise LD matrix showed that two main blocks of LD are present within the CAPN5 gene. Both polymorphisms in the cluster 1 (rs948976 and rs4945140) are in complete LD (D' = –1, P < 0.001), and, on the other hand, both polymorphisms in the cluster 2 (rs2233546 and rs2233549) are also in complete LD (D' = –1, P = 0.002). The two blocks are in partial LD with each other (D' = –0.72, P < 0.001). D' Lewontin defines the range from –1 to +1; the value D' is positive when the rare alleles of each polymorphism are preferentially associated and negative when the rare and frequent alleles are associated (Lewontin and Kojima, 1960).
Haplotype construction comprised of SNPs across four loci within the genomic region of the CAPN5 gene, and haplotype-based association analysis was performed using Thesias software. Only a small proportion of haplotypes really occur when the SNPs are in high LD. In our population, we detect nine unique haplotypes, labelled A-I, among cases and controls combined. Apart from the haplotype comprised of the wild-type allele at each of the polymorphic loci (haplotype D, AGCG), there were eight haplotypes made up of various combinations and permutations of sequence variant and wild-type sequence at each locus. We found a great difference in haplotypes distribution between PCOS and controls (
2 = 29.15 with 8 d.f., P = 0.00029).
The haplotypes H (GGCA) and I (GGTG) were overrepresented in patients when compared with controls, conferring genetic predisposition to PCOS (P = 0.009, OR = 2.0 and P = 0.001, OR = 3.32, respectively, Table III).
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A series of haplotypic background tests were also performed for each of the four polymorphic loci. We analysed the allele effect of each polymorphism on the phenotype according to a haplotype background that only differs at the position of the given polymorphism. In these comparisons, we observed that the rs2233549 polymorphism was only associated with PCOS when it appeared together with the haplotypic background GGC_, haplotypes G/H: GGCG/GGCA (OR = 2.32, P = 0.02, Table IV). The strongest effect is exerted by the rs2233546 in the GG_G context, haplotypes G/I: GGCG/GGTG (P = 0.006, OR = 3.34, Table IV). These results revealed that there are two marker groups that are associated with PCOS, and, interestingly, we observed that the three haplotypes included in these groups are G, H and I (GGCG, GGCA and GGTG) that share a common consensus haplotype, GG_ _, at the first SNP cluster located at intron 1 (rs948976 and rs4945140).
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The association between diplotypes (haplotypes combination) and PCOS was also analysed. There were 31 different diplotypes for cases and controls (Figure 2). Inspection of the diplotypes in these two groups showed that all combinations of haplotypes that were homozygous GG at cluster 1: G/G, G/H, G/I, H/H and H/I were associated with PCOS. The G/G combination is associated with PCOS conferring protective effect (P = 0.01, OR = 0.17), whereas the joint effect of the remaining combinations is overrepresented among PCOS, conferring genetic predisposition to PCOS (P = 0.007, OR = 3.40). Again, the three haplotypes implicated are G, H and I (GGCG, GGCA and GGTG) that share the common consensus haplotype. These results strongly support the presence of a founder mutation in the background of the GG_ _ consensus haplotype related to PCOS risk susceptibility.
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Finally, we performed studies to explore the role of CAPN5 gene alleles in the presence of phenotypic characteristic in PCOS patients, as previously reported by us for CAPN10 gene (Gonzalez et al., 2003). We decided to compare the CAPN5 gene haplotype distribution between groups of patients, divided depending on the presence/absence of specific phenotypic features (menstrual disturbances, hirsutism, acne, infertility, hypercholesterolaemia, hypertension, diabetes and obesity) and familial antecedents of essential hypertension (HTA), diabetes mellitus (NIDDM), obesity and other complex diseases related to PCOS. Figure 3 shows the results of this study. We have only used haplotypes with a frequency greater than 3% to minimize loss of power. Surprisingly, we detected that the presence of familial antecedents of obesity and HTA in PCOS women was associated with a non-random distribution of CAPN5 haplotypes when compared with and without obesity and HTA antecedents (P = 0.02, 2 = 14.7; P = 0.001, 2 = 21.4, respectively).
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On the other hand, we detected association of several clinical characteristics in PCOS with specific CAPN5 haplotypes (Table V). We found association between CAPN5 gene and PCOS patients with amenorrhoea (haplotype H, GGCA, P = 0.01, OR = 0.25), acne (haplotype G, GGCG, P = 0.04, OR = 2.13 and haplotype D, AGCG, P = 0.04, OR = 0.53), obesity antecedents (haplotype G, GGCG, P = 0.02, OR = 4.76; haplotype A, AACG, P = 0.01, OR = 0.31 and haplotype B, AACA, P = 0.003, OR = 7.90), hypertension antecedents (haplotype G, GGCG, P = 0.009, OR = 3.30 and haplotype B, AACA, P = 0.04, OR = 4.95) and aggregation of NIDDM (haplotype I, GGTG, P = 0.04, OR = 4.95).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflicts of interestAcknowledgementsReferences |
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The genetic basis of PCOS is currently not well understood (Franks et al., 1995
). However, it seems that the rule for most PCOS patients will be the coexistence of both environmental and genetic factors interacting in a complex fashion to provoke the PCOS phenotype (Crosignani and Nicolosi, 2001). This aetiology model is applied for almost all common diseases such as schizophrenia, dementia, cardiovascular diseases or diabetes mellitus (Legro et al., 1998). The aim of the present study was to investigate the role of CAPN5 in PCOS susceptibility. To our knowledge, this is the first population-based study that describes SNPs within CAPN5 gene and their potential contribution to PCOS risk. We identified four SNP markers within CAPN5 gene, two in the cluster 1 (comprising the 5' of the gene) and two in the cluster 2 (comprising partial coding region OMP gene in intron 3 of CAPN5 gene).
The pairwise LD matrix of the CAPN5 gene polymorphisms showed that there were two main blocks of LD within the gene: the cluster 1 block (rs948976 and rs4945140) and the cluster 2 block (rs2233546 and rs2233549). These two blocks of consistently high LD are interspersed by an interval of LD breakdown between physically close polymorphisms (approximately 30 kb). This fact suggests the presence of a ‘hot spot’ or region of frequent recombination within CAPN5 that separates these two blocks or clusters. Data provided by Celera Map (www.allsnps.com/SNPbrowser) confirm our hypothesis.
We achieved a construction of haplotypes taking into account different combinations of the four SNPs within the CAPN5 gene. We have analysed haplotype distribution between PCOS population compared to 606 control individuals, and we have found a strong deviation from PCOS population (2 = 29.15 with 8 d.f., P = 0.00029). This finding provides overwhelming evidence that specific haplotypes of CAPN5 gene confers susceptibility to PCOS in the Spanish population. By a comparison of haplotype frequencies between cases and control, the haplotype H (GGCA) and haplotype I (GGTG) appeared to be overrepresented among PCOS compared to controls. Haplotypic background tests revealed that there were two marker groups (G-I and G-H) associated with PCOS, and all haplotypes implicated (GGCG, GGCA and GGTG) shared the GG_ _ background.
We have also discovered a significant association between diplotypes comprising pairs of CAPN5 haplotypes distribution among cases and controls. We observed that all haplotype combinations associated share the GG_ _ background, in which, we suspect should be located a founder mutation responsible for the observed association. However, we cannot exclude the possibility that the founder mutation could be located in another gene in close LD with CAPN5. Further analysis using high-density markers spaced among the region would allow us to perform the positional cloning of the sequence variant leading to the expression of the PCOS phenotype.
Women affected by PCOS often present abnormalities of glucose metabolism and lipid profile and have an increased risk of type 2 diabetes and cardiovascular disease overtime. There are strong evidences that cardiovascular disease, PCOS and metabolic syndrome share genetic factors. We have discovered a significant association between CAPN5 gene and clinical characteristics associated with PCOS, as previously reported in CAPN10 gene (Gonzalez et al., 2003). This genetic association could be of relevance to the clinical management of PCOS patients and the increase of genetic risk to cardiovascular diseases in PCOS women. Moreover, given the haplotype–phenotype results, the CAPN5 gene, just like CAPN10 gene, seems to be highly pleiotropic, modulating different biochemical pathways adding complexity to the genetic basis of PCOS. In any case, to determine the precise role of CAPN5 gene in some phenotypic characteristics of PCOS patients, new molecular genetic studies in specific cohorts of patients must be performed. In addition, it will be necessary to improve the study with other associated complex diseases with PCOS, such as diabetes, metabolic syndrome, obesity, hypertension and cardiovascular disease, since these pathologies share clinical findings, biochemical pathways and genetic risk factors with PCOS (Hernandez et al., 1992; Colilla et al., 2001; Gonzalez et al., 2003).
Our results suggest that CAPN5 is a gene related to PCOS and some phenotypic characteristics associated with PCOS in Spanish population. A larger case-control series as well as similar studies in populations of different ethnicity and geographical location are required to corroborate our findings.
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Conflicts of interest |
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The authors M.E.Saez, J.J.Galan, L.M.Real, A.Ruiz and R.Ramilrez-Lorca have declared that conflicts of interest exist. Some of the work described here is subject to patent filings for diagnostics purposes.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflicts of interestAcknowledgementsReferences |
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We are deeply grateful to PCOS patients and controls for participation in this study. We are very grateful to Sonsoles Vidal, Mayte Pizarro and Dr Ana Bernal for patient and sample management. We thank Ana Salinas for technical support and Eva Molero for informatics support during this work. Neocodex have been partially funded by the Ministerio de Ciencia y Tecnología of Spain (FIT-010000- 2003-36, FIT-010000-2003-89, FIT-0100002003–70, PTQ2002-0206, PTQ2003-0549, PTQ2003-0546 and PTQ2003-0783).
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Submitted on August 23, 2005; resubmitted on October 28, 2005; accepted on November 11, 2005.
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