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The gene SRY (sex determining region of the Y), located at the distal region of the short arm of the Y chromosome, is necessary for male sex determination in mammals. SRY initiates the cascade of steps necessary to form a testis from an undifferentiated gonad. The SRY gene encodes an HMG (High Mobility Group) protein which may act as a transcription factor by binding to double stranded DNA and then bending the DNA. Mutations in SRY have been identified in some subjects with 46,XY pure gonadal dysgenesis. However the role for other autosomal and X-linked genes in testis determination is evident by the presence of a normal SRY gene in the majority of females with 46,XY pure gonadal dysgenesis and the lack of SRY in a minority of males with 46,XY maleness.

We previously reported a subject with 46,XX true hermaphroditism who had a 46,X,del(X) karyotype and Y-chromosomal sequences in genomic DNA. We hypothesized that the Y-chromosomal sequences were translocated to the deleted X chromosome and that the incomplete testis determination of this individual was the result of inactivation of the translocated X chromosome. In situ hybridization studies demonstrated that the Y-chromosomal sequences were located on the distal portion of the short arm of the deleted X chromosome. Investigation of the replication of the X chromosome, using a modified R-banding technique and localization of Y-chromosomal sequences by in situ hybridization, showed that the translocated X chromosome was late replicating in all 100 EBV-transformed lymphoblasts that were examined. By contrast, when cells from a subject with 46,XX maleness were studied, the translocated X chromosome was late replicating in only 21 of 47 cells. As the late-replicating X chromosome is presumed to be the inactive X chromosome, selection of cells in which the Y-bearing X chromosome has been inactivated may play a role in the incomplete testis determination in subjects with "Y-positive" 46,XX true hermaphroditism.

The condition of 46,XX maleness is characterized by testicular development in subjects who have two X chromosomes but who lack a normal Y chromosome. Several etiologies have been proposed to explain 46,XX maleness: 1) translocation of the testis-determining factor from the Y to the X chromosome, 2) mutation in an autosomal or X chromosome gene which permits testicular determination in the absence of TDF, and 3) undetected mosaicism with a Y-bearing cell line. We evaluated 10 affected subjects who were ascertained for different reasons and who had several distinct phenotypes. Six subjects had inherited sequences from the short arm of the Y chromosome including the sex-determining region Y gene (SRY). Five of the subjects were pubertal at the time of evaluation and had a phenotype similar to that of Klinefelter syndrome with evidence of Sertoli cell and Leydig cell dysfunction. One subject had evidence from Southern blot analysis and in situ hybridization for the presence of an intact Y chromosome in approximately 1% of cells. Three subjects lacked Y sequences by Southern blot analysis and by polymerase chain reaction amplification of SRY. These subjects were ascertained in the newborn period because of congenital anomalies. One had multiple anomalies including cardiac abnormalities; one had cardiac anomalies alone; and one had ambiguous genitalia. Our data confirm the genetic heterogeneity of 46,XX maleness, in which some subjects have SRY while other subjects lack it. In addition, there is phenotypic heterogeneity among subjects who lack SRY suggesting that there is also genetic heterogeneity within this subgroup.

We evaluated a patient with partial gonadal dysgenesis including a right dysgenetic testis and a left streak gonad with rudimentary fallopian tube and uterus. She had ambiguous external genitalia and was raised female. Although her height is normal (25th centile at age 12 years), she has some findings of Ullrich-Turner syndrome. Her karyotype was reported to be 46,X,+marker; subsequent molecular investigations showed the marker to be the short arm of the Y chromosome. Genomic DNA, isolated from leukocytes of the patient and her father, was digested with a variety of restriction endonucleases and subjected to Southern blot analysis. A positive hybridization signal was obtained with probes for the short arm of the Y chromosome (pRsY0.55, SRY, ZFY, 47Z, pY-190, and YC-2) in DNA from the patient, indicating the presence of most if not all of the short arm, while long arm probes (HinfA and pY3.4) indicated that at least 75% of the long arm of the Y chromosome was missing. The gene responsible for testicular determination (TDF) is on the distal portion of the short arm of the Y chromosome; Yq has no known influence on sex determination. Hence, the deletion of the long arm of the Y chromosome cannot explain the gonadal dysgenesis in this patient. One explanation for the gonadal dysgenesis and Ullrich-Turner phenotype in the patient could be undetected 45,X/46,X,+marY mosaicism but no such mosaicism was observed in peripheral lymphocytes. Several investigators have suggested the presence of an "anti-Turner" gene near TDF. Hence it is possible that the clinical phenotype in our patient results from a Y chromosomal defect in sequences flanking TDF, which reduces the function of both TDF and the "anti-Turner" genes.

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.