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Specific CAPN10 Gene Haplotypes Influence the Clinical Profile of Polycystic Ovary Patients

Centro Avanzado de Fertilidad (CAF) (A.G., E.A., A.Ro., M.J.A., M.J.F., P.V., O.F.), Unidad de Reproducción y Genética Humana, Instituto Medico Serman, 11405 Jerez de la Frontera, Cádiz, Spain; Unidad Materno-Infantil (A.G.), Hospital Virgen de las Montañas, 11650 Villamartin, Cádiz, Spain; Dpto. Genómica Estructural (R.R., J.J.G., L.M.R., A.Ru.), neoCodex, 41020 Sevilla, Spain; and Servicio de Obstetricia y Ginecología (J.A.H.), Hospital SAS, 11403 Jerez de la Frontera, Spain

Address all correspondence and requests for reprints to: Dr. Alejandro González, Centro Avanzado de Fertilidad, Unidad de Reproducción y Genética Humana, Instituto Medico Serman, Jerez de la Frontera, Cádiz, Spain. E-mail: alejandro{at}caf-jerez.com.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Recently, several research groups have evaluated CAPN10 gene in polycystic ovarian syndrome (PCOS) patients and other phenotypes, including hirsutism or intermediate phenotypes of PCOS. Molecular genetic analysis of CAPN10 gene indicates that different alleles may play a role in PCOS susceptibility and could be associated with idiopathic hirsutism. However, these observations are not exempt from controversy, because independent studies cannot replicate these preliminary findings.

We present a haplotype-phenotype correlation study of CAPN10 haplotypes in 148 women showing ecographically detected polycystic ovaries (PCO) combined with one or more of these clinical symptoms: amenorrhea or severe oligomenorrhea, hyperandrogenism, and anovulatory infertility, as well as 93 unrelated controls. We have reconstructed and analyzed 482 CAPN10 haplotypes in patients and controls. We detected the association of UCSNP-44 allele with PCO phenotype in the Spanish population (P = 0.02). In addition, we identified several CAPN10 alleles associated to phenotypic differences observed between PCO patients, such as the presence of hypercholesterolemia (haplotype 1121, P = 0.005), presence of hyperandrogenic features (P = 0.05), and familial cancer incidence (haplotype 1111, P = 0.0005). Our results confirm the association of UCSNP-44 allele with PCO phenotype in the Spanish population. Moreover, we have identified novel candidate risk alleles and genotypes, within CAPN10 gene, that could be associated with important phenotypic and prognosis differences observed in PCOS patients.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
THE POLYCYSTIC OVARY syndrome (PCOS) is a common endocrinopathy that is found in about 5–10% of women of reproductive age (1). Specifically in Spain, a prospective study reported an overall 6.5% prevalence of PCOS (2). Defining PCOS has generated controversies among clinicians for many years (3). In fact, definition consensus of PCOS, proposed by the National Institutes of Health conference (1990), 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 (4, 5). A definition consensus has been proposed recently, in which ultrasound morphology should be performed 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 (6).

Previous studies suggest that genetic factors play a major role in the etiology of PCOS (4, 7). However, the mode of inheritance of PCOS remains unknown, and recent studies indicate that this disorder could be a complex trait (8). This means that several genes are interacting with environmental factors to provoke the phenotype (9). In contrast, biochemical parameters, including fasting insulin levels or hyperandrogenemia, 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 (5).

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 (see Ref.5 and bibliography therein). Depending on the series, PCOS is associated with a 2- to 7-fold risk of type 2 diabetes mellitus (T2DM) (10, 11). Previous epidemiological and genetic studies have revealed that PCOS and T2DM could share genetic susceptibility factors associated in both pathologies. Using this working hypothesis, several studies have suggested that genes related to T2DM may play a role in PCOS pathogenesis (12, 13, 14, 15, 16, 17, 18, 19, 20).

CAPN10 gene is T2DM locus previously positionally cloned on 2q chromosome (21). CAPN10 alleles are associated with T2DM in several populations (21, 22, 23). Specifically, the calpain 10 gene (CAPN10), encoding a ubiquitous member of the calpain-like cysteine protease family, was positionally cloned within NIDDM1 region at 2q37 (21). Association studies using intragenic markers of CAPN10 gene have revealed that CAPN10 alleles may contribute to genetic predisposition to T2DM in different populations (21, 22, 23) and also could modify proinsulin processing and insulin secretion in nondiabetic patients. Although the precise role of calpain 10 in T2DM remains unknown, UCSNP-43 polymorphism within intron 3 of CAPN10 gene seems to be associated with reduced muscle mRNA levels of CAPN10.

More recently, four independent research groups have evaluated the CAPN10 gene in PCOS patients and other related clinical charts, including intermediate phenotypes of PCOS. Molecular genetic analysis of CAPN10 gene in PCOS patients indicates that CAPN10 alleles may play a role in PCOS susceptibility (18, 19) and could be associated with idiopathic hirsutism (20). However, these observations are not exempt from controversy, because independent studies cannot replicate these preliminary findings (24).

To further analyze the role of CAPN10 gene in PCOS, we present a haplotype-phenotype correlation study of CAPN10 alleles in 148 women showing ecographically detected PCO combined with one or more clinical features of PCOS. In our extension analysis, we reconstructed and analyzed 482 CAPN10 haplotypes in patients and controls. We confirm the association of UCSNP-44 allele with PCO in Spanish women (19). Moreover, we have identified novel candidate risk alleles or genotypes, within CAPN10 gene, that could be associated with important phenotypic and prognosis differences observed between PCOS patients, such as presence of hypercholesterolemia, presence of hyperandrogenic features, or cancer risk. Our results support a role for CAPN10 gene in PCOS pathogenesis.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Clinical definitions

PCO was defined by the presence of bilateral PCO on ultrasound scan: more than eight follicles with a diameter more than 2 mm, increased volume of stroma, and increased ovarian volume (>9 ml) (25)

PCOS was defined, according to Homburg (2002) (6), by the presence of bilateral PCO and: 1) amenorrhea and/or oligomenorrhea (menstrual cycles >35 d); 2) clinical hyperandrogenism: hirsutism following Ferriman and Galleway criteria, acne, alopecia; 3) anovulatory infertility; or 4) biochemical hyperandrogenism: increased testosterone (T) levels (>3 nM).

Patients with the following exclusion criteria were removed: hyperprolactinemia, thyroid disorders, and nonclassic 21-hydroxylase deficiency (25).

Patients

The study population consisted of 148 unrelated women with morphologically detected PCO combined with one or more of these clinical symptoms: amenorrhea, severe oligomenorrhea, hyperandrogenism, or anovulatory infertility. We also included 93 unrelated healthy women. The ethnicity of all probands and women controls was Caucasian (white europid).

Selected probands were examined by one of the study investigators (A. Gonzalez). To perform phenotype-genotype correlations (and in accord with clinical findings during patient examination and interrogation), we decided to divide the patients into three groups: Group I consisted of 74 patients with amenorrhea, severe oligomenorrhea or anovulatory infertility, PCO ecographically detected plus hirsutism (clinical hyperandrogenism). This group of patients presented the endocrine syndrome of PCOS. Group II comprised 52 patients with amenorrhea, severe oligomenorrhea or anovulatory infertility plus PCO ecographically detected without the endocrine syndrome of PCOS. Group III contained 22 patients with confirmed PCO but insufficient clinical data to determine the status of the patient (this group was removed during phenotype-genotype analysis). The clinical profile of the population studied is summarized in Table 1.

DNA extraction and SNP detection

To isolate germline DNA from leukocytes, we obtained 5 ml peripheral blood from each patient and control. DNA extraction was performed according to standard procedures using Nucleospin Blood Kit (Macherey-Nagel, Düren, Germany). To perform PCRs, we prepared aliquots of DNA at a concentration of 5 ng/µl. The rest of the stock was cryopreserved at -20 C. SNPs nomenclature and genotype definitions of the present work are in accordance with Horikawa et al. (21). CAPN10 gene intronic markers UCSNPs-44, -43, -19, and -63 were genotyped using real-time PCR protocols previously reported by us (19).

Haplotype analysis

Haplotype construction, using intronic markers previously described (USNPs-44, -43,-19 and -63) was performed in accordance with previous reports (21, 22). As previously stated by other research groups, we found strong linkage disequilibrium between the four genotyped SNPs. In this way, we found five different haplotypes that account for more than 98% of CAPN10 alleles analyzed (Table 2). The haplotype nomenclature employed is in accordance with Evans et al. (22).

Allelic frequencies and haplotypes observed in patients and controls were compared using standard Pearson ² tests. To perform statistical analysis of genotypes, heterozygous and homozygous individuals for the p allele of each SNP were grouped (when necessary) to avoid values lower than 5 in cells. All statistical calculations were performed using Statcalc software (EpiInfo 5.1, Center for Disease Control, Atlanta, GA) or Analyse-It software (Ver. 1.65, Analyse-It Software, Ltd., Leeds, UK). To compare haplotype distribution in patients, grouped by any clinical parameter, the standard ² test was performed. To compare quantitative variables in designed groups, the mean values of the parameters were compared using t test or Kruskall-Wallis tests (when necessary, data not normally distributed) with analysis software (EpiInfo 5.1).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
CAPN10 haplotypes analysis in PCOS patients vs. unrelated controls

Haplotypes, comprised of SNPs across four loci within the genomic region of the CAPN10 gene, could be inferred from the haplotype compositions previously observed (21, 22) and analyzing homozygous individuals in our population. We determined the CAPN10 haplotypes for 146 of 148 PCOS patients and 93 unrelated healthy women. As previously observed, there were five unique haplotypes (1111, 2111, 1221, 1121, and 1112) that account for almost all the individuals analyzed (Table 2). In accordance with our previous results, one haplotype (2111), comprising UCSNP-44 C variant, is overrepresented in PCO patients when compared with healthy women (² = 4.95, P = 0.02). This result supports a role of CAPN10 UCSNP-44 allele in PCOS susceptibility (19) (Table 3 and Fig. 1). Interestingly, haplotype analysis, comparing group I vs. group II patients, does not exhibit major differences between PCOS subphenotypes in 2111 haplotype frequency. This result indicates that 2111 allele is associated with PCOS phenotype itself with or without hirsutism (P = 0.68). In both groups, haplotype 2111 (comprising UCSNP-44 C variant) is overrepresented compared with control population; however, the deviation is only statistically significant in hirsute women (group I, P = 0.05). This result suggests that UCSNP-44 allele seems to be related directly with the ovarian phenotype of the patients, independently of the array of additional symptoms that define the PCOS phenotype. However, the frequency of UCSNP-44 is slightly increased in hirsute patients (group I). This may indicate that UCSNP-44 could be more relevant in hirsute women. Alternatively, the lack of significant results between controls and nonhirsute women for UCSNP-44 might be simply attributable to smaller sample size of the nonhirsute subgroup.

View larger version (42K): [in this window] [in a new window]   FIG. 1. Comparison of haplotype frequencies among group I, group II, and healthy women. Group 1 vs. Group 2: 2 = 2.25, P = 0.68 (4 degrees of freedom). Group 1 vs. healthy women: 2 = 9.05, P = 0.05 (4 degrees of freedom). Group 2 vs. healthy women: 2 = 3.40, P = 0.49 (4 degrees of freedom).

To explore the role of CAPN10 gene alleles in the presence of phenotypic characteristics in PCOS patients, we decided to compare the CAPN10 gene haplotype distribution between groups of patients, divided depending on the presence/absence of specific phenotypic features (i.e. obesity, presence of acne, infertility, hypercholesterolemia). The study was performed initially in all patients (Fig. 2). Depending on suggestive results, we decided to stratify the PCOS population to compare the distribution of haplotypes in hirsute/nonhirsute patients (groups I and II) (Table 4).

View larger version (65K): [in this window] [in a new window]   FIG. 2. Haplotype-phenotype correlations between CAPN10 alleles and clinical findings observed in PCOS patients. Each bar compares the distribution of independent CAPN10 haplotypes regarding the presence or absence of a specific trait or antecedent. An identical distribution of any haplotype when comparing the presence/absence of a trait should be represented as 50% for each color. P values are calculated using 2 tests with 4 degrees of freedom. Aggregation indicates more than three independent cases of any specific trait or diseases confirmed in the first- or second-degree relatives.

View larger version (37K): [in this window] [in a new window]   FIG. 3. Haplotype distribution (A) and UCSNP-19 marker genotype analysis (B) in the group I (PCOS-hirsute women). Patients analyzed are separated by the presence/absence of high blood levels of cholesterol.

During our study, patient interrogation includes the registry of familial antecedents (2-degree relatives) of cancer, essential hypertension, diabetes mellitus, obesity, and other complex diseases related to PCOS (Fig. 2). Surprisingly, we detected that the presence of familial antecedents of cancer in PCOS women was associated with a nonrandom distribution of CAPN10 haplotypes, when compared with patients with and without cancer antecedents (² = 19.7, P = 0.0005). Moreover, considering only familial aggregation of cancer cases in PCOS relatives (more than three cancer cases in 2-degree relatives), the statistical significance of this association persists (² = 11.03, P = 0.02, Fig. 2).

Our data suggest that Haplotype 1111, comprising wild-type variant of each SNP studied, is notably overrepresented in PCOS patients with familial aggregation of cancer cases (29%) vs. the rest of PCOS patients (14%). These results suggest an increased genetic predisposition to cancer in the families of PCOS patients carrying the 1111 haplotype. Because cancer incidence is higher in PCOS patients (26) and we detect a slight correlation between 1111 haplotype and the presence of hirsutism in PCO patients (not shown), we decided to stratify the PCOS patient, depending on this phenotypic trait. The association between haplotype 1111 and cancer predisposition persists in the PCOS-hirsute patient group alone (group I, ² = 10.38, P = 0.01, with 4 degrees of freedom) (Table 4). This result indicates that 1111 haplotype is associated with familial cancer risk only in PCOS women that present clinical hirsutism. The rest of familial antecedents of complex diseases do not seem to be strongly associated with specific CAPN10 haplotypes. However, nominal P values for familial history of acne and obesity can be observed. In contrast with elevated cholesterol levels, there is no concordance between the effect of CAPN10 haplotypes in patients studied and familiar antecedents explored (see Fig. 2). Given the P values obtained and the discordance observed, we have not further investigated these nominal associations.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Defining the PCOS has generated controversies among clinicians for many years (3). Clinically, PCOS can be divided into three components, i.e. hyperandrogenic, anovulatory, and dysmetabolic. None are specific for PCOS, and we speculated that each component might be related with independent genetic risk factors (27). Moreover, morphologically, ovarian ultrasonography has been used in the last decade as a new diagnostic tool. However, the sonographic definition of PCO is also controversial, mainly because of a lack of consensus about normative data (3). In fact, PCO is detected, using ultrasonography, in about 23% of women without clinical symptoms of PCOS (7). This observation could indicate that PCO stigma might have independent genetic factors related to it. In contrast, previous studies suggest that PCO and PCOS subjects, with menstrual cycle irregularities, have equivalent disturbances in relation to insulin and glucose metabolism, as well as other lipid alterations. This observation supports the idea that both clinical presentations (PCOS and PCO) could have similar metabolic sequelae and genetic risk factors involved (28). However, no specific genetic dissection has been performed in this way.

During this work, we have recruited 148 patients that fit so-called European PCOS definitions (6), However, on the basis of our results, we split the sample into two groups (groups I and II). Evidently, CAPN10 gene haplotypes work only with the National Institutes of Health consensus definition of PCOS (including the clinical signs and symptoms of hyperandrogenism in the definition). This may imply that there are at least two subgroups of patients in the wide definition of PCOS proposed by Homburg (2002) (6).

The calpain 10 gene (CAPN10), encoding a ubiquitous member of the calpain-like cysteine protease family, was positionally cloned within the NIDDM1 region (21). Association studies using intragenic markers of CAPN10 gene have revealed that different alleles may contribute to genetic predisposition to T2DM in several populations (21, 22, 23). Moreover, the CAPN10 gene has been widely evaluated during the last 2 yr in other phenotypes and traits, such as PCOS, idiopathic hirsutism, insulin resistance, free fatty acid levels, glucose disposal, lipolysis, blood glucose levels, and serum cholesterol levels (18, 19, 24, 29, 30, 31, 32).

Preliminary studies of CAPN10 gene in PCOS patients from the United Kingdom provide the first evidence of CAPN10 involvement in PCOS, suggesting a statistically significant association between the UCSNP-44 allele and PCOS susceptibility (33). However, the same research team has not confirmed these results in an extension analysis recently reported (24). On the other hand, Ehrmann et al. (18) identified a CAPN10 haplotype combination (genotype 112/121) that seems to be associated with a 2-fold increase risk of PCOS in both African-Americans and Caucasian PCOS patients. Supporting the CAPN10 gene involvement in PCOS, our preliminary results suggested that CAPN10 UCSNP-44 allele was associated with PCOS in the Spanish population (19).

Our extension analysis, performed in 148 patients, supports the genetic association between CAPN10 UCSNP-44 marker and the susceptibility to PCO in Spanish’s women. However, we cannot exclude whether this allele is associated to PCO stigma only or to PCOS. We have observed an overrepresentation of the allelic frequency of this marker in hirsute and nonhirsute women (group I and II, respectively), although we obtained statistically significant values only for the PCOS-hirsute subgroup (Fig. 1). This data may imply that UCSNP-44 is relevant to group I phenotype only. Against this hypothesis, we have not found differences when comparing UCSNP-44 marker in group I vs. group II patients. For this reason, the lack of significance in nonhirsute women might simply be attributable to the smaller size of the population. Anyway, further molecular analysis, increasing sample size, will be necessary to elucidate the precise role of CAPN10 in ovarian physiology.

An independent study in the Spanish population has suggested that CAPN10 UCSNP-43 allele somehow influences the hirsutism score in hyperandrogenic patients. In addition, another CAPN10 allele (UCSNP-45) could be associated with idiopathic hirsutism (20). Unfortunately, the UCSNP-45 marker has not been included in our extension analysis, and we cannot provide new information for this allele. However, analyzing the haplotype composition in hirsute patients (group I), we determined that haplotypes 2111 and 1221 are overrepresented compared with healthy women, whereas 1121 haplotype frequency is clearly reduced compared with controls (P = 0.05, Fig. 1). Interestingly, haplotype 1221 was associated previously with the hirsutism score of patients by Escobar et al. (20).

In accordance with a previous report (32), we have observed that patients with hypercholesterolemia may have specific CAPN10 genotypes. Our result suggests that haplotype 1121, comprising the UCSNP-19 ins allele, is associated with the presence of high levels of cholesterol in PCOS patients (P = 0.005, Fig. 3). Although Daimon et al. (32) analyzed only two CAPN10 markers (UCSNP-44 and -43), the haplotype detected by us is in accordance with the genetic results obtained in Japanese patients. Daimon et al. (32) annotated the association between 11/11 genotype with high levels of cholesterol; this genotype is the same reported by us, because it is included in the genotype 1121/1121.

The genetic association observed in our patients seems to be restricted only to group I women. This finding supports the idea that lipid disturbances and cardiovascular risk are related to clinical hyperandrogenism as previously stated (34, 35, 36). This genetic association could be of relevance to the clinical management of PCOS patients, and the calculation of genetic risk to cardiovascular diseases in PCOS women. Anyway, because of the small sample size, we feel that new meta-analyses must confirm these observations.

Regarding the cancer risk in PCOS relatives, unexpected results were obtained for haplotype 1111. We have discovered a significant association between this haplotype and the frequency of cancer incidence in first- and second-degree relatives of PCOS patients (P = 0.0005, Fig. 2). Again, this association seems to be consistent only in group I women. Strikingly, the relationships between PCOS and some gynecological tumors have been previously stated (37, 38, 39, 40). In any case, to determine the precise role of CAPN10 haplotypes in cancer susceptibility, new molecular genetic studies, in specific cohorts of cancer patients, must be performed. For this reason, we annotated this indirect observation with caution.

In the last decade, an increasing interest in the research on genetic factors related to PCOS has been evidenced. However, incontrovertible results have not been obtained at any time. Some authors suggest that differences in patient recruitment and the lack of consensus in PCOS definitions contribute to arriving at different conclusions in genetic studies. However, genetic heterogeneity for PCOS is also suspected (5). Our results support that CAPN10 gene may create or add to PCOS phenotype. Several CAPN10 haplotypes seem to be molecular markers associated with PCOS susceptibility. In addition, the clinical profile of hyperandrogenic PCOS women seems to be modulated by other CAPN10 haplotypes. However, the precise mechanism of these genetic associations remains unknown. Moreover, given the Haplotype-phenotype results, the CAPN10 gene seems to be highly pleotropic, modulating different biochemical pathways and adding complexity to the genetic basis of PCOS. To perform a proper dissection of this syndrome, we propose exploring each major sign or symptom of PCOS separately; moreover, hormone levels could be influenced by independent quantitative trait loci contributing to each specific endocrine parameter (41).

	Acknowledgments

 
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.

	Footnotes

 
NeoCodex have been partially funded by the Ministerio de Ciencia y Tecnología of Spain (FIT-010000-2002-43, FIT-010000-2002-64, and PTQ-2002-0206).

Abbreviations: PCO, Polycystic ovaries; PCOS, polycystic ovarian syndrome; SNP, single nucleotide polymorphisms; T, testosterone; T2DM, type 2 diabetes mellitus.

Received February 25, 2003.

Accepted August 7, 2003.

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