Linkage analysis of a kindred with inherited 46,XY partial gonadal dysgenesis.

We have reported a kindred in which 46,XY gonadal dysgenesis was inherited in an X-linked (or autosomal dominant sex-limited) manner and in which affected subjects did not have a large duplication of the short arm of the X-chromosome. In the present study we used linkage and sequence analyses to test the role of X-linked and various autosomal genes in the etiology of the familial 46,XY partial gonadal dysgenesis. For analysis of X-linkage, 28 microsatellite polymorphisms and 1 restriction fragment length polymorphism were studied. The genotypes of informative family members were determined at each locus, and data were analyzed. Despite the large number of loci tested, our studies did not establish linkage between the trait and an X-chromosomal locus. With respect to the study of autosomal genes, linkage analysis using a polymorphism within the 3'-untranslated region of the WT1 gene excluded involvement of WT-1 in the etiology of the abnormal gonadal differentiation of the family in this study. Similarly, linkage analysis using four microsatellites on the distal short arm of chromosome 9 was not consistent with linkage. Linkage analysis of a locus close to the SOX9 gene as well as analysis of the coding region of the SOX9 gene suggested that this gene was not associated with the trait in the affected subjects we studied. Our data suggest the role of an autosomal gene in the abnormal gonadal differentiation in the family in the study, but do not formally exclude the role of an X-chromosome gene.

The role of SRY in mammalian sex determination.

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.

Loss of sequences 3' to the testis-determining gene, SRY, including the Y pseudoautosomal boundary associated with partial testicular determination.

The condition termed 46,XY complete gonadal dysgenesis is characterized by a completely female phenotype and streak gonads. In contrast, subjects with 46,XY partial gonadal dysgenesis and those with embryonic testicular regression sequence usually present ambiguous genitalia and a mix of Müllerian and Wolffian structures. In 46,XY partial gonadal dysgenesis gonadal histology shows evidence of incomplete testis determination. In 46,XY embryonic testicular regression sequence there is lack of gonadal tissue on both sides. Various lines of evidence suggest that embryonic testicular regression sequence is a variant form of 46,XY gonadal dysgenesis. The sex-determining region Y chromosome gene (SRY) encodes sequences for the testis-determining factor. To date germ-line mutations in SRY have been reported in approximately 20% of subjects with 46,XY complete gonadal dysgenesis. However, no germ-line mutations of SRY have been reported in subjects with the partial forms. We studied 20 subjects who presented either 46,XY partial gonadal dysgenesis or 46,XY embryonic testicular regression sequence. We examined the SRY gene and the minimum region of Y-specific DNA known to confer a male phenotype. The SRY-open reading frame (ORF) was normal in all subjects. However a de novo interstitial deletion 3' to the SRY-ORF was found in one subject. Although it is possible that the deletion was unrelated to the subject's phenotype, we propose that the deletion was responsible for the abnormal gonadal development by diminishing expression of SRY. We suggest that the deletion resulted either in the loss of sequences necessary for normal SRY expression or in a position effect that altered SRY expression. This case provides further evidence that deletions of the Y chromosome outside the SRY-ORF can result in either complete or incomplete sex reversal.

Combined Leydig cell and Sertoli cell dysfunction in 46,XX males lacking the sex determining region Y gene.

We have evaluated 3 individuals with a rare form of 46,XX sex reversal. All of them had ambiguous external genitalia and mixed wolffian and müllerian structures, indicating both Leydig cell and Sertoli cell dysfunction, similar to that of patients with true hermaphroditism. However, gonadal tissue was not ovotesticular but testicular with varying degrees of dysgenesis. SRY sequences were absent in genomic DNA from peripheral leukocytes in all 3 subjects. Y centromere sequences were also absent, indicating that testis development did not occur because of a low level mosaicism of Y bearing cells. The subjects in this report demonstrate that there is a continuum in the extent of testis determination in SRY-negative 46,XX sex reversal, ranging from nearly normal to minimal testicular development.

Nonrandom inactivation of the Y-bearing X chromosome in a 46,XX individual: evidence for the etiology of 46,XX true hermaphroditism.

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.

Embryonic testicular regression sequence: a part of the clinical spectrum of 46,XY gonadal dysgenesis.

We report on a group of 9 subjects who had a 46,XY karyotype, ambiguous genitalia, abnormalities of sexual duct formation, and lack of gonadal tissue on one or both sides. This is sometimes referred to as "embryonic testicular regression." Previous investigators have suggested that this condition results from loss of testes at a critical stage in development. We examined the possibility that the "embryonic testicular regression" is part of the clinical spectrum of 46,XY gonadal dysgenesis. Four subjects totally lacked gonadal tissue, three of them having ambiguous genitalia, and one a micropenis. The development of incongruous sexual ducts (presence of Müllerian ducts in the subject with micropenis, and absence of Müllerian and Wolffian ducts in two subjects with ambiguous genitalia) suggests that the embryonic gonads were intrinsically functionally abnormal before their disappearance. Five subjects had unilateral gonadal tissue, ambiguous genitalia, and a mix of Wolffian and Müllerian structures. The development of incongruous sexual ducts in 3 of them, the presence of ambiguous external genitalia in 5, and the presence of abnormal gonadal histology in 2 patients all indicate an underlying abnormality of gonadal differentiation in these subjects. The occurrence of testicular regression in several subjects in the family of one patient suggests a genetic basis for the condition. The presence of multiple congenital anomalies in other subjects in our study suggests either a mutation in a single gene that functions in several developmental pathways, or a defect of multiple genes that might be the result of a chromosome deletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Report of a kindred with X-linked (or autosomal dominant sex-limited) 46,XY partial gonadal dysgenesis.

The condition termed 46,XY complete gonadal dysgenesis is characterized by the lack of testicular determination with resulting streak gonads, normal Mullerian structures, and female external genitalia. In the partial form, there is incomplete testicular determination with a wide range in the degree of ambiguous genitalia and sexual duct development. We evaluated a kindred in which a partial form of 46,XY gonadal dysgenesis occurred in four subjects from two generations. Pedigree analysis indicated an X-linked or possibly an autosomal sex-limited mode of inheritance. All affected subjects were ascertained because of ambiguous genitalia with minimal virilization. At 10 days of age, the proband had a subnormal plasma level of testosterone, and at 4 months, there was no rise in plasma T after stimulation with hCG. At laparotomy, a dysgenetic gonad was found on the right side, but no gonad was found on the left side. A vas deferens was present on the right, indicating the presence of functional Leydig cells early in fetal life. In the other affected subjects, gonadal tissue was also limited to one side of the abdomen and showed poorly developed seminiferous tubules. The sex-determining region Y gene, which encodes the testis-determining factor, was present and unaltered in the genomic DNA of all affected subjects. Duplication of the distal short arm of the X-chromosome has been associated with 46,XY complete gonadal dysgenesis in some patients. In our studies, Southern blot analysis revealed that sequences of the distal short arm of the X-chromosome (DXS9 to DXS84) were present in single copy, excluding a large duplication in this area of the X. Several kindreds with familial 46,XY complete gonadal dysgenesis have been reported; five of them had evidence of an X-linked mode of inheritance. Our study of a kindred with 46,XY partial gonadal dysgenesis further supports the role of an X chromosome gene in testicular determination. Evidence of some fetal Leydig cell function in the affected subjects of our report suggests that mutations of the putative X-chromosome gene can result in a partial as well as complete defect in testicular determination.

The role of the sex-determining region Y gene in the etiology of 46,XX maleness.

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.

Partial gonadal dysgenesis in a patient with a marker Y chromosome.

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

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.

Clinical and pathologic spectrum of 46,XY gonadal dysgenesis: its relevance to the understanding of sex differentiation.

The condition termed "46,XY gonadal dysgenesis" is characterized by a 46,XY karyotype and incomplete testicular determination. It is likely the result of a mutation in the gene for the testicular determination factor or in another gene involved in the early stages of testicular differentiation. In view of the present interest in the identification of gene(s) initiating the differentiation of the embryonic gonads into testes, we have reviewed the phenotype of 15 patients with 46,XY gonadal dysgenesis to use this information for future molecular studies. Seven patients presented a complete form, 46,XY pure gonadal dysgenesis, including streak gonads, normal Müllerian structures, and normal female external genitalia. The structure of the streak gonads in these patients presented some variation. Eight patients presented an incomplete form, 46,XY partial gonadal dysgenesis, with ambiguous external genitalia and partial development of Müllerian and Wolffian structures. Among them, 3 had bilateral dysgenetic testes, and 4 had a streak gonad on one side with a contralateral dysgenetic testis. The streak gonads showed ovarian stroma with occasional primitive sex cords devoid of germ cells. However, a primordial follicle was observed in 1 streak gonad. The dysgenetic testes showed disorganized seminiferous tubules and ovarian stroma. In some patients, the ovarian stroma was intermixed with testicular tissue, while in others, distinct ovarian and testicular portions were present. In 1 patient, the dysgenetic testis contained a focus of well-differentiated ovarian tissue with primordial follicles. Our observations support the hypothesis that streak gonads in 46,XY pure gonadal dysgenesis arise from fetal ovaries and that dysgenetic testes in the partial form in 46,XY partial gonadal dysgenesis develop from ovotestis.(ABSTRACT TRUNCATED AT 250 WORDS)