Molecular analysis in true hermaphroditism: demonstration of low-level hidden mosaicism for Y-derived sequences in 46,XX cases

True hermaphroditism (TH) is an unusual form of sex reversal, characterized by the development of testicular and ovarian tissue in the same subject. Approximately 60% of the patients have a 46,XX karyotype, 33% are mosaics with a second cell line containing a Y chromosome, while the remaining 7% are 46,XY. Molecular analyses have demonstrated that SRY is present in only 10% of TH with a 46,XX karyotype; therefore, in the remaining 90%, mutations at unknown X-linked or autosomal sex determining loci have been proposed as factors responsible for testicular development. True hermaphroditism presents considerable genetic heterogeneity with several molecular anomalies leading to the dual gonadal development as SRY point mutations or SRY hidden gonadal mosaicism. In order to identify genetic defects associated with subjects with the disease, we performed molecular analyses of the SRY gene in DNA from blood leukocytes and gonadal tissue in 12 true hermaphrodites with different karyotypes. Our results using PCR and FISH analyses reveal the presence of hidden mosaicism for SRY or other Y sequences in some patients with XX true hermaphroditism and confirms that mosaicism for SRY limited to the gonads is an alternative mechanism for testicular development in 46,XX true hermaphrodites.     

Investigation of the ZFY gene in XX true hermaphroditism and Swyer syndrome

Summary Four patients with 46,XX true hermaphroditism and one patient with 46,XY pure gonadal dysgenesis (Swyer syndrome) were analyzed with a Y chromosome-derived probe that detects a specific fragment on the short arm of the Y chromosome in the putative testicle-determining region and also a fragment on the short arm of the X chromosome. Normal males and females, an individual with Turner syndrome, and patients with various causes of anomalous gonadal differentiation accompanied by cytogenetically present Y chromosome were used as controls. The Y-specific fragment was not detected in any of the persons with 46,XX true hermaphroditism. However, this fragment was positive in the 46,XY female and in all Y-bearing patients. Cytogenetic and molecular absence of the ZFY sequence in 46,XX true hermaphrodites calls for explanations other than the classic embryogenie theory. The absence of testicular differentiation in the ZFY-positive XY female evidences functionally altered sex determination or, alternatively, defective gonadal receptors.     

The role of the sex-determining region of the Y chromosome (SRY) in the etiology of 46,XX true hermaphroditism

Summary The syndrome of 46,XX true hermaphroditism is a clinical condition in which both ovarian and testicular tissue are found in one individual. Both Mullerian and Wolffian structures are usually present, and external genitalia are often ambiguous. Two alternative mechanisms have been proposed to explain the development of testicular tissue in these subjects: (1) translocation of chromosomal material encoding the testicular determination factor (TDF) from the Y to the X chromosome or to an autosome, or (2) an autosomal dominant mutation that permits testicular determination in the absence of TDF. We have investigated five subjects with 46,XX true hermaphroditism. Four individuals had a normal 46,XX karyotype; one subject (307) had an apparent terminal deletion of the short arm of one X chromosome. Genomic DNA was isolated from these individuals and subjected to Southern blot analysis. Only subject 307 had Y chromosomal sequences that included the pseudoautosomal boundary, SRY (sex-determining region of Y), ZFY (Y gene encoding a zinc finger protein), and DXYS5 (an anonymous locus on the distal short arm of Y) but lacked sequences for DYZ5 (proximal short arm of Y) and for the long arm probes DYZ1 and DYZ2. The genomic DNA of the other four subjects lacked detectable Y chromosomal sequences when assayed either by Southern blotting or after polymerase chain reaction amplification. Our data demonstrate that 46,XX true hermaphroditism is a genetically heterogeneous condition, some subjects having TDF sequences but most not. The 46,XX subjects without SRY may have a mutation of an autosomal gene that permits testicular determination in the absence of TDF.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

The sole presence of the testis-determining region of the Y chromosome (SRY) in 46,XX patients is associated with phenotypic variability.

Four cases of XX patients with testis development are reported. The aim of this study was to describe their clinical features and to see if there was any relationship between phenotypes and the presence of Y material. Several human Y-derived sequences including the SRY probe were used to analyze the DNA of the patients. Yp material including the pseudo-autosomal region and SRY was detected. The cases reported in this study confirm that XX true hermaphrodites cannot be distinguished from XX males on the basis of their genotypes. There is no relationship between clinical and anatomical phenotypes and the presence of Y material. SRY does not warrant a complete and normal testis differentiation. Although similar in some features with Klinefelter's syndrome patients, XX males exhibit specific clinical manifestations due to the lack of Y-specific genes.     

A possible common origin of “Y-negative” human XX males and XX true hermaphrodites

Summary We have studied nine patients aged 1 month to 16 years with 46, XX karyotypes and testicular tissue. Some of these patients were followed through puberty. Phenotypically, two presented normal and seven abnormal external genitalia (AG). Among this latter group, four showed hypospadias and three true hermaphroditism (TH). The endocrine data were similar in all three groups: testosterone levels were within normal limits during puberty, decreasing in adulthood; gonadotrophin levels were above the control values at mid puberty. Histologies of the two sub groups of AG patients were identical up to 5 years of age and presented differences when compared with controls, regardless of the ovarian part of the ovotestis. However, in patients older than 8 years, germ cells disappeared and dysgenesis became obvious. In one patient, the ovarian zone of the gonad was detected only after complete serial sections of the removed gonad were examined. Southern blot analysis with Y-DNA probes displayed Y-specific material for the classic 46 XX males and a lack of such sequences for all patients with AG and TH. Based on these findings, we postulate that 46, XX males with AG and 46, XX TH may represent altenative manifestations of the same genetic defect. These data together with those concerning familial cases of 46, XX males with AG and 46, XX TH suggest an autosomally (or pseudoautosomally) determined mechanism.     

Polymorphic detection of a parthenogenetic maternal and double paternal contribution to a 46,XX/46,XY hermaphrodite.

True hermaphroditism in humans usually is associated with a 46,XX karyotype or with mosaicism in which admixtures of cells with an XX and an XY karyotype are seen. However, the mechanisms that cause such mosaicisms are poorly understood. To date, with rare exceptions, analyses of hermaphrodites have been limited mostly to cytogenetic investigations. In this report, we describe a 5-year-old patient with true hermaphroditism and a 46,XX/46,XY karyotype (ratio 38:12) in lymphocytes, suggesting involvement of two fertilization events. Microsatellite DNA polymorphisms distributed throughout the genome were analyzed, to investigate the origin of the cell lines concerned. The results are consistent with double paternal and single maternal genetic contributions. Possible mechanisms that would explain these findings are discussed. The most likely mechanism involves a single haploid ovum dividing parthenogenetically into two haploid ova, followed by double fertilization and fusion of the two zygotes into a single individual, at the early embryonic stage.     

Absence of Y-specific DNA sequences in human 46,XX true hermaphrodites and in 45,X mixed gonadal dysgenesis

Summary A search for Y-specific DNA sequences has been performed in a sample of seven 46,XX true hermaphrodites and one 45,X mixed gonadal dysgenesis case and compared with a sample of 11 XX males. Using six Y-specific DNA probes no hybridization signal was obtained in the hermaphrodite group; in contrast, all XX males gave a positive signal with at least one probe. This difference is statistically highly significant. We conclude that the aetiology of true hermaphroditism is different from that of the XX male syndrome. As all cases of the hermaphrodite group are positive for the serological sex-specific antigen (Sxs) it is concluded that this antigen can be present even in the absence of Y-specific DNA.     

Absence of the candidate male sex-determining gene dmrt1b(Y) of medaka from other fish species

Although the sex-determining genes are known in mammals, Drosophila, and C. elegans, little is known in other animals. Fishes are an attractive group of organisms for studying the evolution of sex determination because they show an amazing variety of mechanisms, ranging from environmental sex determination and different forms of hermaphroditism to classical sex chromosomal XX/XY or WZ/ZZ systems and modifications thereof. In the fish medaka, dmrt1b(Y) has recently been found to be the candidate male sex-determining gene. It is a duplicate of the autosomal dmrt1a gene, a gene acting in the sex determination/differentiation cascade of flies, worms, and mammals. Because in birds dmrt1 is located on the Z-chromosome, both findings led to the suggestion that dmrt1b(Y) is a "non-mammalian Sry" with an even more widespread distribution. However, although Sry was found to be the male sex-determining gene in the mouse and some other mammalian species, in some it is absent and has obviously been replaced by other genes that now fulfil the same function. We have asked if the same might be true of the dmrt1b(Y) gene. We find that the gene duplication generating dmrt1b(Y) occurred recently during the evolution of the genus Oryzias. The gene is absent from all other fish species studied. Therefore, it may not be the male-sex determining gene in all fishes.     

True hermaphroditism: a new case with complex mosaicism

True hermaphroditism is a very rare disorder of human sexual differentiation. In the medical literature, more than 450 cases are described, and about 250 true hermaphrodites have been subjected to chromosome studies. A 21-year-old "man" was examined because of genital and phenotypic abnormalities: clinical, surgical and laboratory investigations showed a true hermaphroditism, with a quadruple mosaicism 45,X/46,XX/46,XY/47,XXY. We believe that this is the first case in which this peculiar type of multiple mosaicism has been documented.     

Investigation of three XX males by cytogenetic and DNA analyses

Summary We report cytogenetic and DNA studies in three XX males. Two males seemed to have extra chromosomal material on the tip of one X chromosome. All three males were shown to have Y chromosome material as indicated by hybridization of Y-specific DNA probes to genomic DNA. One male was unusual in that as he showed the 15-kb fragment detected by pDP34 that is thought to map close to the Y centromere. It is suggested that this finding might point to an inversion on the Y chromosome.     

An extended long-range restriction map of the human sex-determining region on Yp, including ZFY, finds marked homology on Xp and no detectable Y sequences in an XX male.

We have used pulsed-field gel electrophoresis to study the short arm of the Y chromosome by using a pseudoautosomal probe (MIC2Y) and adjacent Y-specific sequences 27a and 47z (DSXY5) in XX males and XY females, in order to detect chromosomal breakpoints which may have given rise to these individuals. The preliminary published long-range restriction map was used as a basis for this study. Our data confirm the reported fragment sizes and resolve some discrepancies. In addition, the recently cloned ZFY locus, pDP1007, the putative sex-determining locus, has been used to extend this long-range restriction map on Yp. Thus far, the X and Y copy of this sequence appear to have conserved GC islands around this locus, since it is found on a 280-kb fragment in males and females by using SacII, BssHII, NarI, and NotI. Only two Y-specific sequences of 50 and 70 kb have been detected at the pulsed-field level by using SfiI and NaeI, respectively. No translocation breakpoints have been detected in any of the patients studied. One XX male, GM1889, however, does not have any of the Y-specific fragments detected using conventional or pulsed-field gel electrophoresis. This is one of the few typical XX males who therefore does not have the ZFY copy of the TDF clone. Since all the other XX males hybridized to 47z, which is centromeric to ZFY, a series of DNA loci that are centromeric to 47z need to be studied in order to detect chromosomal breakpoints.     

Sry-negative XX true hermaphroditism in a roe deer

A two-year-old roe deer was brought down in the course of a hunt in the north of Spain (Asturias). On physical examination the individual presented well-developed bared antlers, but surprisingly a female external genitalia. Several anatomical, histological and genetic analyses were performed in order to explain the observed phenotype. Necropsy evidenced ovary-like structures with follicles on the surface; histological analyses of testes evidenced positive immunolabel against testosterone in Leydig cells; genetic analyses showed that the sex of the individual was consistent with a female individual. PCR analysis failed to detect SRY sequences; no PIS deletion, which is responsible for XX sex-reversal in goats, was detected. On the basis of its presumptive normal female sexual karyotype (XX) and the presence of two functional abdominal bilateral testes and ovaries, the roe deer was finally diagnosed as possessing an XX hermaphroditism syndrome. However, as in many other cases, the specific reason for the occurrence of this case of hermaphroditism could not be determined.     

SRY-negative true hermaphrodites and an XX male in two generations of the same family

Two 46,XX true hermaphrodites and one XX male without genital ambiguities are reported. They coexist in two generations of the same pedigree, with paternal transmission and in the absence of SRY (sex-determining region, Y chromosome). These familial cases provide evidence to support the hypothesis that these disorders are alternative manifestations of the same genetic defect, probably an autosomal dominant mutation (with incomplete penetrance) or an X-linked mutation (limited by the presence of the Y chromosome).     

Familial true hermaphroditism: paternal and maternal transmission of true hermaphroditism (46,XX) and XX maleness in the absence of Y-chromosomal sequences

We report on 46,XX true hermaphroditism and 46,XX maleness coexisting in the same pedigree, with maternal as well as paternal transmission of the disorder. Molecular genetic analysis showed that both hermaphrodites as well as the 46,XX male were negative for Y-chromosomal sequences. Thus, this pedigree is highly informative and allows the following conclusions: first, the maternal as well as paternal transmission of the disorder allows the possibility of an autosomal dominant as well as an X-chromosomal dominant mode of inheritance; second, testicular determination in the absence of Y-specific sequences in familial 46,XX true hermaphrodites as well as in 46,XX males seems to be due to the varying expression of the same genetic defect; and third, there is incomplete penetrance of the defect.     

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein.     

Accidental X-Y recombination and the aetiology of XX males and true hermaphrodites

Accidental recombination between the differential segments of the X and Y chromosomes in man occasionally allows transfer of Y-linked sequences to the X chromosome leading to testis differentiation in so-called XX males. Loss of the same sequences by X-Y interchange allows female differentiation in a small proportion of individuals with XY gonadal dysgenesis. A candidate gene responsible for primary sex determination has recently been cloned from within this part of the Y chromosome by Page and his colleagues. The observation that a homologue of this gene is present on the short arm of the X chromosome and is subject to X-inactivation, raises the intriguing possibility that sex determination in man is a quantitative trait. Males have two active doses of the gonad determining gene, and females have one dose. This hypothesis has been tested in a series of XX males, XY females and XX true hermaphrodites by using a genomic probe, CMPXY1, obtained by probing a Y-specific DNA library with synthetic oligonucleotides based on the predicted amino-acid sequence of the sex-determining protein. The findings in most cases are consistent with the hypothesis of homologous gonad-determining genes, GDX and GDY, carried by the X and Y chromosomes respectively. It is postulated that in sporadic or familial XX true hermaphrodites one of the GDX loci escapes X-inactivation because of mutation or chromosomal rearrangement, resulting in mosaicism for testis and ovary-determining cell lines in somatic cells. Y-negative XX males belong to the same clinical spectrum as XX true hermaphrodites, and gonadal dysgenesis in some XY females may be due to sporadic or familial mutations of GDX.     

True hermaphroditism in a child with a chimeric XX/XY chromosome, a double erythrocyte population and an unusual Lewis (a+ b+) phenotype

A 2 years-old Korean girl is seen because of ambiguous external genitalia. Surgical exploration shows the right gonad to be an ovary and the left one to be an ovotestis, thus demonstrating a true hermaphroditism. Cytogenetic studies of peripheral lymphocytes reveal a mixture of 46,XX and 46,XY cells, with a predominant XX cell line. The patient's red cells are composed of two distinct populations differing in three genetically independent blood group systems. The ratios of the two cell lines in various tissues, especially among the cells secreting Lewis antigens, appear to be very different and suggest several hypothesis to explain the highly unusual red cell Lewis phenotype Le (a+ b+). We conclude to a dispermic chimera, however the adopted status of this child prevents any identification of the maternal or paternal contributions. Because of the physical aspect it was decided to remove the ovotestis, to repair the external genitalia and to bring up this child as a female.     

H-Y antigen and sexual dysgenesis in man

H-Y antigen is a surface component associated with the heterogametic sex of various species and supposed to induce testicular differentiation. Genes controlling directly or not the expression of H-Y antigen and testicular differentiation have been localized on Y as well as on X chromosome and even autosomal chromosome. However the genetical localization of the H-Y structural gene remains unknown. We analysed the expression of H-Y antigen in three types of sexual dysgenesis (males bearing XX caryotype, testicular feminization syndrome and one case of hermaphroditism) to clarify the function and the genetics of this antigen.     

Genetic analysis of 38XX males with genital ambiguities and true hermaphrodites in pigs

In pig, the frequency of intersexuality ranges from 0.1 to O.6%, depending on the breed. In a closed pig herd at INRA an intersex condition was observed in 0.75% of ‘females’. The present study describes 11 animals with a 38XX karyotype and the presence of testicular tissue. Phenotypically, all presented with abnormal external or/and internal genitalia. Southern blot analysis with Y-specific probes (SRY and ZFY) revealed the absence of Y material in all animals tested. By polymerase chain reaction (PCR) amplification, 10 of 11 intersex pigs lacked the SRY gene in gonad DNA. These data are compatible with an autosomally (or pseudoautosomally) determined mechanism. Moreover, analysis of familial cases seemed to indicate that 38XX male pseudohermaprodites and 38XX true hermaphrodites may represent alternative manifestations of the same genetic defect.