Familial XX chromosomal maleness does not arise from a Y chromosomal translocation

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Familial XX chromosomal maleness does not arise from a Y chromosomal translocation Harry Ostrer, MD, Gabriela Wright, MS, Mark Clayton, Bs, Nicos Skordis, MD, a n d Margaret H, MacGillivray, MD From the Departments of Pediatrics, Biochemistry and Molecular Biology, and Pathology, and the R, C. Philips Research and Education Unit, University of Florida College of Medicine, Gainesville, and the Division of Pediatric Endocrinology, Children's Hospital of Buffalo

To determine the mechanism for the coexistence of XX chromosomal maleness and true hermaphroditism in the same family, we performed cytogenetic and molecular genetic analyses, using DNA probes from the short arm of the Y chromosome. These studies excluded the following possible mechanisms: (4) an inherited, mitotically unstable Y chromosome that results in chromosomal mosaicism, (2) an inherited Y-to-X or Y-autosomal translocation, (3) recurrent Y-to-X translocation, and (4) incomplete inactivation of the X chromosomal homolog for the testicu!ar determining factor. We c o n c l u d e that the disorder of sexual differentiation observed in this family can be best explained by a dominant autosomal gene with variable expressivity. (J PEDIATR1989;114:977-

82)

The understanding of mammalian sexual development has been advanced by the analysis of individuals with aberrant gonadal and genital differentiation (see de la Chapell 1 for review). The region of the Y chromosome that is involved in the initial testicular induction event has been characterized in considerable detail by the application of DNA probe analysis to males with a 46,XX karyotype and to females with gonadal dysgenesis and Y chromosomal structural abnormalities,a3 Recently a gene from this region with the characteristics of a DNA binding protein has been proposed as the testicular-determining factor? How such a gene may operate remains an open question, but the observation that this gene has a homolog on the X chromosome suggests the possibility that, as for some invertebrates, mammalian sexual differentiation may be a gene dosage phenomenon?

Supported in part by a Basil O'Connor Starter Grant from the March of Dimes Birth Defects Foundation (No. 5-585) and by the State of Florida Department of Health and Human Services. Submitted for publication July 18, 1988; accepted Feb. 7, 1989. Reprint requests: Harry Ostrer, MD, Division of Genetics, Department of Pediatrics, University of Florida College of Medicine, Box J-296, Gainesville, FL 32610.

Recently, some of us reported a family with coexisting XX chromosomal maleness and true hermaphroditism? The pattern of inheritance was incompatible with either an inherited mosaicism, an inherited Y-to-X translocation, or an inherited X-linked gene. The latter two possibilities were excluded by the observation that some obligate carriers for such a translocation were phenotypic females with no signs of sexual ambiguity. By cytogenetic analysis, we did not demonstrate Y chromosomal mosaicism in blood and gonads from two XX-male brothers nor in blood DNA TDF

Deoxyribonucleicacid Testicular determining factor

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from their true hermaphrodite paternal uncle. From the combined pedigree and cytogenetic analysis, we concluded that these findings could be explained by activation of a dominant autosomal gene or by a Y-autosomal translocation. Although not considered at the time, the findings were also compatible with recurrent Y-to-X translocation, such as occurs in the sex-reversed mouse2 Such a mechanism may explain three related XX-males in another family who were found to have varying portions of the Y chromosome? The experiments described here were designed to ask 977

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The JournalofPediatrics June 1989

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Fig. I. A, Map of Y chromosomal DNA probes used in this study and their location relative to one another and to TDF. Distances shown on this map are not exact. Pseudoautosomal region of Y chromosome is exchanged between X and Y chromosomes during male meiosis. Frequency with which recombination has been observed between these pseudoautosomal loci and TDF is also shown. B, Inversion polymorphismof Y chromosome, resulting from breakpoints proximal to TDF and distal to 27a. This results in moving 27a adjacent to 47z and placing TDF just below pseudoautosomal region. two different questions. First, could the recurring XX maleness and true hermaphroditism be explained on the basis of an inherited Y-autosomal translocation? Second, could this be explained on the basis of a recurring Y-to-X translocation during male meiosis, such as occurs in the sex-reversed mouse? Both questions could be readily addressed with the use of DNA probes. METHODS Patients. DNA from two XX male brothers, their normal sister, and their parents were studied. Both brothers had chordee, hypospadias, and a bifid scrotum containing gonads. Biopsy of the gonads revealed them to be hypoplastic testes. Samples from other family members, including the grandparents and the XX hermaphrodite uncle, could not be obtained for DNA study. DNA from a normal male and a normal female with no known sexual dysfunction was included for comparison. Probes. DNA probes that mapped to the distal short arm of the human Y chromosome were used (Fig. 1, A). These probes included 47z, which is proximal to the

testicular determining factor, and 27a, which is distal. They also included pSG1, l13D, 601, and 29C1, probes that map to the pseudoautosomal region of X and Y chromosomes, the region that is exchanged between these chromosomes during male meiosis.911 Molecular techniques. DNA was prepared from venous blood, cut with restriction enzymes, electrophoresed, and blotted onto nylon m6mbranes ("Southern blotting") by means of standard techniques.!2 Restriction enzyme digestion was performed with either EcoRI or TaqI by the methods recommended by the manufacturer (Bethesda Research Laboratories, Gaithersburg, Md., or Boehringer Mannheim Diagnostics, Indianapolis, Ind.). DNA probes were radio!abeled by the primer extension method I3 and hybridized to the blots under conditions previously described.2.9,11,

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RESULTS To determine whether there was an inherited Y-autosomal translocation, we studied the family by means of probes 47z and 27a, which flank the testicular-determining

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Familial X X chromosomal maleness

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region on the short arm of the Y chromosome. The presence of Y-specific restriction fragments is diagnostic for this region of the chromosome. As shown in Figs. 2 and 3, Y-specific bands were seen only for the normal m a l e s - the father and the control subject. This excludes the possibility of an inherited Y-autosomal translocation with a breakpoint proximal to TDF. To determine whether there was a recurring Y-to-X transloeation, we studied these subjects by using probes from the pseudoautosomal region? Inversion polymorphisms of the Y chromosome have been reported to place T D F in a distal location on the chromosome, just proximal to the pseudoautosomal region (Fig. l, B). If such an inversion were present in this family, then the inverted Y chromosome may have become a target for recurrent Y-to-X interchange, with T D F being translocated onto the X chromosome. The results are indicated in the Table and in Figs. 4 and 5. Because the grandparents were not available for study, an arbitrary assignment of parental alleles was made on the basis of the band sizes that were observed in the autoradiograms. An example is shown for the probe 29C1, which maps to the subtelomerie region of the Y chromosome (Fig. 4). The father had major restriction fragments at 3.9, 3.6, 3.45, and 3.1 kb. Based on inheritance, these were divided into two alleles: allele A, 3.6, 3.45, and 3.1 kb; and allele B, 3.9 kb. (A single allele may comprise more than one band on an autoradiogram because this subtelomeric region of the sex chromosomes has had variable numbers of reduplications.) Similarly, the mother's alleles were divided into two groups: allele C, 4.4 kb; and allele D, 2.4 kb. Thus the children's alleles were identified as AC, AC, and BC. Similar assignments are shown for the probes l 1 3 D and 601. The parents were both heterozygous for probe 113D (father, 1, 3; mother, 1, 2) and homozygous for 601 (father, 1, 1; mother, 2, 2). On the basis of this analysis, we assigned coupling phases on the principle of maximum parsimony (i.e., that the fewest recombinations be required to account for the

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Fig. 2. Southern blot analysis of DNA from family with recurrent XX chromosomal maleness and from normal male and normal female. DNA was digested with restriction enzyme TaqI. Southern blot was hybridized with probe 47z. Presence of band at 4.3 kb is diagnostic for presence of that region of human Y chromosome. Lanes: a, normal female; b, mother (I-2); c, molecular weight markers; d, daughter (II-1); e, XX-male son (II-2);f XX-male son (II-3); g, father (I-l); and h, normal male.

results) (Fig. 5). From this analysis, the father's chromosomes were characterized as A 3 l and B l l (29C1, l13D, and 601) and the mother's as C22 and D12. Thus the children's chromosomes were determined to be A31 and C22 (II-1), A31 and C12 (II-2), and B l l and C22 (II-3), respectively. These assignments demonstrate that a recombinational event occurred between 29C1 and 113D during the maternal meiosis that resulted in II-2. In addition, when the inheritance of the pseudoautosomal region is compare d in the two X X chromosomal males, they are found to have inherited completely different paternal alleles. Thus, if their X X maleness arose as the result of an aberrant Y to X interchange, putting T D F onto the X

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Fig. 3. Southern blot analysis of DNA from family with recurrent chromosomal maleness and from normal male and normal female. DNA was digested with restriction enzyme Taql. Southern blot was hybridized with the probe 27a. Presence of band at 2.2 kb is diagnostic for presence of that region of human Y chromosome. Lanes: a, normal female; b, mother (I-2); c, daughter (II-l); d, molecular weight markers; e, XX-male son (II-2);f, XX-male son (II-3); g, father (I-1); and h, normal male.

chromosome, then a second recombinational event would have h a d to occur on this derived X chromosome for one of these males to restore the original X chromosomal alleles. T h e likelihood of two recombinational events has been previously d e m o n s t r a t e d to be very small. 15 A D N A probe for the testicular d e t e r m i n i n g gene itself was not available, so the possibility of a more complex translocation, involving only this gene, could not be excluded. T h e likelihood of such an event is extremely small. DISCUSSION As noted previously, X X chromosomal maleness a n d true h e r m a p h r o d i t i s m may be different phenotypic mani-

Fig. 4. Southern blot analysis of DNA from family with recurrent XX chromosomal maleness and from normal male and normal female. DNA was digested with restriction enzyme TaqI. Southern blot was hybridized with probe 29C1. Alleles were assigned as follows: A = 3.6, 3.45, and 3.1 kb; B = 3.9 kb; C = 4.4 kb; and D = 2.4 kb. Lanes: a, normal female; b, mother (I-2); c, daughter (II-1), d, molecular weight markers; e, XX-male son (II-2);f XX-male son (II-3); g, father (I-1); and h, normal male. Note that normal male and normal female have alleles that differ from those segregating in this family.

festations of the same genetic disorder, 6 which can be explained by genetic factors operating on the primordial cells o f the gonad to promote local sexual differentiation. One possible m e c h a n i s m is the presence of an unstable Y chromosome in the presence of two intact X chromosomes. T h e d o m i n a n t gene on the Y c h r o m o s o m e promotes testicular differentiation, and loss of t h a t chromosome results in ovarian differentiation. In this family, there

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appears to be no evidence for Y chromosomal material associated with testicular determination of XX maleness, as determined by either cytogenetic or molecular genetic techniques. A second mechanism is variable or incomplete X chromosomal inactivation. The recently identified candidate T D F gene was found to have a homolog on the X chromosome. Although not yet demonstrated, this Xlinked gene may be expressed and thus may be subject to inactivation. According to this hypothesis, in an undifferentiated gonad with two X chromosomes, only one copy is expressed, and it instructs the gonad to differentiate into an ovary. As with XY individuals, if two copies of the T D F gene are expressed, then the gonad will differentiate into a testis. If the X chromosomal inactivation mechanism is partial or incomplete, then the equivalent of two genes may be expressed into some cells and the equivalent of only one gene may be expressed in others. The result could be true hermaphroditism. The pattern of inheritance for such a mechanism would be X linked. This mechanism was excluded in this family by the daughter's inheriting the X chromosome that would contain such a mutation and yet not having sexual ambiguity. A "leaky" X chromosomal inactivation mechanism may explain other cases of XX true hermaphroditism, without requiring gene duplication, as was proposed in a recent theory? A third possibility is activation of a dominant autosomal gene acting in concert with a threshold phenomenon. As a result of stochastic gene expression, the level of the gene product may vary from one cell to another. Thus, in some cells with a higher level of gene expression, testicular differentiation may occur. In those cells with a lower level of expression, ovarian development may occur. For XX maleness to have occurred, all the precursor cells must have surpassed the threshold of testicular differentiation. For XX true hermaphroditism to have occurred, only some of the precursor cells would have had to surpass the threshold. Analysis of other pedigrees with recurrent XX maleness or with XX maleness coexisting with XX true hermaphroditism fits this model. 16 Models associated with Y-autosomal or Y-to-X transmission have been excluded from the results of this study, in that neither of the XX males had evidence for DNA markers flanking TDF. In addition, each of these individuals inherited different pseudoautosomal alleles, demonstrating different recombinational events. Furthermore, the phenotype is not compatible with Y-to-X or Yautosomal translocations. Both groups of individuals have a well-characterized phenotype, which includes short stature and small testes, without evidence of sexual ambiguity. 1 The sexual ambiguity in this family is not compatible with a Y chromosomal translocation? 6 Demonstrating a dominant autosomal gene in this family would prove to be very difficult. Although markers have

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Fig. 5. Pedigree for nuclear family with recurrent XX chromosomal maleness, demonstrating linkage phases for alleles in pseudoautosomal region. These loci are 29C1, l13D, and 601. Results are most compatible with a single recombination event during maternal meiosis that resulted in II-2. They demonstrate that the XX-male sons inherited different paternal pseudoautosomal alleles.

been demonstrated for all of the human chromosomes, 17 regions on some chromosomes have not been mapped. Without cytogenetic markers suggesting a possible location for such an autosomal gene, estimates by others suggest that in excess of 200 markers would have to be tested to find at least one that may demonstrate linkage. TM Proving linkage to a D N A marker with a high level of confidence would be difficult, given the size of this family. The observation that the T D F candidate gene encodes a D N A binding protein suggests that the product of this gene may be used as a probe to clone its own target. For the time being, efforts at understanding gonadal differentiation are best focused on genes on the sex chromosomes, which may explain many of the disorders of sexual differentiation that are observed in clinical practice. We thank Jean Weissenbach, Peter Goodfellow, and Howard Cooke for providing recombinant DNA probes. REFERENCES

1. De la Chapelle A. Genetic and molecular studies on 46,XX and 45,X males. Cold Spring Harbor Symp Quant Biol 1986;51:249. 2. Vergnaud G, Page DC, Simmler M-C, et al. A deletion map of the human Y chromosome based on DNA hybridization. Am J Hum Genet 1986;38:109. 3. Affara NA, Ferguson-Smith MA, Magenis RE, et al. Mapping the testis determinants by analysis of Y-specific sequences in males with apparent XX and XO karyotypes and

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females with XY karyotypes. Nucleic Acids Res 1987; 15:7325. Page DC, Mosher R, Simpson EM. The sex-determining region of the human Y chromosome encodes a finger protein. Cell 1987;51:1091. German J. Gonadal dimorphism explained as a dosage of a locus on the sex chromosomes, the gonad-differentiation locus (GDL). Am J Hum Genet 1988;42:44. Skordis NA, Stetka DG, MacGillivray MH, Greenberg SP. Familial 46,XX males coexisting with familial 46,XX true hermaphrodites in same pedigree. J PEDIATR 1987;l 10:244. Singh L, Jones KW. Sex reversal in the mouse (Mus musculus) is caused by a recurrent nonreciprocal crossover involving the X and an aberrant Y chromosome. Cell 1982; 28:205. Page DC, de la Chapelle A, Weissenbaeh J. Chromosome Y-specific DNA in related human XX males. Nature 1985;315:224. Cooke H J, Brown WRA, Rappold GA. Hypervariable telomeric sequences from the human sex chromosomes are pseudoautosomal. Nature 1985;317:687. Simmler M-C, Rouyer F, Vergnaud G, et al. Pseudoautosomal DNA sequences in the pairing region of human sex chromosomes. Nature 1985;317:692.

11. Goodfellow PJ, Darling SM, Thomas NS, Goodfel!ow PN. A pseudoautosomal gene in man. Science 1986;234:740. 12. Matteson KJ, Ostrer H, Chakravarti A, et al. A study of restriction fragment length polymorphism at the human alpha-l-antitrypsin locus. Hum Genet 1985;69:263. 13. Feinberg AP, Vogelstein B. Addendum: a technique for radiolabeling DNA restriction fragments to high specific activity. Ann Bioehem 1984;137:266. 14. Pritehard CA, Goodfellow P J, Goodfellow PN. Isolation of a sequence which maps close to human sex determining gene. Nucleic Acids Res 1987;15:6159. 15. Rouyer F, Simmler M-C, Johnsson C, et al. A gradient of sex linkage in the pseudoautosomal region of human sex chromosomes. Nature 1986;319:291. 16. De la Chapelle A. The Y chromosomal and autosomal testicular determining genes. Development 1987;101 (suppl):33. 17. Donis-Keller H, Green P, Helms C et al. A genetic linkage map of the human genome. Cell 1987;51:319. 18. Botstein D, White RL, Skolnick M, Davis RW. Construction of a linkage map in man using restriction fragment length polymorphism. Am J Hum Genet 1980;32:314.

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