Analysis of the Sex-determining Region of the Y Chromosome (SRY) in a Case of 46, XX True Hermaphrodite

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1 Clin Pediatr Endocrinol 1994; 3(2): Copyright (C) 1994 by The Japanese Society for Pediatric Endocrinology Analysis of the Sex-determining Region of the Y Chromosome (SRY) in a Case of 46, XX True Hermaphrodite Takashi Okabe, Masayoshi Miura, Mika Oonari, Naofumi Kitano, Mayumi Taniuchi, Tomoji Nozaki and Masatoshi Miyamoto Department of Pediatrics (TO, MM, MO, NK), Department of Pediatric Surgery (MT, TN, MMiya), Toyama City Hospital, Toyama, Japan Abstract We present here an 18-month-old 46, XX true hermaphrodite with hypospadias and bifid scrotum. The patient's gonadotropin levels before and after the LH-RH test were within the normal range. The serum testosterone level was <5.0 ng/dl and rose to 381 ng/dl after the human chorionic gonadotropin test. The serum estradiol level was <10 pg/ml and did not rise after the human menopausal gonadotropin test. Pelvic magnetic resonance imaging (MRI) revealed a vagina-like structure. Bilateral gonads were palpable in the scrotum, and histologically, they were ovotestes. We could find no sequence of sex-determining region Y (SRY) in the ovotestes, perineal skin, or leukocytes by polymerase chain reaction (PCR) methods. Other Y-specific DNA sequences were also not detected. These findings suggest that some ) XX ( true hermaphroditism is caused by a very low proportion of Y-containing cells, which cannot be detected by PCR methods. Alternatively, a mutation of one of the down-stream genes of SRY could be involved in the pathogenesis. Key words: sex-determining region Y, true hermaphroditism, polymerase chain reaction Introduction The gene encoding the testis-determining factor (TDF) has recently been reported. This was called the SRY, located within a 35-kilobase interval on the short arm of the Y chromosome close to the boundary of the pseudoautosomal region (1). Received: March 23, 1994 Accepted: June 7, 1994 Correspondence: Dr. Takashi Okabe, Department of Pediatrics, Toyama City Hospital, 292 Imaizumi, Toyama-shi, Toyama 939 Japan Most )0( males without ambiguous genitalia possess various segments of the short arms of the Y chromosome, including the SRY (2). However, the mechanism of development of XX true hermaphrodites is less well understood than that of XX males. In this report, an XX true hermaphrodite was tested for the presence of sequences of SRY by examining blood lymphocytes, perineal skin and ovotestes with PCR methods. 91

2 Vol.3/No.2 Okabe et al. Case Report The case, T.H., an 18-month-old boy, was referred to us because of ambiguous genitalia. Past history revealed that he was the first offspring of a 26-year-old mother and was born at 36 weeks gestation by normal delivery. Family history was unremarkable with no consanguinity in his parents. His younger brother, 4 months old, had normal genitalia. On physical examination, his height and weight were 80.0 cm (-0.42 SD) and 12.2 kg (+1.36 SD). He had a small penis, with stretched length of 1.5 cm, hypospadias and a bifid scrotum. Small gonads, which were about 1 ml in volume, were palpable in the scrotum (Fig. 1). Routine blood examinations (cell count and chemistry) and urinalysis were normal. Chromosomal analysis showed a 46, XX karyotype. Endocrinological examination showed that basal levels of adrenocortical hormones and sex steroids were normal (Table 1). Basal and peak levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were normal by the LH-RH test. The serum testosterone was <5.0 ng/dl and rose to 381 ng/dl after the human chorionic gonadotropin (HCG) test. The serum estradiol level was <10 pg/ml and did not rise after the human menopausal gonadotropin (HMG) test (Table 2). Table 1 Endocrinological data Fig. 1 External genitalia of 11-month-old patient. 92

3 December 1994 SHY in a Case of 46, XX True Hermaphrodite Table 2 Results of various endocrinological loading tests Pelvic MRI showed a vagina-like structure, but did not demonstrate ovaries or uterus. A voiding cystourethrogram disclosed a normal bladder and urethra, without any urogenital sinus. When he was two years old, bilateral gonadal biopsies were performed. Histological findings in both gonads showed ovotestes, with primordial follicles in the ovarian region (Fig. 2) and immature seminiferous tubules in the testicular region (Fig. 3). DNA analyses were done with the blood Fig. 2 Ovarian region of the ovotestis shows primordial follicles. Reduced from ~200. Fig. 3 Testicular region of the ovotestis shows immature seminiferous tubules. Reduced from ~

4 Vol.3/No.2 Okabe et al. lymphocytes, perineal skin and ovotestes. Interestingly, we could find no sequence of SRY in these specimens by PCR methods (Fig. 4). Other Y-specific DNA sequences detected. sequences Discussion In most XX male patients, were also not Y-specific DNA of the short arm of the Y chromosome are detected on one X chromosome, suggesting aberrant X-Y terminal interchanges during paternal meiosis (3). Nakagome et al. (4) reported an XX true hermaphrodite who had Y- specific DNA sequences including SRY in blood lymphocytes. However, McElreavey et al. (5) analyzed 30 cases of 46, XX true hermaphro- ditism, and reported that SRY were present in the lymphocytes in only 3 cases and absent in the remaining 27. They suggested that the phenotype in SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism giving rise to both testicular and ovarian tissues, and that the remaining SRY-negative cases may be explained by hidden XX/XY or XX/XXY mosaicism limited to the gonads. We examined an XX true hermaphrodite for the presence of sequences of SRY, using not only blood lymphocytes but also perineal skin and ovotestes, but Y-specific DNA sequences including SRY were not detected in these specimens. These findings suggest that some XX true hermaphroditism is caused by a very low proportion of Y containing cells, which cannot be detected by PCR methods, or by a mutation of one of the down-stream genes of SRY, which by itself may be insufficient for complete testicular formation. Acknowledgments The authors are grateful to Prof. Y. Nakagome, Department of Human Genetics, University of Tokyo, for DNA analysis by PCR methods. References 1. Sinclair AH, Berta P, Palmer M, Hawkins JR, Griffiths BL, Smith MJ, et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990; 346: Fig. 4 PCR analysis of SRY sequences. 2. Numabe H, Nagafuchi S, Nakahori Y, 94

5 December 1994 SRY in a Case of 46, XX True Hermaphrodite Tamura T, Kiuchi H, Namiki M, et al. DNA analyses of )0C. and XX-hypospadiac males. Hum Genet 1992; 90: Page DC. Sex reversal: deletion mapping the male-determining function of the human Y chromosome. Cold Spring Harb Symp Quant Biol 1986; 51: Nakagome Y, Seki S, Fukutani K, Nagafuchi S, Nakahori Y, Tamura T. PCR detection of distal Yp sequences in an XX true hermaphrodite. Am J Med Genet 1991; 41: McElreavey K, Rappaport R, Vilain E, Abbas N, Richaud F, Jacob SL, et al. A minority of 46, XX true hermaphrodites are positive for the Y-DNA sequence including SRY. Hum Genet 1992; 90: