XX Male: A report of two unrelated cases and review of literature. Sawsan J. Abulhasan, Laila A. Bastaki, Fawziah M. Mohammed, Kamal K.Naguib, Sayed AR. Gouda, Jaber Al-Tahoo, Sadika A. Al-Awadi. From the Kuwait Medical Genetics Center, Kuwait. Abstract: Two unrelated phenotypically males with 46,XX karyotype are presented. The first patient is 5.5 year old who presented with congenital undescended testis while the other patient is 35 year old who has infertility since 6 years. Hormonal profile of the second patient is consistent with hypergonadotrophic hypogonadism with low testosterone level. The first patient's profile revealed a picture consistent with 21-hydroxylase deficiency. FISH technique using (Quint-Essential-Y-specific DNA probe) specific for Yp11.2 region was applied on metaphase spreads revealed the presence of Yp11.2 sequence on the short arm of the X chromosome in the second patient while it was absent in the first patient. DNA analysis of both patients using SRY gene amplification revealed the existence of SRY gene in the second patient and absence of the gene in the first patient. The possible underlying mechanisms for sex reverse in both cases are discussed. Introduction: hybridization, detection and amplification were done according to the manufactures instruction XX males are characterized by the presence of with slight modifications. Slides were counter testes and external male genitalia despite a stained with propidium iodide (0.6 µg/mL) in 46,XX karyotype(1) In about 80% of these antiffade solution (0.3 µg/mL). Hybridized cells individuals the male differentiation of the gonads were examined under a Zeiss axiophote can be ascribed to the presence of Y specific microscope equipped with a fluorescence filter DNA including the SRY gene which encodes the sets. testes determining factor.(1-3) Most affected individuals have normal male external genitalia, PCR: but about 10% have hypospadias.(4) Subjects are Genomic DNA prepared from peripheral blood considered to have 46,XX true hermaphroditism samples(5) was amplified by polymerase chain if they have ovarian and testicular tissue. Those reaction (PCR) in a Perkin-Elmer thermal cycle. individuals usually have ambiguous genitalia and The presence of a SRY 609-bp sequence mixed Wolffian and Müllerian structures located to inside the conserved high mobility reflecting the extent of testicular development. group (HMG) box defined by the primers Here in we are reporting two unrelated males XES7/XES2(6). The presence of 170-bp portion of with 46,XX karyotype. The molecular study and the Y centromeric sequence (DYZ3) was defined the underlying mechanisms of sex reverse in by the primers Y /Y (7). PCR products were both cases are discussed. 1 2 revealed in 1% agarose gels. Subjects and Methods: Case report I: FISH: The proband a phenotypically male child aged 2 patients with 46,XX maleness were five and half year old attended the Kuwait ascertained clinically and cytogenetically. Medical Genetics Centre because of bilateral Chromosomal study using peripheral blood and undescended testes. He is the product of the 4th conventional cytogenetic technique was carried pregnancy to first cousin phenotypically normal out for both patients. Studies of fluorescence in parents having 4 phenotypically normal situ hybridizations (FISH) were performed using- daughters in addition to the proband. Pedigree commercially available digoxigenin labeled DNA study preconceptional, 1st and 2nd trimesters probe (Quint essential Y probe) specific for histories were irrelevant. The pregnancy was Yp11.2 locus and X/Y cocktail probe for X ended spontaneously at full term, with an cen/Yq12) direct-labeled with fluorescent average birth weight. The proband was (ONCOR Gailhersburge MD). Probes incubated for 9 days due to cyanosis and Alexandria Journal of Pediatrics, Volume 13, Number 2, July 1999 327 respiratory distress. At birth, the external showed 46,XX karyotype. FISH technique using genitalia showed bilateral undescended testes, Quint-Essential Y specific for Yp11.2 locus and normal sized phallus and empty scrotum. Body X/Y cocktail probe for X cen/Yq12) were applied organs examinations were normal. Hormonal on metaphase spreads and interphase nuclei profile showed high 17 hydroxyprogesterone at 4 and revealed the presence of Yp11.2 DNA weeks old (<75 nmol/L), high DHEAS (15.6 material on short arm of one X chromosome in nmol/L), ± normal testosterone (1.9 nmol/L), high all cells (Figure1). Accordingly, the karyotype Deoxycortisol 98 nmol/L, high LH and FSH, will be 46,X,-X,+ t(Xp;Yp). DNA study revealed normal growth hormone and normal thyroid the presence of SRY gene and absence of Y hormones. Reevaluation of 17 OH progesterone centromeric sequence (Figure 2A, B). at 9 months and 2½ year old was consistently high. Biochemical study showed normal Na and Discussion: high K levels. Pelvic U/S at different occasions In order for an infant to develop as showed neither uterus nor ovaries or testes. phenotypically complete male or female a Bone age study at 2 3/12 year was of 3½ year. cascade of complex molecular and morphologic Routine chromosomal study using conventional events must occur at the appropriate time and in technique showed 46,XX karyotype. FISH the correct sequence during ontogeny(8). The technique was applied on metaphase spreads male embryo's genetic sex is determined by its and interphase nuclei using whole chromosome chromosomal constituents the most important of painting probe showed presence of two normal which is the sex determining gene or testes size X chromosomes. To exclude X/Y or determining factor (TDF) on the Y chromosome. Y/autosomes translocation, Quint-Essential Y Male gonadal sex or testes formations are specific for Yp11.2 locus and X/Y cocktail probe subsequently thought to be determined by this for X cen/Yq12) were applied on metaphase gene and by other secondary pathways. spreads and interphase nuclei and revealed Since the 1950's it has been known that sex in absence of Y materials in this patient. man and in other mammals, is determined by the Chromosomal study and FISH technique were presence or absence of the Y chromosome.(9) It applied to his parents and sisters and revealed was believed that one or more genes on the Y no phenotypic/genotypic discrepancy. DNA study chromosome induce the undifferentiated gonad revealed the absence of both SRY gene and Y to become a testes, although the subsequent sex centromeric sequence (Figure 2A, B). differentiation is under the hormonal control of the testes. Case report II: The concept that genes located on the Y The proband is a Kuwaiti male aged 35 years old chromosome play an essential role in human sex who is married to non-consanguineous determination is stressed by the existence of phenotypically normal wife. He attended the various sex chromosomal aberrations. These centre because of infertility since 6 years. He conditions include, the 46,XX male syndrome has 5 married sibs of both sexes who have true hermaphroditism, 46,XY gonadal dysgenesis normal children. Pedigree study was irrelevant. and various Y chromosomal aberrations that On examination his weight was 75.5 kg, height results in female development.(2) XX male 179 cm, occipito-frontal circumference 54 cm, syndrome is a rare disorder with an estimated hand length 19 cm, palm length 10.5 cm and incidence varying from 1:9000 to 1:20000/ middle finger 8.5 cm. He has no craniofacial newborn males(2,10,11). The diagnosis is most dysmorphia. External genitalia examination often made in the post pubertal period. revealed small penis, small descended testes, Among prenatally diagnosed sample the incidence male distributed pubic, axillary and facial hair. of XX males was found to be 1.73% (2/112). Gynecomastia was detected. Hormonal profile Phenotypically two distinct subgroups have been showed the following results. SHBG 25.9 nmol/L, recognized among the subjects with 46,XX sex testosterone 3.99 nmol/L, LH 22.5 nmol/L, FSH reversal. Most affected individuals have normal 38.9 nmol/L, E2 89.84 nmol/L and TSH normal. male external genitalia, small azoospermic testes, Semen analysis showed azoospermia. but about 10% have hypospadias cryptorchidism Antimicrosal antibodies were negative while and a more prominent gynecomastia especially in antithyroglobulin antibodies were positive, anti these with Y (-ve) XX males.(4,12-13) Most normal DNA -ve and positive ANA. Chromosomal study appearance subjects with 46,XX maleness using peripheral blood and trypsin G banding contained sequences for SRY. By contrast, most Alex J Pediatr, 13 (2), July 1999 328 46,XX males with abnormal genitalia lack present but was not detected or the mechanism of sequences for SRY.(14) Similarly, almost all subjects maleness was Y independent. with 46,XX true hermaphroditism lack sequences for In the present report the probands had 46,XX sex SRY in genomic DNA. (15-16) reversal with positive SRY gene in case II while absence of SRY gene was found in case I. In case Many cases of XX maleness have been reported in I we failed to detect SRY sequence in genomic DNA literature.(17-20) Only less than 25% of the cases by PCR amplification. In addition he lacked proved to have X-Y translocation.(20-22) Other causes sequences for the Y centromere excluding the could be attributed as XX maleness such as loss of possibility of low-grade mosaicism with a Y bearing Y chromosome (originally was XXY Klinefelter cell line. Individuals with 46,XX sex reversal who syndrome), XX maleness due to Y-autosome