Androgen Receptor Gene CAG Repeat Polymorphism and X ...

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Androgen Receptor Gene CAG Repeat Polymorphism and X-Chromosome Inactivation in Children with Premature Adrenarche Saila Lappalainen, Pauliina Utriainen, Tiina Kuulasmaa, Raimo Voutilainen, and Jarmo Jaä¨skelaïnen Department of Pediatrics, Kuopio University and University Hospital, 70211 Kuopio, Finland

Context: There is variation in the adrenal androgen levels and clinical findings of children with premature adrenarche (PA). Objectives: We hypothesized that androgen sensitivity, indicated by the length of CAG repeat in the X-chromosomal androgen receptor (AR) gene has a role in the polygenic pathogenesis of PA. Design and Patients: We performed a cross-sectional association study among 73 Finnish Caucasian children with PA (10 boys and 63 girls) and 97 age- and gender-matched healthy controls (18 boys and 79 girls). Main Outcome Measures: AR gene methylation-weighted CAGn(mwCAGn) via CAGn length and X-chromosome inactivation analysis and clinical phenotype were determined. Setting: The study took place at a university hospital. Results: PA subjects had significantly shorter mwCAGn than controls [mean difference (95% confidence interval); 0.76 (0.14 –1.38); P ⫽ 0.017]. AR gene mwCAGn did not correlate with androgen or SHBG levels in either group. In children with PA, mwCAGn correlated positively with body mass index (BMI) (␶ ⫽ 0.19; P ⫽ 0.02). The mean of mwCAGn was significantly shorter in PA children with lower BMI compared with PA children with higher BMI [BMI SD score ⬍ 0.79, n ⫽ 35, vs. BMI SD score ⬎ 0.79, n ⫽ 36; 1.13 (0.38 –1.87), P ⫽ 0.004] and in PA children with lower BMI compared with healthy children with same BMI (P ⫽ 0.004). Conclusions: The AR gene CAGn polymorphism may have a significant role in the pathogenesis of PA, especially in lean children. (J Clin Endocrinol Metab 93: 1304 –1309, 2008)

remature adrenarche (PA) is defined as the appearance of androgenic signs before the age of 8 yr in girls and 9 yr in boys. PA has been connected with the risk of developing ovarian hyperandrogenism and features of metabolic syndrome (1–3). It has been suggested that children with PA need to be followed up to rule out and even prevent the development of these long-term sequelae (4). The clinical findings of PA range from the appearance of pubic or axillary hair to acne, adult-type body odor, and oily hair, and girls are affected more frequently than boys (5, 6). Interestingly, not all children with premature pubarche (PP) have adrenal androgen levels elevated above the normal levels for age.

P

Thus, the development of pubic hair is dependent on an interplay between the plasma androgen level and other biological factors that affect the response of the pilosebaceous unit to androgens (7). However, these biological factors are not fully understood. One of the suggested biological factors is the androgen receptor (AR), through which the androgens mediate their effects. The liganded AR binds to regulatory DNA elements in the promoter region of target genes to influence the transcription rate through interaction with cofactors and transcription machinery (8). The X-chromosomal AR gene contains a highly polymorphic region with variable number of CAG repeats (CAGn) that en-

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Abbreviations: AR, Androgen receptor; BMI, body mass index; CI, confidence interval; DHEAS, dehydroepiandrosterone sulfate; HOMA-IR, homeostasis model assessment for insulin resistance; ISIcomp, insulin sensitivity index; mwCAGn, methylation-weighted CAG repeat; PA, premature adrenarche; PCOS, polycystic ovary syndrome; PP, premature pubarche; SDS, SD score.

Printed in U.S.A. Copyright © 2008 by The Endocrine Society doi: 10.1210/jc.2007-2707 Received December 7, 2007. Accepted January 25, 2008. First Published Online February 5, 2008

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J Clin Endocrinol Metab. April 2008, 93(4):1304 –1309

J Clin Endocrinol Metab, April 2008, 93(4):1304 –1309

codes a polyglutamine tract in the N-terminal transactivation domain of the receptor (9, 10). In vitro studies demonstrate an inverse relationship between AR transcriptional activity and the number of CAGn (11, 12). Many studies indicate that CAGn lengths correlate in both men and women to variations in androgen-sensitive disease processes such as breast and prostate cancer, infertility, and polycystic ovary syndrome (PCOS) (13–17). Two studies have demonstrated that Mediterranean girls with PP have about one repeat shorter mean CAGn than healthy controls (18, 19). In addition, there was an association of the shorter AR gene CAGn with an increased risk of subsequent ovarian hyperandrogenism in Spanish PP girls (18). However, none of the studies took the X-chromosome inactivation into account. One X-chromosome becomes inactive in every female cell. X-inactivation converts one of the two X-chromosomes into transcriptionally inactive highly condensed heterochromatin through a series of events including coating of the X-chromosome by Xist RNA, DNA methylation, and histone modification. X-inactivation occurs shortly after the implantation of female embryos or during the induction of cell differentiation, and the maintenance of stable X-inactivation requires synergistic actions of several epigenetic mechanisms. X-inactivation offers a possible epigenetic mechanism, through which environmental conditions influence gene expression for instance during gestation (20, 21). Methylation of HpaII sites close to the AR gene CAGn correlates with X-inactivation (22). Our aim was to determine whether there is a difference in AR gene CAGn between children with PA and healthy controls after the consideration of X-chromosome inactivation. In addition, we examined the relationships between CAGn and the clinicalmetabolic phenotypes of PA in prepubertal children.


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stage was determined to exclude the presence of central puberty by a single investigator (P.U.). Birth weight, birth length, and gestational age data were obtained from hospital records. The birth measures were converted to SD scores (SDS) by plotting them on the growth charts and adjusting the birth measures for gender and duration of gestation (24). Height was measured with a calibrated Harpenden stadiometer three times and recorded to the nearest 0.1 cm. Weight was recorded to the nearest 0.1 kg. Body mass index (BMI) was calculated according to the formula [weight (kilograms)/height squared (square meters)], and BMI SDS was determined by British reference values (25).

Endocrine-metabolic assessment An iv cannula was placed for sampling. Baseline levels of plasma glucose, serum insulin, serum dehydroepiandrosterone sulfate (DHEAS), androstenedione, and SHBG were measured in all subjects after an overnight fast between 0900 and 1000 h. After separation, all serum samples were immediately frozen and stored at ⫺70 C until assayed. An oral glucose tolerance test was performed by administering 1.75 g/kg glucose (maximum 75 g) to each subject with samples for glucose and insulin analyses taken at 30, 60, 90, and 120 min. Homeostasis model assessment for insulin resistance (HOMA-IR) was calculated according to the formula: fasting plasma glucose (millimoles per liter) ⫻ fasting serum insulin (milliunits per liter)/22.5 (26). Insulin sensitivity index (ISIcomp) was calculated according to the formula: 10,000/ 公[fasting glucose (milligrams per deciliter) ⫻ fasting insulin (microunits per liter) ⫻ mean glucose (milligrams per deciliter) ⫻ mean insulin (microunits per liter)] (27). The assays for plasma glucose and cholesterol, serum insulin, SHBG, DHEAS, and androstenedione have been reported previously (3, 23).

Genotyping DNA was isolated from full blood samples using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). DNA from all subjects in the study was used to amplify the polymorphic 5⬘-terminal poly-CAG repeat region of the human AR, and the samples were run on a denaturing gel by automated fluorescence detection as described previously (28). Ten percent of the samples were cross-checked by direct sequencing.

X-chromosome inactivation analysis

Subjects and Methods Subjects The study group comprised 170 Finnish children. For the subjects with PA, the criteria for entry into the study were any clinical sign(s) of adrenarche, including pubic/axillary hair, acne, adult-type body odor, and oily hair before the age of 8 yr in girls and 9 yr in boys. All eligible children were invited to the study between October 2004 and January 2006 from our Hospital District in Eastern Finland. Seventy-five eligible children were found, and 73 (97.3%) of them were willing to participate (63 girls and 10 boys). Steroidogenic enzyme defects and virilizing tumors were excluded biochemically and by adrenal ultrasonography. Altogether, 97 healthy age- and gender-matched controls (79 girls and 18 boys) from a random sample of children from the same district, obtained from the Finnish population register, participated. At examination, girls in both groups had to be less than 9 yr and boys less than 10 yr of age. Children with central puberty, any endocrine disorder, or long-term medication were excluded from both groups. The study protocol was approved by the Research Ethics Committee of Kuopio University Hospital. Informed written consent from parents and assent from children were obtained for participation in the study, including collection and genotyping of DNA samples. The recruitment of the subjects has been documented previously in more detail (23).

Clinical assessment The appearance time of the adrenarcheal signs was obtained by interviewing the parents. The children were examined, and the Tanner

An X-chromosome inactivation assay based on the AR gene methylation pattern using lymphocyte DNA has been described previously (22). Methylation of HpaII sites close to the AR gene CAGn correlates with X-chromosome inactivation; the sites are methylated on the inactive X-chromosome. Methylation-sensitive restriction enzyme HpaII digests only the unmethylated (active X-chromosome) DNA, which is thereby unavailable for the following PCR amplification. Post-digestion PCR products therefore represent methylated (inactive X-chromosome) DNA sequences only. We used the technique modified from that previously described (17). The concentrations of 12 control and two PA DNA samples were too low for analysis. The remaining heterozygous samples of girls, including 60 (95%) PA and 58 (73%) controls were analyzed. For each DNA sample, two separate reactions were prepared: 2 ␮g DNA digested with 10 U HpaII (New England Biolabs, Beverly, MA) at 37 C for 12 h and a parallel reaction performed without the enzyme. Reactions were stopped with a final enzyme denaturation step at 95 C for 5 min. Samples were prepared in 20-␮l reaction volume. One hundred nanograms of both digested and undigested DNA were amplified by PCR, and 1.5 ␮l of the PCR products was run on denaturing gel as in the genotyping analysis. Total fluorescent peak areas for both alleles were determined for digested and undigested samples. All samples were analyzed in nondigested and digested conditions at least in duplicate. For each individual, the mean of these measurements was used for statistical analysis. To compensate for the unequal amplification of alleles, values for the digested samples were normalized with those for the undigested samples with calculations as described previously (29). Inactivation of shorter alleles was calculated with formula (p1d/p1u)/(p1d/p1u ⫹ p2d/p2u), in

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which p1d and p2d represent the peak areas of HpaII-digested alleles, and p1u and p2u are the corresponding peak areas of the undigested alleles. Methylation-weighted biallelic means of CAGn (mwCAGn) were achieved by multiplying each allele in a genotype pair by its percentage of total expression (100 minus inactivity percent) and summing the two adjusted repeat values as described previously (17). Individuals homozygous at the AR gene CAGn locus (nine controls and one PA subject) and boys were included in the mwCAGn analyses because variation in the allele expression would not alter the mean value of alleles of equivalent repeat number. Skewing of X-chromosome inactivation was determined as 80% or higher percentage for one allele to be active as discussed previously by Naumova et al. (30).

Statistical analysis The distribution of AR gene CAGn and mwCAGn was compared between the PA and control groups by t test. The t test was used when comparing clinical and biochemical characteristics between the PA and control groups. If distribution was not interpreted as normal, raw data were log-transformed before using the t test, and results are presented as geometric means with 95% confidence intervals (CI). For parameters remaining nonnormally distributed after log-transformation, the MannWhitney U nonparametric test was used. The differences in the CAGn distribution and the X-chromosome inactivation between the PA and control groups were tested using Fisher’s exact test. The difference between the arithmetic mean of CAGn and the mwCAGn was tested with paired-samples t test. The strength of the relationship between mwCAGn and the clinical measurements was estimated by Kendall’s rank correlation in the PA group and in the control group. The relationship between mwCAGn and ISIcomp was estimated using multiple linear regression allowing for BMI SDS. P ⬍ 0.05 was considered statistically significant. All statistical analyses were performed with SPSS 14.0 statistical package (SPSS Inc., Chicago, IL).

Results CAG allele distribution and X-chromosome inactivation analysis The clinical characteristics of the PA and control groups are shown in Table 1. A total of 73 children with PA (63 girls and 10 boys) and their 97 control subjects (79 girls and 18 boys) were genotyped for the CAGn polymorphism of the AR gene. CAGn ranged from 14 –25 in the PA children and from 13–28 in the TABLE 1.


control children. Ten girls exhibited a homozygous genotype of AR gene CAGn. They and the boys were included in the distribution comparison of mwCAGn because variation of allele expression would not alter the mean value of alleles. Altogether, 71 PA subjects and 85 controls were included in the distribution comparison of AR gene mwCAGn. The arithmetic mean of AR gene CAGn was significantly shorter in the PA compared with the control group [PA vs. controls: mean (SD), 20.7 (1.8) vs. 21.4 (2.3); mean difference (95% CI), 0.72 (0.09 –1.34); P ⫽ 0.025]. Furthermore, the difference between the groups became stronger when the X-inactivation was taken into account in mwCAGn [PA vs. controls: 20.7 (1.7) vs. 21.4 (2.3); mean difference (95% CI), 0.76 (0.14 –1.38); P ⫽ 0.017; Fig. 1]. When the mwCAGn were divided into three groups (CAGn ⱕ 18, 18 ⬍ CAGn ⬎ 25, and CAGn ⱖ 25), the distribution was significantly different in the PA group compared with the control group (P ⫽ 0.009; Table 2). Nine controls but none of the PA subjects had mwCAGn longer than 25 repeats. The distribution of mwCAGn was similar in the control group compared with that of CAGn of a previously reported Finnish male control population (31). The methylation-weighted and arithmetic means of CAGn were significantly different in the total group of 156 children. X-inactivation changed the mean length of CAGn significantly toward the longer CAGn [mean difference (95% CI), 0.08 (0.01– 0.15); P ⫽ 0.019]. The incidence of nonrandom X-inactivation (alleles, ⱖ60% inactive) was similar in the PA and the control group (39% PA, 35% controls; P ⫽ 0.70). In addition, we found no evidence of abnormal incidence of skewed X-inactivation (alleles, ⱖ80% inactive) in either group (3.3% PA, 1.7% controls; P ⬎ 0.99). The entire group of children was divided into two groups by whether DHEAS was less than or more than 1.0 ␮mol/liter, respecting biochemical adrenarche. There was no significant difference in the mwCAGn between the groups with lower and higher DHEAS levels [DHEAS ⬍ 1 ␮mol/liter, n ⫽ 73, vs. DHEAS ⬎ 1 ␮mol/liter, n ⫽ 83; mean (95% CI), 21.1 (20.7– 21.6) vs. 21.1 (20.7–21.5); P ⫽ 0.9].

Clinical and hormonal characteristics in children with PA as compared with healthy controls Mean (95% CI) Variable

Controls (n ⴝ 85)

PA subjects (n ⴝ 71)

P valuea

Gender (boys/girls) Age (yr) Birth weight (SDS) Birth length (SDS) BMI (SDS) HOMA-IRc ISIcomp Cholesterol (mmol/liter) DHEAS (␮mol/liter)c ⌬4-A (nmol/liter)c SHBGc (nmol/liter)

18/67 7.6 (7.4 to 7.7) 0.2 (⫺0.05 to 0.4) 0.2 (0.03 to 0.4) 0.4 (0.1 to 0.6) 0.9 (0.8 to 1.0) 1.1 (1.0 to 1.2) 4.2 (4.1 to 4.3) 0.8 (0.7 to 0.9) 1.3 (1.2 to 1.5) 96 (89 to 104)

10/61 7.4 (7.2 to 7.7) ⫺0.02 (⫺0.3 to 0.3) ⫺0.03 (⫺0.3 to 0.2) 1.0 (0.7 to 1.3) 1.1 (1.0 to 1.2) 0.9 (0.8 to 1.0) 4.2 (4.1 to 4.4) 1.7 (1.5 to 2.0) 2.4 (2.1 to 2.7) 73 (65 to 82)

0.290b 0.282 0.080b 0.001 0.002 ⬍0.001 0.802 ⬍0.001 ⬍0.001 ⬍0.001

⌬4-A, Androstenedione. a

Values taken from t test unless indicated otherwise.

b

Analyzed with Mann-Whitney U test.

c

Raw data were log-transformed before using the t test, and results are presented as geometric means with 95% CI.



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tively with BMI SDS, there were significant differences between BMI SDS groups in the PA subjects [BMI ⬍ 0.79 SDS vs. BMI ⬎ 0.79 SDS; 1.0 (0.84 –1.2) vs. 0.69 (0.59 – 0.80); P ⫽ 0.002] and control subjects [1.2 (1.1–1.3) vs. 0.89 (0.72– 1.07); P ⫽ 0.002]. The difference in ISIcomp between PA and control subjects reached statistical significance in the both BMI groups (BMI ⬍ 0.79 SDS, P ⫽ 0.028; BMI ⬎ 0.79 SDS, P ⫽ 0.029).

Discussion FIG. 1. Methylation-weighted biallelic mean of AR gene CAGn length in 71 subjects with PA and their 85 healthy controls.

CAG repeat length and clinical and biochemical characteristics The PA subjects had significantly higher BMI, HOMA-IR, and adrenal androgen levels and significantly lower ISIcomp and SHBG levels (Table 1). In the PA group, the mwCAGn correlated positively to subject’s age (␶ ⫽ 0.27; P ⫽ 0.026) and BMI (␶ ⫽ 0.31; P ⫽ 0.009) and negatively to ISIcomp (␶ ⫽ ⫺0.30; P ⫽ 0.014). When the relationship between mwCAGn and ISIcomp was estimated using multiple linear regression allowing for BMI SDS, the effect of mwCAGn became insignificant [coefficient (95% CI), ⫺0.02 (⫺0.04 to 0.01); P ⫽ 0.168]. In the control group, there were significant negative correlations between mwCAGn and birth weight SDS (␶ ⫽ ⫺0.15; P ⫽ 0.038), birth length SDS (␶ ⫽ ⫺0.17; P ⫽ 0.025), and total cholesterol level (␶ ⫽ ⫺0.16; P ⫽ 0.037). The mwCAGn did not correlate significantly with HOMA-IR, adrenal androgen, or SHBG levels in either group. The PA group was divided into two equal sized groups by the median BMI of 0.79 SDS (BMI ⬍ 0.79 SDS, n ⫽ 35; BMI ⬎ 0.79 SDS, n ⫽ 36). The PA subjects with lower BMI SDS had significantly shorter mwCAGn in the AR gene [mean difference (95% CI), 1.13 (0.38 –1.87); P ⫽ 0.004]. The PA subjects with lower BMI SDS had significantly shorter mwCAGn than the controls with the same BMI SDS [PA, n ⫽ 35, vs. controls, n ⫽ 59; difference, 1.2 (0.4 –2.0); P ⫽ 0.004]. There were no significant differences in mwCAGn between the PA subjects with higher BMI SDS and controls with the same BMI SDS [PA, n ⫽ 36, vs. controls, n ⫽ 26; difference, 0.5 (⫺0.6 to 1.5); P ⫽ 0.4] or between the controls with lower and higher BMI SDS [BMI ⬍ 0.79 SDS, n ⫽ 59, vs. BMI ⬎ 0.79 SDS, n ⫽ 26; ⫺0.4 (⫺1.4 – 0.7); P ⫽ 0.5]. Because ISIcomp correlates nega-

Our aim was to determine whether androgen sensitivity, indicated by AR gene CAGn, and X-chromosome inactivation have a role in the polygenic pathogenesis of PA and to correlate androgen sensitivity with clinical and biochemical findings of PA in prepubertal children. Our PA subjects had significantly shorter mean AR gene CAGn than the controls, and the difference between the groups was even stronger when the X-inactivation was taken into account. However, AR gene CAGn did not correlate with adrenal androgen or SHBG levels in either group. In the PA children, mwCAGn correlated positively to BMI SDS. The PA children with lower BMI were found to have more sensitive AR than those with higher BMI and the controls with low BMI. Girls with PP and elevated DHEAS levels at the time of PP diagnosis have shorter mean AR CAGn in Catalonia (18). We took X-chromosome inactivation into account and found the mean difference of 0.7 repeat in CAGn between PA children and their healthy controls, which is consistent with the finding of the Spanish study. The postmenarcheal Catalan PP girls with mean CAGn less than 20 repeats had higher testosterone levels and more pronounced signs of hyperandrogenism (18). We did not find any correlation of mwCAGn with adrenal androgen levels in prepubertal PA children or healthy controls, which suggests that AR gene CAGn has no role in the feedback regulation of adrenal androgen secretion. We did find a significant correlation of mwCAGn with BMI SDS and were able to indicate more sensitive AR in lean PA children. A positive correlation of the CAGn with body fat mass has been found in healthy males as well (32). The postmenarcheal Catalan PP girls studied previously had normal BMI, and there was no significant difference in BMI between them and their controls (18). The results of the previous association studies investigating the relationships between AR gene CAGn and clinical parameters are contradictory in hirsute women and women with PCOS (16, 28, 33, 34). There is only one Australian study that has taken X-inactivation into account in PCOS women; it described pref-

TABLE 2. Distribution of AR gene methylation-weighted CAGn (mwCAGn) length in children with PA and their gender- and age-matched controls AR gene mwCAGn length

a

Population

25

P valuea

PA subjects (n ⫽ 71) Controls (n ⫽ 85)

4 (5.6%) 3 (3.5%)

67 (94.4%) 73 (85.9%)

0 (0.0%) 9 (10.6%)

0.009

From Fisher’s exact test.

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erential expression of the allele with longer CAGn resulting in higher proportion of subjects with mwCAGn more than 22 repeats in women with PCOS compared with healthy controls. The authors found a positive correlation between mwCAGn and testosterone, but not with BMI, in women with PCOS (17). In our study, there was a significant difference between the arithmetic mean of CAGn and the mwCAGn, indicating the importance of considering X-inactivation in the analyses of X-chromosomal genes. X-inactivation has been shown to have an influence on the clinical presentation of PCOS, in which hyperandrogenism plays a crucial role. Sister pairs in families with PCOS have been examined for the patterns of X-inactivation. In the majority of cases in which the sister pairs have the same genotype but different clinical presentations, a different pattern of X-inactivation is evident (35). Our study design did not enable testing differences in X-inactivation between family members. We did not find any differences in the incidence of nonrandom or skewed X-inactivation between our PA and control groups. A small Italian study of women with idiopathic hirsutism suggests that skewing of X-inactivation leads to preferential expression of the AR gene with shorter CAGn (36). The results are questioned by a Spanish study with larger sample size demonstrating neither skewing of X-inactivation nor difference in CAGn among the groups of women with hyperandrogenic and idiopathic hirsutism and healthy controls (37). On the other hand, a similar methylation pattern of the AR gene has been reported in pubic hairs of Italian prepubertal girls with PP compared with girls with Tanner stage II pubertal development, and the methylation of the AR gene in peripheral leukocytes was significantly lower in girls with PP than in control prepubertal girls (19). However, the role of AR gene methylation in receptor activation and androgen sensitivity has not been examined in more detail. We had no possibility to investigate X-inactivation in peripheral target tissues and cannot state whether it is different from that in leukocytes. The differences in X-inactivation between different tissues and the mechanisms leading to secondary skewing of X-inactivation are not fully understood. Testing androgen sensitivity and X-inactivation in peripheral blood may best reflect the overall situation in androgen target tissues. Although the difference of 0.7 repeat in AR gene CAGn between the PA and control groups is not big, its clinical relevance may be considerable. A recent metaanalysis investigated the studies on the association of male infertility with AR gene CAGn and revealed a statistically significant difference (standardized mean difference with 95% CI) of 0.31 (0.14 – 0.47) in AR gene CAGn between the cases and controls (15). The shorter mean CAGn in Catalan PP girls is related to the risk of adolescent ovarian hyperandrogenism (18). The longitudinal follow-up will show whether the length of AR gene CAGn is related to the risk of ovarian hyperandrogenism in this Finnish cohort. Furthermore, nonobese Catalan PP girls had abnormal lipid pattern and hyperinsulinism both prepubertally and throughout puberty, whereas studies on other populations have failed to find the association (2, 38). In a pilot study, the antiandrogen flutamide reduced hirsutism and androgen and triglyceride levels but failed to decrease hyperinsulinemia in adolescent girls with ovarian


hyperandrogenism after PP (39). Furthermore, low-dose flutamide in combination with metformin treatment achieved greater reduction in adiposity of postmenarcheal PP girls with shorter CAGn mean length in comparison with subjects with CAGn mean length longer than 20 (40). It is well known that healthy obese children have elevated adrenal androgen levels compared with lean children (41). Based on the results of our previous study, Finnish PA girls have increased prevalence of childhood metabolic syndrome mainly due to their overweight and hyperinsulinism. High insulin concentrations were rare in lean PA subjects (3). Although previous studies and our analyses have indicated hyperinsulinism in the normal-weight PA subjects, the hyperinsulinism is more evident in PA subjects with higher BMI. Now we have demonstrated that PA children with lower BMI had more active AR, which offers a tempting mechanism to explain PA in lean children. Hyperinsulinism may be the key inducer of PA in overweight children. In conclusion, X-inactivation is important to take into account in studying X-chromosomal AR gene CAGn, but X-chromosome inactivation itself seems to play no significant role in the pathogenesis of PA. Children with PA have shorter AR gene mwCAGn than healthy controls. In prepubertal PA children, mwCAGn correlates positively with BMI SDS. PA children with lower BMI have more sensitive AR in comparison with PA subjects with higher BMI and with controls with the same BMI. AR gene CAGn may have a significant role in the polygenic pathogenesis of PA in lean children, whereas hyperinsulinism is important in overweight children.

Acknowledgments Ms. Mari Tuovinen and Ms. Minna Heiskanen are thanked for their skillful assistance. Address all correspondence and requests for reprints to: Saila Lappalainen, M.D., Department of Pediatrics, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio, Finland. E-mail: [email protected]. This work was supported by grants from Kuopio University Hospital, Pediatric Research Foundation, Academy of Finland, The Finnish Medical Foundation, and Sigrid Juse´lius Foundation. Disclosure Statement: The authors have nothing to declare.

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