J Med Genet: first published as 10.1136/jmg.2.3.205 on 1 September 1965. Downloaded from Case Reports J. med. Genet. (I965). 2, 205. Ovarian Dysgenesis and Presumed Isochromosome of the Long Arm of X MICHAEL J. REE General Practitioner, Keynsham, Somerset Turner's syndrome has long been associated Case Reports with primary amenorrhoea, webbing of the neck, Case I. The second child of unrelated parents born cubitus valgus, and short stature (Turner, 1938). in November I948, birthweight 6j lb. (2-9 kg.), was The majority of these cases are negative first seen by me in February I964. Although a scanty and have an XO complement. Ovarian menstrual discharge had occurred some six months maldevelopment is frequently a feature of this previously a regular cycle had never been established. condition (Albright, Smith, and Fraser, I942), as Her mother was 37 and her father 4I years old when she somatic abnormalities. was born. She had a brother who was three years her is the presence of multiple senior. Both parents and her brother were pheno- Chromatin positive cases have been reported (De typically normal. Her mother's height was 5 ft. 5 in. la Chapelle, I962; Lindsten, I963; Williams, Engel, (i65 cm.), and her father's height was 6 ft. (I83 cm.). and Forbes, I964) bearing some of the features of Her brother was 6 ft. 2 in. (I88 cm.). the classicalP Turner's syndrome. Stunting of Detailed inquiry into the maternal and paternal family stature is nearly always evident, but webbing of the history revealed no evidence of thyroid disease or father suffered from intermittent neck and other somatic abnormalities are less diabetes mellitus. Her copyright. commonly found. Most of these subjects have claudication and on one occasion his serum cholesterol been shown to be chromosomal mosaics XO/XX. level had been 305 mg./ioo ml. Both his parents were A much rarer group is thought to be associated stated to have died from coronary thrombosis, as did his eldest brother at the age of 50 years. with an X iso complement. was short ft. in. (i44-2 cm.)) The isochromosome is metacentric and may be The patient (Fig. i) (4 81 formed by replication of the long arm of the X chromosome.. The exact mechanism is not known though there are several theories (Jacobs, Harnden, http://jmg.bmj.com/ Buckton, Court Brown, King, McBride, Mac- Gregor, and Maclean, I96I; Lindsten, Fraccaro, Ikkos, Kaijser, Klinger, and Luft, I963). The latter authors suggest that the isochromosome may result from a misdivision of the which divides transversely instead of longitudinally. They postulate that this is more likely to happen at the first reduction division of in spermatogonia on September 28, 2021 by guest. Protected owing to the possible difficulties in pairing up the morphologically dissimilar X and Y . Published material records i6 cases of X iso X chromosome complement in which there was no evidence of mosaicism (Jacobs et al., 196I, Hamer- ton, Jagiello, and Kirman, I962; Forbes and Engel, I963; Lindsten et al., I963; Lindsten, I963; Sparkes and Molutsky, I963; Williams et al., I964). This paper records two further cases of primary ovarian failure due to presumed isochromosome of the long arm of X. FIG. I. The patient aged 15. Note diminutive stature, short neck, Received March 22, I965. cubitus valgus, and scanty pubic hair. 205

M.G.-4 J Med Genet: first published as 10.1136/jmg.2.3.205 on 1 September 1965. Downloaded from

206 Michael J. Ree tures. There was a well-marked cubitus valgus. The fingers were rather short, but the nail development was normal. A transverse palmar crease was present on the left hand but not on the right. No evidence of colour blind- ness was found (Ishihara plates) and the visual acuity in both eyes was 6/I2. The visual fields were normal. Both femoral pulses were palpable. The blood pressure was I45/85 mm. Hg. The electrocardiogram was normal. Intelligence was apparently average. She attended a Secondary Modern School and was in a 'C' stream. In a recent terminal examination she was placed 5th in a class of 29 children. In certain subjects she was reported to be above average ability.

RADIOGRAPHIC AND LABORATORY INVESTIGATIONS. Radiographs of the hands, wrists, and knees showed epiphysial development to be within normal limits. The skull and sella turcica were normal. Fifty oral cells were examined from buccal smears, 54% of which showed a single nuclear sex chromatin mass. Examination of stained neutrophil leucocytes was made for nuclear appendages. 400 cells were counted of which 64 (i6%) contained 'large drumsticks' FIG. 2. Photograph demonstrating normal breast contour, infantile (Fig. 3) and 69 (17%) contained 'small clubs'. nipples, and absent axillary hair. Some pigmented naevi are evident. The 24-hour urinary excretion of I7-Qxosteroids and 17-hydroxycorticosteroids was 4-75 mg. and 8-85 mg., and rather obese. The neck was short but there was no respectively. Serum protein electrophoresis showed a was infantile: the labia normal pattern. The serum sodium, potassium, chloride, webbing. The external genitalia copyright. majora and clitoris were absent, and the labia minora calcium, and inorganic phosphorus levels were within was very poorly developed. Vaginal examination was normal limits, as was the blood urea. The serum alka- not possible but a probe could be passed for a distance of line phosphatase was I9 King Armstrong units (normal I in. (2-54 cm.) into the poorly developed vagina. On range 2-I2 units per IOO ml.). The serum cholesterol rectal examination a small uterus was palpable. There content estimated on two occasions was 260 mg./Ioo ml. was some scanty pubic hair but no axillary hair. The and 263 mg./ioo ml., respectively. Repeated estima- lower jaw tended to recede, and the palate was high and tions of blood sugar levels ranged between 65 mg. and arched. There were multiple pigmented naevi particu- 8o mg./ioo ml., and routine analysis of the urine larly evident on the face. The breast contour was revealed no abnormality. The haemoglobin content,http://jmg.bmj.com/ normal, and there was slight pigmentation of the total white cell count, and differential count were areolar, but Montgomery's tubercles were absent. The normal, and haemoglobin electrophoresis showed nipples (Fig. 2) were poorly-developed infantile struc- normal adult type haemoglobin. Dr. Ruth Sanger examined samples of blood from the subject and her parents for the sex-linked Xga blood group. All were Xg (a+). The erythrocyte glucose 6 phosphate dehydrogenase (G6PD) activity was 2400 units/ml. of red cell mass. No thyroid auto-antibodies could be detected in the on September 28, 2021 by guest. Protected subject's serum or in that of her parents using the latex particle technique, but a weak positive titre (1/25) was found when the subject's serum was examined by the tanned red cell haemagglutination test. The 4-hour uptake of 25 microcuries of 18I1 was 35% of the dose, and the 48-hour uptake was 52%. The 48-hour protein bound iodine (PBI) was 0o7% of the dose per litre of plasma. (Normal range 0-03 to o03% of the dose per litre of plasma.)

CHROMOSOME STUDIES. This work together with the autoradiographic findings was undertaken by Dr. F. Lewis of Southmead Hospital, Bristol. The chromo- FIG. 3. Neutrophil polymorph with large drumstick. (x Isoo.) somal pattern was studied on growing cells of the peri- J Med Genet: first published as 10.1136/jmg.2.3.205 on 1 September 1965. Downloaded from

Ovarian Dysgenesis and Presumed Isochromosome of the Long Arm of X 207

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2o8 Michael J. Ree TABLE I

pheral blood by a modification of the method of Moor- with H.3 thymidine in the S phase of DNA synthesis head, Nowell, Mellman, Battipps, and Hungerford (Muldal, Gilbert, Lajtha, Lindsten, Rowley, and Frac- (I960). The results, together with the buccal smear caro, I963; Giannelli, I963) were performed on 20 cells analysis, are summarized in Table I. in which one heavily labelled chromosome could be Subject. The modal cell showed 22 pairs of demonstrated. In each case this was morphologically and an abnormal sex chromosome in all the spreads consistent with a presumptive isochromosome of X. examined. In place of the second X chromosome was a Mother. 30 cells in metaphase were examined and large chromosome which paired in size with chromo- I3 cells fully analysed by photography. There was no some number 3 (Denver Classification, I960). The two significant abnormality in the cells fully analysed. apparently equal arms were similar in size to the long Father. A sample of 30 cells in metaphase was arm of the other X. This abnormal chromosome was examined. Of these 29 contained 46 chromosomes, the present also in the two hypomodal cells (Fig. 4). Auto- karyotype being consistent with the normal male of 22 radiographic studies on cultured leucocytes labelled pairs of autosomes and sex chromosomes X and Y. copyright. http://jmg.bmj.com/

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(b) FIG. 5. Main lines of dermal ridge patterns on finger-tips, palms, and soles. J Med Genet: first published as 10.1136/jmg.2.3.205 on 1 September 1965. Downloaded from

Ovarian Dysgenesis and Presumed Isochromosome of the Long Arm of X 209 DERMATOGLYPHIC ANALYSIS. Dermatoglyphic pat- urinalysis was normal. The urinary 17-oxosteroid terns (Fig. 5) were examined by Prof. L. S. Penrose of excretion was 6-4 mg./24 hours, and the 17-hydroxy- The Galton Laboratory, University College, London. corticosteroid excretion was 6-8 mg./24 hours. The Finger-tip patterns show fairly high intensity. The urinary 24-hour excretion of creatinine was o-8 g. total ridge count (that is the sum of the highest count on each finger) was i82. Comparison with counts obtained CHROMOSOME STUDIES. Chromosomal analysis (Dr. F. on the average for Turner's syndrome and related condi- Lewis) was performed on cultured leucocytes. The tions, and for normal female controls is shown in Table II patient had a modal cell line of 46 chromosomes, com- where measurements on the palms are also given. There prising 22 pairs of autosomes, a single X chromosome, were strong hallucal patterns on the feet, which were and a large medial chromosome resembling an iso- of the normal type. chromosome of the long arm of X. II TABLE I.

Total Ridge a-b Ridge Maximal atd Count (right Count (right Angle (right and left) and left) and left) Subject I82 83 950 Turner's syndrome and related conditions I67 96 I07° k Female controls 130 84 880 ( TREATMENT. Intermittent treatment with O-OI mg. Ti ethinyl oestradiol per day resulted in withdrawal bleeds which simulated a normal menstrual flow. As t;N i > 4.~~~~~~1ifSI yet there has been no improvement in the development ~.p: of the external genitalia. issS r M:. .o.: copyright. Case 2. This patient was seen in April I964, at the . \ .tUR age of 30, by Mrs. M. Bennett at Southmead Hospital, i W,c Bristol, for investigation of amenorrhoea. Some I7 * years previously three or four scanty periods had k, Yf° occurred. Most of her life had been spent abroad, and 0- _11 _ _,. 1|_ she had received, on occasions, various forms of medica- 111! >_ -U tion with resultant withdrawal bleeding. She had two - i= l l - - -- brothers aged 45 and 33 years, and two sisters 36 and FIG. 6. The second patient aged 28. Short stature, cubitus valgus, 34 years old, respectively. and multiple pigmented naevi are evident. http://jmg.bmj.com/ of dimunitive stature 8iP:e- The patient was (Fig. 6), height 4 ft. 7,1- in. (I4I cm.), and weighed 5 st. I I lb. (36 8 kg.). There was a wide carrying angle of the arm. There was Discussion and Conclusions no axillary hair but some pubic hair of pubertal distri- The two patients described show the same clinical bution. Some pigmented naevi were present on the features as subjects with a presumed isochromosome skin of her chest and face. The breasts were reasonably of the long arm of X described by previous authors. well developed, but the nipples were infantile in appear- Stunting of stature is invariable, as is sexual infanti- ance. The labia were poorly developed and the vagina lism. The majority of cases so far reported may be on September 28, 2021 by guest. Protected was small. Examination under anaesthetic revealed a with the of small atrophic uterus from which no curettings could be classified type ovarian dysgenesis, with- obtained. The ovaries were not palpable. The cardio- out webbing of the neck, and of small stature, as vascular system was normal and the blood pressure was suggested by Polani (I96I), but a patient described I30/70 mm. Hg. by Hamerton et al. (I962) was a low-grade mental defective, had severe somatic abnormalities, and RADIOGRAPHIC AND LABORATORY INVESTIGATIONS. had webbing of the neck. Radiography showed a normal sella turcica. Epiphyses In view of the report by Forbes and Engel (I963) in the wrist were fused. The pelvis was normal as was on the high incidence of diabetes mellitus in patients a chest radiograph. Gynaecography failed to reveal with gonadal dysgenesis and in their close relatives, ovarian shadows. care was taken to find evidence diabetes Vaginal smears showed that 90% of the cells had particular of large well-formed nuclei, and Io% showed a variable mellitus in the paternal and maternal relatives of degree of pyknosis. the first subject; with negative results. Repeated The haemoglobin was 13 9 g./IOO ml. (94%). Routine examinations for sugar content were made on J Med Genet: first published as 10.1136/jmg.2.3.205 on 1 September 1965. Downloaded from

210 Michael J. Ree samples of urine and blood obtained both from the compared with 23 and 24 years, respectively, in the subject and from her parents. No abnormal findings subjects reported by Sparkes and Motulsky, and were reported. 34 and 31 years in the subjects described by Autoradiographic studies performed on cultured Williams et al. leucocytes from subjects with an X iso X chromo- Other workers suggested that abnormalities of some complement show that it is invariably the the X chromosome were directly responsible for isochromosome that is inactivated (Muldal et al., abnormal antibody formation. According to Burch I963). This apparent contradiction of the Lyon (I963), cell bound antibody may be synthesized via (I96I) hypothesis has been explained by Gartler messenger RNA coded from an autosomal locus, and Sparkes (I963) by assuming that at some stage and a locus on one X chromosome; the former of early embryonic life the cells whose normal X synthesizing the 'light' polypeptide, and the latter chromosomes are inactivated will die, the survivors the 'heavy' polypeptide chains. On theoretical being those cells in which the isochromosome is grounds it was suggested (Burch and Rowell, I963) inactivated. The final result, therefore, should be that as well as somatic autosomal mutations, four similar to those individuals having an XO chromo- mutant inherited genes on the X chromosome may some composition, in which the single X chromo- feature in the aetiology of Hashimoto's thyroiditis. some is either inactivated, in which case the cell If one assumes that either the isochromosome of the dies, or remains extended and genetically active, long arm of X is not completely inactivated, or that ensuing clones being normal. in stem cells of the lymphoid series inactivation There is some evidence (Reed, Simpson, and does not occur, and that the isochromosome is isoal- Chown, I963) suggesting that in normal individuals lelic (i.e. the arms are genetically identical), it inactivation of the X chromosome may not be would be necessary to postulate the inheritance of complete, and it is ofinterest to speculate ifinactiva- only two mutant genes on one parental X chromo- tion of the isochromosome may be likewise incom- some before isochromosome formation during plete. meiosis. If this were correct, and as yet there is no Some authorities consider that abnormal experimental evidence, it may help to explain the may be produced by abnormal immunological high incidence of active thyroiditis in subjects withcopyright. mechanisms (Falikow, I964). The finding of a an X iso X chromosome complement. It would high frequency of thyroid auto-antibodies (Engel, also be necessary to postulate that the isochromo- Forbes, Mantooth, and Socolow, I963) in patients some is not completely genetically inert. with the more usual form of ovarian dysgenesis Perhaps both theories are acceptable. A high (XO, and XO/XX mosaics) supports this view. In circulating level of auto-antibodies may produce patients having an X iso X chromosome comple- chromosomal abnormalities, but if by chance ab- there is a incidence of thyroiditis normalities of the X chromosome occur, abnormal ment, high http://jmg.bmj.com/ (Williams et al., I964; Sparkes and Motulsky, I963). antibody production may result. The study and Although there is as yet no evidence of thyroiditis follow-up of similar patients and their relatives may in the first patient reported in this communication, shed more light on this problem. she does have a weak thyroglobulin antibody titre, though no thyroid auto-antibodies could be Summary demonstrated in her parents' sera. In Hashimoto's Reports are given of 2 patients with gonadal thyroiditis there may be a normal 131I uptake, but dysgenesis due to presumed isochromosome forma-

PBI is often raised (Murray, I964). This is due tion of the long arm of X. The clinical features on September 28, 2021 by guest. Protected to (a) impaired iodine binding resulting in a release agree with those described by previous authors. of unbound iodide from the thyroid cells, the so- The association between abnormalities of the X called discharge phenomenon; (b) the leakage of chromosome and antibody production is discussed. abnormal, butanol insoluble, iodoproteins or iodo- peptides directly into the circulation; and (c) a destructive reduction of the total thyroid iodine My thanks are due to Mrs. M. Bennett for her permission to include the second case, and for her pool. The disorder usually becomes manifest in constant encouragement; Dr. F. Lewis, Southmead middle-aged women when hypothyroidism is Hospital, Bristol, for the chromosomal and autoradio- associated with a rapidly enlarging goitre. Some graphic work; Dr. Ruth Sanger, M.R.C. Blood Group cases of autoimmune thyroiditis remain euthyroid. Research Unit, the Lister Institute, London, who reported on the Xge blood groups; Professor L. S. It is of interest to note that the subject had a Penrose, the Galton Laboratory, University College, normal ""'I uptake but a raised PBI. Her age was London, for the dermatoglyphic analysis; Dr. R. iS years when these investigations were carried out Gibson, Area Central Laboratory, Bath, for the bio- J Med Genet: first published as 10.1136/jmg.2.3.205 on 1 September 1965. Downloaded from

Ovarian Dysgenesis and Presumed Isochromosome oJ the Long Arm of X 211 chemical investigations; and Dr. G. Foss, United Hamerton, J. L., Jagiello, G. M., and Kirman, B. H. (1962). Sex Bristol Hospitals for reading the manuscript and for chromosome abnormalities in a population of mentally defective many helpful suggestions. children. Brit. med J., I, 220. was a from the Emily Jacobs, P., Harnden, D. G., Buckton, K. E., Court Brown, W. L., The study supported by Grant King, M. J., McBride, J. A., MacGregor, T. N., and Maclean, Showering Trust. N. (I96I). Cytogenetic studies in primary amenorrhoea. Lancet, I, iI83. REFERENCES Lindsten, J. (I963). The Nature and Origin of X Chromosome Aber- rations in Turner's Syndrome. A Cytogenetical and Clinical Study Albright, F., Smith, P. H., and Fraser, R. (1942). A syndrome of 57 Patients. Almqvist and Wiksell, Stockholm. characterized by primary ovarian insufficiency and decreased -, Fraccaro, M., Ikkos, D., Kaijser, K., Klinger, H. P., and stature. Report of I i cases with a digression on hormonal control Luft, R. (I963). Presumptive iso-chromosomes for the long arm axillary and hair. Amer. J. med. Sci., 204, 625. of pubic of the X in man. Analysis of five families. Ann. hum. Genet., 26, Burch, P. R. J. I963). Autoimmunity: some aetiological aspects. 383. Lancet, I, 1253. Lyon, M. F. (I961). Gene action in the X-chromosome of the -, and Rowell, N. R. (I963). Autoimmunity. Aetiological mouse (Mus. musculus L.). Nature (Lond.), 190, 372. aspects of chronic discoid and systemic lupus erythematosus, systemic sclerosis, and Hashimoto's thyroiditis. Some immuno- Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battipps, D. M., logical implications. ibid., 2, 507. and Hungerford, D. A. (I960). Chromosome preparations of Denver Classification (I960). A proposed standard system of leukocytes cultured from human peripheral blood. Exp. Cell nomenclature of human mitotic chromosomes. ibid., I, 1O63. Res., 20, 6I3. De la Chapelle, A. (I962). Cytogenetical and clinical observations Muldal, S., Gilbert, C. W., Lajtha, L. G., Lindsten, J., Rowley, J., M. Tritiated in an in female gonadal dysgenesis. Acta endocr. (Kbh.), 40, Suppl. 65. and Fraccaro, (I963). thymidine incorporation Engel, C., Forbes, A. P., Mantooth, L. C., and Socolow, E. L. isochromosome for the long arm of the X chromosome in man. (I963) Abstracts, Annual Meeting, American Society of Human Lancet, I, 86I. , New York, July I8-21, P. i8. Murray, I. P. C. (I964). Thyroid Disorders; A Guide to Diagnosis Fialkow, P. J. (I964). Autoimmunity: a predisposing factor to and Treatment. Pitman, London. chromosomal aberrations? Lancet, I, 474. Polani, P. E. (196I). Turner's syndrome and allied conditions. Forbes, A. P., and Engel, E. (I963). The high incidence of diabetes Brit. med. Bull., 17, 200. mellitus in 4I patients with gonadal dysgenesis, and their close Reed, T. E., Simpson, N. E., and Chown, B. (I963). The Lyon relatives. Metabolism, 12, 428. hypothesis. Lancet, 2, 467. Gartler, S. M., and Sparkes, R. S. (I963). The Lyon-Beutler Sparkes, R. S., and Motulsky, A. G. (I963). Hashimoto's disease hypothesis and isochromosome X patients with the Turner in Turner's syndrome with isochromosome X. ibid., I, 947. syndrome. Lancet, 2, 4II . Turner, H. H. (1938). A syndrome of infantilism, congenital webbed Giannelli, F. (1963). The pattern of X-chromosome deoxyribonuc- neck, and cubitus valgus. Endocrinology, 23, 566. leic acid synthesis in two women with abnormal sex-chromo- Williams, E. D., Engel, E., and Forbes, A. P. (I964). Thyroiditis some complements. ibid., I, 863. and gonadal dysgenesis. New Engl.J. Med., 270, 805. copyright. http://jmg.bmj.com/ on September 28, 2021 by guest. Protected