Article 55

Atypical Polycystic Ovary Syndrome – A Genetic Analysis

Authors

M. Scholten1, C. Vilser1, A. Weise2, A. Baniahmad2

Affiliations

1

Key words ▶ atypical PCOS ● ▶ androgen levels ● ▶ chromosome banding ● ▶ androgen receptor gene ● sequencing ▶ virilization ●

Abstract

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1387735 Published online: August 22, 2014 Exp Clin Endocrinol Diabetes 2015; 123: 55–60 © J. A. Barth Verlag in Georg Thieme Verlag KG ­Stuttgart · New York ISSN 0947-7349 Correspondence Dr. med. M. Scholten Department of Pediatrics Jena University Hospital Kochstraße 2 07740 Jena Germany Tel.:  + 49/3641/938 369 Fax:  + 49/3641/938 234 [email protected]. de

 Department of Pediatrics, Jena University Hospital, Jena, Germany  Institute of Human Genetics, Jena University Hospital, Jena, Germany



Background/aims:  Although polycystic ovary syndrome (PCOS) is a common endocrinopathy the pathogenesis is not entirely understood. Typically, high androgen levels are associated with increased virilization. We report 2 rare groups of patients with either unexpectedly high testosterone levels despite low virilization as well as patients with low testosterone levels despite high grade of virilization. One possibility for the atypical PCOS may be based on an altered androgen receptor (AR) signaling. Methods:  6 patients and when available the parents were included in this study. Alterations of the metaphase chromosomes by GTG staining, the length of both the trinucleotide CAG- and GGC-repeats of the androgen receptor (AR) gene

Abbreviations



AR DHEA-S FG GTGbanding PCOS SGA

androgen receptor dehydroepiandrosterone – sulphate Ferriman-Gallwey index score Giemsa-banding polycystic ovary syndrome small for gestational age.

Introduction



PCOS is one of the most common endocrinopathies in women; the prevalence in women is estimated to be around 7 % [1, 2], initially described by Stein and Leventhal in 1935 [3]. Meanwhile, there have been established different criteria to confirm PCOS: The NIH-Criteria from 1990, the revised Rotterdam criteria from 2003 [4] and the AES-criteria from 2006 [5]. Although PCOS presents as a heterogeneous clinical picture, generally the grade of virilization is positively

was determined by PCR, further the entire AR gene was sequenced and analyzed. Results:  The GTG banding revealed no chromosomal alterations and the range of CAG- and GGC-repeat lengths are within the normal range. Interestingly, by sequencing of the entire AR gene few genetic mutations were identified. Conclusion:  The detected mutations do not alter the AR protein sequence but they change the codon usage towards less frequent codons that potentially may alter AR protein levels and androgen signaling. In addition to this, we postulate also other causes for manifestation of atypical PCOS, which may include AR-coregulators or epigenetic alterations. To our knowledge this is the first report of combining chromosomal analysis of PCOS patients with full sequencing of the human AR gene and linking codon usage to PCOS.

correlated with the excess of androgens. We, however, observe that some patients have a high grade of virilization despite low androgen levels and vice versa. Therefore, we postulated that these patients belong to a special subgroup of PCOS, the atypical PCOS. In addition to factors involved in steroid biosynthesis, changes of the trinucleotide repeat number in the androgen receptor gene were linked to PCOS [6–12]. In patients with atypical androgen levels we hypothesized an alterations in the androgen receptor gene sequence that may change androgen signaling. Therefore following aspects were analyzed: (I) chromosomal alterations including aneuploidy and GTG banding pattern; (II) changes of the trinucleotid lengths of both GGC- and CAG- trinucleotide expansions in exon 1 of the AR alleles; (III) other changes in the DNA sequence of the AR. We recruited 6 patients with rare atypical PCOS, and when available, the parents of the patients were also included in the study. After written consent we took the medical history including

Scholten M et al. Atypical Polycystic Ovary Syndrome …  Exp Clin Endocrinol Diabetes 2015; 123: 55–60

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received 15.03.2014 first decision 20.06.2014 accepted 04.08.2014

2

56 Article

Material and Methods



Prior to the study the ethical approval was obtained from the ethical committee of Jena University Hospital. All patients and relatives signed a written consent to take part in the study. We examined 6 patients and 8 relatives during routine presentation in the endocrinological outpatient department of the Jena University Pediatric Hospital. Based on the rare occurrence of untypical PCOS, the number of included patients is very limited. For all patients, a thorough clinical examination was performed. Hirsutism was assessed according to the Ferriman-Gallwey index score [13] dividing the patients into 2 groups with either low grade of virilization (FG up to 3) or high grade of virilization (FG 7 or more). This is in line with other authors [14]. The BMI was calculated by weight [kg]/height [m]2 and the percentile was calculated according to Kromeyer-Hauschild et al. 2001 [15] revealing obesity only in one patient (BMI above the 95th percentile). PCOS was diagnosed according to the revised Rotterdam criteria 2003 [4]: 2 out of the following criteria: (I) oligo- or anovulation; (II) clinical and/or biochemical signs of hyperandrogenism (III) polycystic ovaries. The Rotterdam criteria are commonly used in defining PCOS [16–18]. All patients showed either clinical or biochemical signs of hyperandrogenism. Patient 5 fulfilled the criteria for polycystic ovaries, all other patients presented with amenorrhea.

Overview

Patient 1 (13 11/12 years):  Secondary amenorrhea for 9 months, menarche 2 years before presentation, minute acne, no hirsutism, Ferriman-Gallwey index Score (FG) = 0. High testosterone levels. Patient 2 (16 8/12 years):  Secondary amenorrhea and obesity. Ovary volumes (10.8 and 10.4 ml) above 95th percentile compared to age-matched standards [19], follicles on both sides. Oily hair and slight acne, no hirsutism, FG = 0. High testosterone levels. Patient 3 (15 0/12 years):  Amenorrhea for 12 months, severe obesity. Slight acne on back, face and thorax and very discrete hirsutism on the upper lip and below the umbilicus, FG = 2–3. High testosterone levels.

95th percentile with several follicles up to 15 mm diameter [19]. Low testosterone levels.

Patient 6 (14 0/12 years):  Obesity and hirsutism. Last menstrual cycle was 3 months ago. Slight acne on back, thorax and face. Distinct hair growth on upper legs and arms and rhomboid pubic hair, FG = 7. Low testosterone levels. She was diagnosed ▶  Fig. 1). with a heterozygous Factor-V-Leiden like her mother ( ●

Sonography

Transabdominal ultrasound was performed to obtain ovary size and appearance.

Hormone measurements

From venous blood, the level of total testosterone, free testosterone, androstenedion and DHEA-S was measured by Siemens Advia Centaur chemiluminescence immunoassay and by Beckman coulter radioimmunoassay. In literature, free testosterone is described to correlate well to hirsutism in adults [20]. Due to high age-dependent variability of free testosterone in juvenile patients total testosterone was chosen for evaluating hyperandrogenemia [21]. Late onset congenital adrenal hyperplasia as a differential diagnosis in hyperandrogenism was excluded by measuring 17-OH-progesterone and adrenocoticotropic hormone levels. Hyperandrogenemia was assessed as testosterone levels > 2.2 nmol/l according to internal standards, the androgen action was assessed by the grade of hirsutism according to the score of Ferriman and Gallwey (FG) and/or acne [13]. Additional causes for amenorrhea were assessed. In all obese patients, hyperinsulinemia was excluded by measuring insulin levels. In lean patients hyperinsulinemia was excluded by the absence of clinical signs of hyperinsulinemia such as acanthosis nigricans. In no patient clinical evidence was found for Cushing’s syndrome, hypothyroidism or prolactinoma.

Chromosomal analysis

Using cytogenetic standard protocols lymphocyte cultures were incubated for 72 h at 37 °C in RPMI 1640 medium supplemented with 10 % fetal calf serum and 1 % phytohemoagglutinin (PHA-L). PHA-L (0.24 mg/ml) was dissolved in sterile distilled water and added 24 h before harvesting. Colcemide (0.1 µg/ml) and ethidiumbromide (0.001 %) were added 90 min before culture termination. Chromosome preparation was done according to standard protocols [22, 23]. The chromosomes were GTG banded and

Patient 4 (13 7/12 years):  Primary amenorrhea when considering first breast development at the age of 8 years and the significantly accelerated bone age between 15 and 16 years. Slight hirsutism on upper and lower legs, FG = 3, minute acne in face. High testosterone levels despite antiandrogen treatment with a contraceptive drug containing 2 mg cyproterone acetate and 0.035 mg ethinylestradiol (Diane, Bayer Vital). Upon treatment, regular menses started immediately. Patient 5 (15 1/12 years):  Severe acne in face and on back, distinctive hirsutism on upper lips, back, upper and lower abdomen, FG = 9. High basal LH (23.2 U/l) and high LH increase up to 165 U/l in LHRH-test. Volume of ovaries (12 and 14 ml) above

Fig. 1  Patient 6 exhibits increased hair growth on her hands. FerrimanGallwey-Score (FG) = 7.

Scholten M et al. Atypical Polycystic Ovary Syndrome …  Exp Clin Endocrinol Diabetes 2015; 123: 55–60

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birth weight, virilization during pregnancy, performed body examinations and analyzed peripheral blood samples for androgens and gonadotrophic hormones.

Article 57

DNA extraction

DNA was extracted from venous blood by the QIAamp DNA Mini Kit according to manufacturer’s protocol.

PCR

Primers were designed by using a web resource [24] and subsequent improvements of primer sequences. The primers were obtained from Eurogentec at a final concentration of 100 µM in H2O. Qiagen® Multiplex PCR Kit: Standard protocol; Q-Mix only

1

2

3

4

in primers for exon 1 due to high CG-content in parts of exon 1. Cycler PeqLab primus 96: 15 min initialization 95 °C, 40 cycles with 30 s denaturation 94–95 °C, 90 s annealing 55–64 °C (depending on primer pair), 90 s extension 72 °C. 10 min final elongation 72 °C. PCR products were verified by agarose gel and ▶  Table 1). finally by sequencing ( ●

Sequencing

PCR products were purified by Primers QiaQuick PCR purification kit according to manufacturer’s protocol (Qiagen). Labeling: BigDye Terminator v3.1 Cycle Sequencing Kit (Invitrogen): 1 µl Ready Reaction Mix, 1 µl Big Dye Sequencing Buffer, 0.32 µl single Primer (10 µM), 6.68 µl ddH2O, 1 µl purified DNA (3–10 ng); Cycler conditions (PeqLab primus 96): 96 °C 1 min, 24 × 10 s denaturation 96 °C, 5 s annealing 50 °C, 4 min extension 60 °C. Afterwards, the sample was purified via DyeEx 2.0 spin kit (Qiagen), dried in a vacuum centrifuge for 20 min, solved in 10 µl formamide and processed to the sequencing machine (3100-Avant Genetic Analyzer – AbiPrism).

5

Results

▼ 6

7

8

13

14

15

19

20

9

21

10

11

12

16

17

18

22

X

Y

Fig. 2  Exemplified GTG-banding of metaphase chromosomes from patient 6. Normal female 46, XX karyotype. No obvious chromosomal alteration was detected.

Table 1  Primer list for the human androgen receptor gene (5′→3′). Exon (length)

Primer sequence

Exon 1 A 639 bp Exon 1B 817 bp Exon 1C 384 bp Exon 1D 309 bp Exon 2 340 bp Exon 3 389 bp Exon 4 411 bp Exon 5 283 bp Exon 6 353 bp Exon 7 260 bp Exon 8 354 bp

F: gcctgttgaactcttctgagc R: cctcgctcaggatgtctttaag F: ccccactttccccggcttaag R: tgtgaagagagtgtgccagg F: ggagaacccgctggactac R: gactgggatagggcactctgctcacc F: gcggcgaggcgggagctg R: caggggcaatctgagtgttc F: cttgcctatttctgccattc R: gttatttgatagggccttgc F: cccgaagaaagagactctgg R: cggaatggggattggtatag F: gcattgtgtgtttttgaccac R: ggtccataggagcgttcac F: aacccgtcagtacccagactgacc R: gcttcactgtcaccccatcaccatc F: gcaagctcttcttggaaaac R: gaagggaaatgtccaggag F: aacttggtgctttgtctaatgc R: ctgataaagcaccctccatcg F: gaccaaaaatcagaggttgg R: caatagaggaaattccccaag

F = forward; R = reverse The product sizes were adopted from the published sequence and in cases of

The Ferriman-Gallwey-Score (FG) represents the grade of virilization with high values for strong virilization in patient 5 and 6 despite low testosterone levels (  99. 85. 99. 85. 36.

23/24 21/24 19/23 19/21 20/20 20/23 20 22/24 20/24

17/18 16/17 17/18 17/18 17/18 17/18 18 16/17 16/17

NA NA NA NA NP NP NP NA NA

severe none slight back none

2.08

20.4 24.4 25.3 45.5 29.1

48. 51. 94. 99. 73.

19/21 21/28 19/21 19/24 21

16/17 12/16 17/18 17/18 17

NP NP NA NA NA

Acne

3.54 3.65

2.80

1.66

In all cases the amounts of CAG- and GGC-trinucleotide repeats were according to the parents and within the normal range ▶  Table 2). Interestingly, the mothers of the patients exhibiting ( ● a high grade of hirsutism also suffered from increased virilization at the time of presentation indicating a genetic-based inheritance. The androgen receptor gene is located on the X-chromosome. Therefore, for the fathers only one allele is present as shown in ●  ▶  Table 2. We evaluated baldness in males as it could be linked to PCOS in the daughters [26] but included fathers did not suffer from severe baldness indicating a maternal inheritance. Obesity may be also linked to increased hirsutism in PCOS patients [27]. In this group, only patient 3 and the mother of patient 6 suffered from obesity according to the definition of Poskitt (1995) [28]. The affected patient did not show increased grade of hirsutism. Testosterone levels were quantified before therapy except for patient 4, where the elevated testosterone level occurred even during therapy with Diane, a contraceptive ▶  Table 2,  3). drug that contains antiandrogens ( ● Upon sequencing the entire androgen receptor gene, few differences to the standard sequence (NCBI Reference Sequence: NG_009014.2) were identified. In PCOS-patients with high testosterone and low virilization the codon at position 639 was changed from a GAG towards a GAA and the codon at position 1296 was mutated from TCC towards TCT. None of these mutations leads to an altered protein sequence, however, the codon usage of these mutated codons is significantly lower: In humans, 75 % of glutamic acid in final proteins is encoded by GAG vs. 25 % by GAA; and for serine 28 % is encoded by TCC and only 13 % by TCT [29]. This indicates that the mutations result in both cases in codons less often represented in the genome and may lead to a delay or reduced AR protein level. The difference between the ▶  Table 3) may result DNA sequence of patient 2 and her mother ( ● from the inheritance of only one X-chromosome from the mother, the other one is inherited from the father. The change in patient 2 inherited from her father who was not included in the study and the change seen in her mother was not inherited. Taken together, several alterations in the AR gene sequence in some PCOS patients were identified. In PCOS-patients with high testosterone levels but low virilization change in the codon usage towards lower presented codons were identified.

Table 3  Sequence differences from standard AR gene sequence. Patient

Position, changes

2 2M 3 3F 6 6M

Exon 1, 1 296 bp TCC > TCT (Ser > Ser) Exon 1, 639 bp GAG > GAA (Glu > Glu) Exon 1, 639 bp GAG > GAA (Glu > Glu) Exon 1, 639 bp GAG > GAA (Glu > Glu)  − 79 bp 5′of exon 6 T > G  − 79 bp 5′of exon 6 T > G

M = mother; F = father

Discussion



Although there has been much research on PCOS, the pathophysiology is still not yet entirely understood. We focused on virilization of patients with PCOS. Most patients exhibit a correlation between high androgen levels and a higher grade of virilization. In contrast to this, we observed a small number of patients who do not fit into this general pattern. Different mechanisms have been suggested that may lead to increased virilization despite normal androgen levels. Kuttenn et al. (1977) showed that in idiopathic hirsutism the conversion of testosterone to 5-alpha metabolites was increased in skin homogenates [30]. Because of ethical reasons the acquisition of probes was limited in this study to venous blood. Therefore, we were unable to assess e. g. the activity of the 5-alpha reductase activity in the skin or in hair follicles, which may also play a role for virilization. Various clinical factors tend to have an influence on PCOS development – the androgen levels in pregnancy, the birth weight, obesity and baldness of the father. A recent study with rats showed a correlation between significantly increased androgen levels during pregnancy and a phenotype of PCOS in the adult female offspring [31]. Therefore we evaluated hirsutism and acne as an evidence for elevated androgen levels in all mothers of the included patients during pregnancy. Most mothers reported of no signs at all and 2 of only mild symptoms: During pregnancy, the mother of patient 5 exhibited very mild hirsutism of the upper lip and mild hirsutism of the lower legs. The mother of patient 2 exhibited mild acne on the forehead and the

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FG = Ferriman-Gallwey-Score; M = mother; F = father; NA = no obvious alteration; NP = not performed; patients written in bold

Article 59 androgen insensitivity syndrome of variable degree, some in patients with cancer of the prostate, liver, testes, breast or ovaries, some in normal healthy people. Some mutations have been linked to premature ovarian failure [48], indicating that AR dysfunction may play a role in reduced female fertility. Here, we detected some point mutations of the AR gene. These point mutations encode for the same amino acids, however, the codon usage of these mutated codons is significantly lower as compared to the wild type AR sequence. The functional consequences are so far unclear. However, it is known that the codon usage is very important for proper protein biosynthesis while lower codon usage matches available aminoacyl-tRNAs and is associated with changed mRNA stability and lower translation efficiency [49, 50]. Thus, these changes in codon usage may affect also AR mRNA stability or translation efficiencies. Further analyses including e. g. expression profiling and epigenetics in these patients may reveal whether an altered androgen-ARsignaling is responsible for atypical PCOS.

Acknowledgements



This work was supported by grants from the Interdisciplinary Centre for Clinical Research (IZKF), Jena University Hospital.

Conflict of interest: None. References

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chin. This indicates that highly elevated androgen levels during pregnancy, as suggested in the animal experiments to significantly increase the risk of developing PCOS, seem unlikely in the patients. Also, higher prevalence of SGA (small for gestational age, i. e., birth weight below 10th percentile) was described among PCOS-patients [32]. Nevertheless the birth weight and the calculated birth date of only one patient indicated a birth weight below the 10th percentile suggesting there is no general association of SGA being more often represented in patients with atypical PCOS. Percentiles are adopted from Voigt et al. (1996) [33]. 2 studies showed that obese PCOS patients are more likely to develop hirsutism [27, 34]. In this group, only patient 3 and the mother of patient 6 suffered from obesity according to the definition of Poskitt (1995) [28]. The affected patient did not show an increased grade of hirsutism. Therefore, presumably other causes seem to be responsible for atypical PCOS. We evaluated baldness in males as it could be linked to PCOS in the daughters [26] but both included fathers did not suffer from severe baldness. Taken together, none of the mentioned risk factors of developing PCOS seems to be causative in our patient group. The androgen receptor gene is located on the X-chromosome at Xq11-12 [35]. It consists of 8 exons [36] and encodes for an N-terminal domain, a DNA binding domain and a ligand-binding domain [37]. Alterations of the AR gene sequence, especially trinucleotide repeat lengths were linked to PCOS. 2 highly variable regions are located in exon 1 both containing 2 sets of trinucleotid repeats. The CAG-repeats encode for glutamine residues and consist of a normal range of around 22 repeats [38–40]. Findings linking the number of CAG repeats with testosterone levels and manifestation of PCOS are inconsistent: Some studies found an association between elevated testosterone levels and long CAG-repeats [7, 8] or short CAG-repeats, [41] others found no correlation [9, 42]. The CAG-length is described to be negatively correlated with transactivational function of the AR gene [43, 44]. 2 groups reported an increased prevalence of short CAG-repeats in women with PCOS [11, 12]. Van Nieuwerburgh et al. (2008) showed increased androgen levels in patients with shorter CAG-repeats leading to hirsutism and acne [10]. Jääskeläinen et al. (2005) reported that the length of CAGrepeats is not a major determinant of PCOS but may be a modulator in some patients [6]. These findings are consistent with a Chinese group who did a genome-wide association study on Chinese PCOS-patients – no alteration in the AR gene region were found [45]. Only a small number of patients fit into the pattern of atypical PCOS. Therefore the study group is small and the evidence is limited. Interestingly, in our patient group the number of repeats was within the normal range compared to the average in European people: Ferk et al. (2008) found a normal range of CAG repeats of 23.9 ± 3.5 [46] and Skrgatic et al. (2012) a normal range of 21.9 ± 3.2 [7]. The GGC-repeats encode for glycine residues and are also located in exon 1 [47]. In this study, the number of GGC repeats detected was within the normal range. To our knowledge this is the first report to examine the number of GGC-repeats in PCOS patients. As there were no abnormalities within the trinucleotide repeat lengths in these patients with atypical PCOS we decided to examine the entire AR for mutations. To our best knowledge, we are the first group that sequenced the entire androgen receptor gene and performed the karyotype in patients with PCOS. Many mutations of the AR have so far been described in other diseases. Most of them have been reported in patients with

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Atypical polycystic ovary syndrome--a genetic analysis.

Although polycystic ovary syndrome (PCOS) is a common endocrinopathy the pathogenesis is not entirely understood. Typically, high androgen levels are ...
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