Clinical Genetics 1985: 27: 167-174

Tetraploidy in pol ycyst ic

two sisters with the ovary syndrome

ARAM ROJANASAKUL', KARL-HENRIK GUSTAVSON~, HANS LITHELL3 AND SVEN JOHAN NILLIUS' Departments of 'Obstetrics and Gynaecology, Tlinical Genetics, University Hospital and the

Lipoprotein Laboratory and 3Department of Geriatrics, Kungsgardets sjukhus, Uppsala, Sweden

An increased frequency of tetraploid cells in two sisters and their mother with a probably dominantly inherited polycystic ovary syndrome is reported.

Received 18 June, accepted.for publication 17 September 1984

Key words: Chromosomal abnormalities; dominant inheritance; hyperandrogenism; hyperin- sulinaemia; lipid metabolism; lipoprotein lipase; obesity; polycystic ovary syn- drome; tetraploidy.

Polycystic ovarian disease (PCO) is a syn- drome of chronic anovulation and hyper- androgenism often associated with obesity (Goldzieher 1981). A subgroup of this syn- drome is the Stein-Leventhal syndrome. The occurrence of PCO in many members of some families suggests heredity factors in the aetiology of the disease. However, the mode of inheritance has not been estab- lished. Chromosome abnormalities have been described in PCO (Netter et al. 1961a, b, de Grouchy et al. 1961, Bishun & Morton 1964, Hargrave 1965, Cooper et al. 1968, Givens et al. 1971). Recently, Parker et al. (1980) summarized cytogenetic analysis in 189 women with PCO studied between 1961 and 1975. They found that a typical and consistent chromosomal abnormality is not associated with PCO. Here we describe cytogenetic analysis of two sisters with PCO. An increased occurrence of tetra- ploidy was found in the two patients and their mother.

Case Reports Patient 1: (111: 3 in the pedigree), a 19-year- old woman, was referred for treatment of primary anovulatory infertility. She was oligomenorrhoeic since menarche at age 11, markedly hirsute and obese. Her weight increased from 55 kg at age 11 to 100 kg at age 13. She had used a combined oral contraceptive pill for 2 years from the age of 13. (An analysis of the seminal fluid of her male partner was normal). On physical examination she was found to be obese (97 kg, height 163 cm) with excessive hair on her upper lip, chin, chest, abdomen, arms and legs (Fig. 1). Blood pressure was 120/70 mm Hg. Her breasts were well developed with no galactorrhoea. She was clinically euthyroid. Pelvic examination revealed a male escut- cheon with normal-sized clitoris. The uterus was normal. The ovaries could not be pal- pated.

168 R O J A N A S A K U L E T A L .

Patient 2: (111: 4), the 17-year-old sister of patient 1, was referred for obesity, oligome- norrhoea, hirsutism and chronic lower ab- dominal pain. After menarche at age 11, the frequency of her menstrual periods had progressively decreased and she increased in weight. At age 15, she underwent a laparo- tomy because of a left-sided ovarian cyst. The cyst was removed and found to be a simple follicular cyst. Bilateral wedge resec- tion of the slightly enlarged ovaries was performed. Histopathological examination showed changes consistent with PCO. On physical examination, she was found to be obese (86 kg, height 167 cm) with excessive hair on her face, chest, abdomen, arms and legs (Fig. 2). The blood pressure was 110/70 mm Hg. No galactorrhoea could be ex-

pressed from normal breasts. Pelvic exami- nation showed pubic hair of male distribu- tion with normal-sized clitoris. The ovaries were not palpable.

The mother: (11: 3), a 43-year-old woman, had previously been investigated at age 29 for hirsutism, oligomenorrhoea and obesity. Hormonal evaluation showed hyperandro- genism. During the last few years she had been on combined oestrogen-progestogen therapy.

Family History Two other female members of the family of the mother had clinical signs consistent with PCO, e.g. oligomenorrhoea, hirsutism and obesity. However, neither was infertile. The

Fig. 1. Patient 1 (Ill: 3) illustrating marked obesity and hirsutism. hirsutism.

Fig. 2. Patient 2 (111: 4) illustrating marked obesity and

P C O S Y N D R O M E W I T H T E T R A P L O I D Y 169

I

II

m I I

0 0 NORMAL 0 PCO

Fig. 3. Pedigree of affected family.

parents of the cases with PCO are non- consanguineous. The pedigree of the family is shown in Fig. 3.

Material and Methods

Chromosome Analysis Skin biopsies and peripheral venous blood samples were taken from the three PCO patients. From 34 normal individuals, one skin biopsy was obtained. From 50 normal individuals peripheral venous blood in a heparin tube was taken. The skin biopsies were taken with a punch without local anaesthesia. They were immediately placed in culture medium, consisting of Eagle’s MEM (Eagle 1959) with 17.5% pooled AB, Rh-positive sera and addition of penicillin and streptomycin. 100 ml of the medium was buffered with 2 ml of HEPES, pH 7.3. This medium was used both for transport and as a culture medium. The skin biopsies were cut into 0.5 to 2 mm pieces and were spread out with the dermis side down in a Falcon tissue culture flask with a 25 cm2 growth area, where they were allowed to become adherent for 30 min. Two to three ml of the medium were added to each culture, which was grown at 37.0 & 0.5 C in the closed flask. The medium was changed every third day during culture growth. The total culture period was five to six weeks. Subculturing was performed three times during this time.

For the subculturing, the cells were deta- ched with 2 4 drops of trypsin containing 20% versene. One drop of colchicine was added to each culture 2+ h before harvest- ing. Lymphocyte cultures from the patients were set up according to Hungerford (1965). For the chromosome preparation the stan- dard technique was followed. Conventional air-dried slides were prepared and then stained for Giemsa-banding and with the quinacrine mustard method for QM- banding (Caspersson et al. 1970). Cultivated white blood cells were examined for fragile X chromosomes (Gustavson et al. 1981). 100 metaphases were analysed from each biopsy and lymphocyte culture.

Hormone Assay Methods The pituitary and steroid hormones in blood were measured by Ass. Prof. Leif Wide at the Department of Clinical Chemistry, Uni- versity Hospital, Uppsala, Ass. Prof. Kjell Carlstrom at the Hormone Laboratory, De- partment of Obstetrics and Gynaecology, Huddinge Hospital and Mrs. Britt Green at the Primate Laboratory, Department of Ob- stetrics and Gynaecology, University Hos- pital, Uppsala.

Lipid and Lipoprotein Assay Methods The methods used for lipoprotein and lipid quantification, apolipoprotein (Apo) analy- sis, intravenous fat tolerance test (IVFTT) and intravenous glucose tolerance test (IVGTT) have previously been described in detail (Vessby et al. 1980). Lipoprotein lipase activity (LPLA) was determined in tissue biopsy samples from adipose and skeletal muscle tissue (Lithell & Boberg 1977, 1978).

Results

The results of the chromosomal investiga- tion are shown in Table 1. The Frequency of tetraploid cells in lymphocyte cultures was

170 R O J A N A S A K U L E T A L .

Table 1

The incidence of tetraploid cells in cultured fibroblasts and lymphocytes and of cultured lymphocytes with Fra (X) f rom three relatives wi th PCO

Patient

Lymphocytes Tetraploid Tetraploid with Fra (X)/lOO

cells/100 counted cells/100 counted counted lympho- cultured fibroblasts cultured lymphocytes cytes

Patient 1 (Ill: 3) 1st culture 2nd culture

Patient 2 (Ill: 4) 1st culture 2nd culture

The mother (11: 3) 34 normals 50 normals

0 7 1

6 8 2 2

mean I/lOO -

- -

- < 0.2

0

0

0

0

increased in the affected mother and her two affected daughters (1-8 %) as compared with the controls (<0.2%). One of the affected daughters also had an increased frequency of tetraploidy in cultured fibro- blasts. Otherwise the three patients all had a normal diploid chromosome constitution.

A summary of the results of the hormone analyses is shown in Table 2. The LH/FSH ratio was not raised. The oestrogen produc-

Table 2

The hormonal profiles in the two sisters with PCO

Patient 1 Patient 2

Prolactin (pg/l) LH (pgN FSH (pg/l) Estradiol (pmol/l) Estrone (pmol/l) Estrone sulphate (nmolil) Progesterone (pmol/l) 17ct OH Progesterone (nmolil) Testosterone (nmol/l) Androstendione (nmol/l) DHAS (nmol/l) DHAS (nmol/l) SHBG (nmol DHT/I)

7 4.6 1.20 1.27 1.13 1.05 128 104 390* 250' 7.23 4.23 1.22 1.05 3.7* 2.1*

10.8 12.4* 23.2 30.0 7575 7575

4.2' 2.8

8.i** a.a**

tion was low-normal with an abnormally high oestrone/oestradiol ratio. The andro- gen production was increased with raised serum testosterone in patient 1 and raised serum androstenedione in patient 2. There was no evidence of adrenal disease. The results are consistent with PCO.

The results of the metabolic evaluation are shown in Table 3. The two index pa- tients showed values for very low density lipoprotein (VLDL) triglyceride (TG) and cholesterol (Chol) as well as serum TG concentrations outside the normal range for the age and sex group. They were in the upper normal range for low density lipopro- tein (LDL) Chol and in the lower normal range for high density lipoprotein (HDL) Chol. Apo B, which is the major apolipo- protein in VLDL and LDL, was high in both subjects. The low fractional removal rate (K, value) at an IVFTT is consistent with the lipoprotein abnormality and indi- cates, together with the low LPLA values in adipose and muscle tissue, a deficient re- moval of circulating triglycerides. In con- trast, the K value of the IVGTT was normal. The normal fasting blood glucose concentration and the normal glucose toler- ance was achieved by major increases in the

* Elevated values. ** Low values.

P C O S Y N D R O M E W I T H T E T R A P L O I D Y 171

Table 3

Lipid and lipoprotein results for the two patients and reference values for 21 females (mean age 27 years, range 18-35 years). TG and Chol concentrations in VLDL, LDL and HDL and in serum are given. The Apo A-I, A-ll and B (expressed in percent of a reference serum sample) are indicated. Body weight and the K, value of IVFTT and IVGTT, lipoprotein lipase activity (LPLA) in adipose and muscle tissue, fasting blood glucose (fB glucose) and serum insulin values before glucose injection, after four and six minutes after the glucose injection (mean value= peak value) and 60 minutes after

the glucose injection started are also given

Reference values Patient 1 Patient 2 Mean f SD

VLDL TG, mmol/l VLDL Chol, mmoljl LDL TG, mmol/l LDL Chol, mmol/l HDL TG, mmol/l HDL Chol, mmol/l Serum TG, mmoljl Serum Chol, mmol/l Apo A-I, %

Apo B, % Body weight, kg IVFTT K,-value. %/min LPLA, adipose tissue mU/g LPLA, muscle tissue, mU/g IVGTT K-value, %/min fB-glucose, mmol/l Serum insulin, 0 min Serum insulin, peak value Serum insulin, 60 min

APO A-11, %

2.24 0.82 0.34 4.37 0.30 1.01 2.89 6.15 94 107 154 97.3 3,93

17 2.35 3.8 25

158 114

-

2.75 1.14 0.35 4.42 0.30 1.01 3.52 6.63 96 98

180 86.4 1.61 129 19

1.47 4.0 33

108 150

0.43f0.23 0.17 f0.14 0.31 fO.09

0.1 7 f 0.09 1.43 f 0.27 0.94f0.32 4.42f0.92

97f14

2.81 rt0.82

-

a a f i 8 60.8f6.4 7.1 k4 .7

419f 170 52f13

1.8Of 1.2 4.6f0.9 9.3+2.7

41.0f 19.0 13.1 k 6 . 0

serum insulin concentrations before and after glucose injection.

Discussion

The two sisters in this report had typical clinical and endocrinological signs of PCO. Their mother and other relatives on the maternal side also exhibited clinical mani- festation of PCO, although they were not infertile. Givens et al. (1971) studied families where several members were affected and suggested that PCO may be inherited as X- linked transmission, but autosomal domi- nant mode of inheritance cannot be ex- cluded. Cooper et al. (1968) suggested an autosomal dominant mode of inheritance. Several investigators (Byrd et al. 1964, Sten- chever et al. 1968, Gardo et al. 1974),

failed to demonstrate chromosome abnor- malities in PCO. Parker et al. (1980), recent- ly used modern banding techniques in cyto- genetic studies of women with PCO. They found trisomy 14 in 2 to 4% of the cells in 5 out of 15 patients (33%). This high in- cidence of chromosomal aberration in pa- tients with PCO may probably be due to more sensitive methods of investigation.

In our report, a significant increase in the number of tetraploid cells was found in lymphocyte culture of the two sisters and their mother and in fibroblast cultures of one sister. The significance of the associ- ation of tetraploidy in cultured cells with the probably dominantly inherited type of PCO in the present family is uncertain. The fre- quency of tetraploid cells is postnatally nor-

172 R O J A N A S A K U L E T A L .

mally very low in lymphocyte and fibroblast cultures but can be very high in amniotic cell cultures (Milunsky et al. 1971, Therkelsen et al. 1972, Annerkn 1982).

Increased in vitro tetraploidy has been reported in cultured skin fibroblasts and cells from colon from patients with and some family members at risk of Gardner’s syndrome and some other autosomal domi- nantly inherited colon cancer syndromes (Danes 1976, Danes & Lynch 1983). It has also been observed in cultured skin cells from patients with other rare heritable can- cer syndromes and in normal first- and second-degree relatives to patients with lung, breast, genital and gastro-intestinal cancers (Danes & Lynch 1983).

An increased frequency of tetraploid cells has also been found in skin cultures taken from extremities with unilateral longitudinal meromelia of unknown origin and from extremities with reduction malformation due to thalidomide embryopathy (Annerkn 1982).

It is unlikely that PCO is a phenotypic expression of the different chromosomal aberrations reported in these patients. It is however possible, that the increased in vitro tetraploidy in the patients with PCO in the present family as well as the other unspecific chromosomal anomalies reported in a mino- rity of PCO patients are signs of chromoso- mal instability typical of some distinct gene- tic disorders, such as Fanconi’s anemia, Bloom’s syndrome, ataxia telangietasia xeroderma pigmentosum and Gardner’s syndrome, considered to be an expression of the underlying gene defect (Sandberg 1980).

It is also probable that the increased frequency of tetraploid cells in cultured lymphocytes and fibroblasts from our famil- ial PCO cases reflects a disturbance of the mitotic process, expressed in an increased frequency of polyploidy, which might be of pathogenetic importance. This disturbance of the mitotic process could be related to an

underlying gene defect or due to metabolic and/or hormonal influences.

The type of lipoprotein abnormalities seen in the two index patients are often found in obese subjects. However, the lipid and lipoprotein pattern of the two patients are more similar to those of obese male subjects than to those of obese female sub- jects. The metabolic abnormalities are pro- nounced for our patients’ sex and age. We have previously described similar abnormal lipid and lipoprotein profiles in PCO (Nil- lius & Lithell 1983). Furthermore, the high insulin values before and after glucose injec- tion indicate a high peripheral insulin re- sistance, which may be related to the degree and the male type of obesity in our patients. Insulin resistance has previously been de- scribed in association with hyperandrogen- ism (Wortsman & Soler 1982, Barbieri & Ryan 1983, Chang et al. 1983, Pasquali et al. 1983, Shoupe et al. 1983).

To conclude, an increased frequency of tetraploid cells were found in two sisters and their mother, all three with clinical and endocrinological features typical of the polycystic ovary syndrome. The significance of the association of tetraploidy with the probably dominantly inherited syndrome is unknown but could possibly be related to the gene defect.

Acknowledgment

We thank Mrs. Karin Mattsson for excel- lent secretary assistance.

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Address: S. J . Nillius, M.D. , Ph.D. Department of Obstetrics and Gynaecology University Hospital 9 7 5 1 85 Uppsala Sweden