Am J Hum Genet 31:458-468, 1979
Sexual and Somatic Determinants of the Human Y Chromosome: Studies in a 46,XYp- Phenotypic Female RON G. ROSENFELD,1 LUIGI LUZZATTI,I RAYMOND L. HINTZ,' ORLANDO J. MILLER,2 GLORIA C. Koo,3 STEPHEN S. WACHTEL3'4
SUMMARY A case of a 46,XYp - phenotypic female provided an opportunity to evaluate both sexual and somatic determinants of the Y chromosome. The patient had multiple stigmata of Turner syndrome, but normal stature. Laparotomy revealed a normal uterus and tubes, with 1.5 cm undifferentiated gonads. Serological tests for H-Y antigen (ostensibly the product of Y-chromosomal testis-determining genes) indicated absence of the H-Y+ phenotype normally associated with the intact Y chromosome. We conclude that genes exist on the short arm of the human Y chromosome which both suppress some of the somatic stigmata of Turner syndrome and determine normal expression of H-Y antigen and testicular differentiation of the primitive gonad. Our data are consistent with the view that H-Y genes comprise a family of testis-determinants, and that loss of a critical moiety is inconsistent with normal development of the male gonad.
INTRODUCTION
Structural abnormalities of the sex chromosomes provide valuable insight into the location of both gonadal and somatic determinants. This has been true particularly for Received September 28, 1978; revised January 17, 1979. This study was supported in part by research grants from the National Foundation-March of Dimes, grants HD10065, HD 00171, CA 08748, and GM 22966 from the National Institutes of Health, and grant FRA 167 from the American Cancer Society. It was presented in part at the meeting of the American Pediatric Society-Society for Pediatric Research, New York, NY, April 1978. 1 Department of Pediatrics, Stanford University Medical Center, Stanford, CA 94305. 'Departments of Human Genetics and Development, and Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.. Memorial Sloan-Kettering Cancer Center, New York, NY 10021. 4Departments of Pediatrics, New York Hospital-Cornell Medical Center, New York, NY 10021. © 1979 by the American Society of Human Genetics. 0002-9297n9/3104-0008$01.15
458
DETERMINANTS OF THE Y CHROMOSOME
459
the X chromosome, where extensive phenotypic-karyotypic correlations have been employed in the study of gonadal dysgenesis and Turner stigmata [1, 2]. Similar evaluation of the Y chromosome has been limited by the relative scarcity of morphological abnormalities unassociated with mosaicism, and by the inability to satisfactorily characterize Yp and proximal Yq by current banding techniques. Consequently, precise location of the gene(s) responsible for testicular differentiation remains controversial, and the presence of somatic determinants on the human Y chromosome is still speculative. Recent reports have established that a genetic locus on the Y chromosome controls expression of H-Y antigen [3, 4], a cell surface component responsible for rejection of male skin grafts by syngeneic females [5, 6]. H-Y antigen has been detected in the heterogametic sex (XY) of all vertebrates tested [7]. Accordingly, it has been proposed that H-Y antigen is the product of a phylogenetically conserved, Y-linked gene(s) that directs testicular differentiation of the primordial mammalian gonad [8-10]. Localization of the H-Y gene(s) thus becomes important to an understanding of the relationship between structural abnormalities of the Y chromosome and the various abnormalities of sexual development. A short arm deletion of the Y chromosome (46,XYp- ) in a phenotypic female with Turner stigmata provided us with an opportunity to localize genetic determinants of H-Y antigen and testicular differentiation. CASE REPORT
The patient was born at Stanford University Medical Center in 1963 to a 28-year-old primigravida. Pregnancy and delivery were normal. At birth, the infant weighed 3,500 grams, had marked lymphedema of the extremities, increased nuchal skin folds, and normal female external genitalia (figs. 1 and 2). Buccal smears were consistently negative, and initial nonbanded karyotypes from peripheral lymphocytes (orcein stain) were interpreted as 46,XY. Karyotypes of both parents revealed no abnormalities, and Xg(a) typing was consistent with maternal origin of the patient's X chromosome. Laparotomy performed at 3 months revealed a normal uterus and fallopian tubes, and bilateral 1.5 cm gonads, consisting of fibrous stroma with focal aggregations of undifferentiated cells arranged either in cords or circumscribed clusters scattered throughout the stroma (fig. 3). No distinct primordial follicles or seminiferous tubules could be identified. Karyotypic analysis of cells cultured from fascia and one gonad were again interpreted as 46,XY (orcein).
FIG. 1..-Lymphedema of the lower extremities at. birth
460
ROSENFELD ET AL.
FIG. 2. -Increased nuchal skin folds at birth
The patient was re-evaluated at 9 years (fig. 4). She was a phenotypic female with a height at the 25th percentile. External genitalia showed no evidence of masculinization. The neck was characterized by mild webbing and a low posterior hairline. Additional features included cubitus valgus, a broad chest, pigmented nevi, a high-arched palate, and clinodactyly of the fifth digits, with shortening of the middle phalanx but normal metacarpals. MATERIALS AND METHODS
Cytogenetic Studies Initial karyotypes performed in infancy employed conventional acetic orcein staining. Karyotypes of the patient, at 9 years, and her father were done with standard Q- and C-banding. Endocrine Studies Plasma testosterone and estradiol were measured by radioimmunoassay before and after stimulation with human chorionic gonadotropin (2,000 U I.M. q.o.d. x 6 doses) [11, 12]. Assay of plasma FSH and LH was performed by a modification of the radioimmunoassay described by Weinstein and Reitz [13].
FIG. 3. -Appearance of gonadal stroma at 3 months
DETERMINANTS OF THE Y CHROMOSOME
461
FIG. 4. -Patient at 9 years
Serological Studies Expression of H-Y antigen was determined by measuring the ability of patient and control cells to absorb H-Y antibodies from the serum of male-grafted female mice in three assays: (1) the sperm cytotoxicity test [14], (2) the mixed hemadsorption-hybrid antibody (MHA, HA) test [15, 16], and (3) the newly devised protein-A-sheep red blood cell (PA-SRBC) test [17], which uses antibodies of the IgG class only. For each assay, selected pools of H-Y antisera were subdivided into four parts: one part was not absorbed, and the other three were absorbed with cells from normal males, normal females, and the patient, respectively. Positive absorption, indicating the presence of H-Y antigen on absorbing cells, was manifested as a fall in cytotoxic titer of the antiserum (cytotoxicity test) or as a decrease in rosette formation (MHA, HA and PA-SRBC tests), when compared with values obtained after absorption with normal female cells.
FIG. 5. -Q-banding at 9 years, revealing a deletion of the short arm of the Y chromosome.
462
ROSENFELD ET AL.
FIG. 6.-Initial nonbanded chromosome studies at birth showing deletion of Yp. Father has a normal Y Chromosome. RESULTS
Cytogenetic Studies Initial nonbanded chromosome studies from lymphocyte cultures and gonadal biopsy were interpreted as 46,XY. Cytogenetic studies were repeated at 9 years employing Qand C-banding methods. Evaluation of metaphases from 30 peripheral lymphocytes and 50 cultured gonadal fibroblasts indicated a small acrocentric chromosome with bright fluorescence of the terminal long arms, a well-defined centromere, and a clear deletion of the short arms (fig. 5). A morphologically normal short arm could not be detected on any of the patient's Y chromosomes. The autosomes and X chromosome exhibited normal banding. A review of the initial nonbanded karyotypes confirmed the deletion of Yp (fig. 6), and the patient was assigned.a 46,XYp- karyotype. Chromosome analysis of the father revealed a 46,XY karyotype with a morphologically normal Y chromosome (figs. 5 and 6). Endocrine Studies The results of stimulation with chorionic gonadotropin are summarized in table 1. FSH and LH levels were elevated, compatible with values seen in patients with gonadal dysgenesis [18] or anorchia [19]. Baseline levels of testosterone and estradiol were low, consistent with the patient's prepuberal status, but failed to show the expected rise following a two-week course of HCG [20, 21]. Serological Studies (tables 1-3) Presence of H-Y antigen on peripheral leukocytes was determined by absorption in the sperm cytotoxicity test. The normative data in table 2 indicate a significant TABLE I HCG STIMULATION Normal male prepuberal
Normal prepuberal Honnone
FSH (mIU/ml) .................. LH (mIU/ml) ................... Testosterone (ng/dl) ............. Estradiol (pg/ml) ................
NOTE.-Method: HCG 2000U I.M. q.o.d.
Baseline
68 24 5
Sexual and Somatic Determinants of the Human Y Chromosome: Studies in a 46,XYp- Phenotypic Female RON G. ROSENFELD,1 LUIGI LUZZATTI,I RAYMOND L. HINTZ,' ORLANDO J. MILLER,2 GLORIA C. Koo,3 STEPHEN S. WACHTEL3'4
SUMMARY A case of a 46,XYp - phenotypic female provided an opportunity to evaluate both sexual and somatic determinants of the Y chromosome. The patient had multiple stigmata of Turner syndrome, but normal stature. Laparotomy revealed a normal uterus and tubes, with 1.5 cm undifferentiated gonads. Serological tests for H-Y antigen (ostensibly the product of Y-chromosomal testis-determining genes) indicated absence of the H-Y+ phenotype normally associated with the intact Y chromosome. We conclude that genes exist on the short arm of the human Y chromosome which both suppress some of the somatic stigmata of Turner syndrome and determine normal expression of H-Y antigen and testicular differentiation of the primitive gonad. Our data are consistent with the view that H-Y genes comprise a family of testis-determinants, and that loss of a critical moiety is inconsistent with normal development of the male gonad.
INTRODUCTION
Structural abnormalities of the sex chromosomes provide valuable insight into the location of both gonadal and somatic determinants. This has been true particularly for Received September 28, 1978; revised January 17, 1979. This study was supported in part by research grants from the National Foundation-March of Dimes, grants HD10065, HD 00171, CA 08748, and GM 22966 from the National Institutes of Health, and grant FRA 167 from the American Cancer Society. It was presented in part at the meeting of the American Pediatric Society-Society for Pediatric Research, New York, NY, April 1978. 1 Department of Pediatrics, Stanford University Medical Center, Stanford, CA 94305. 'Departments of Human Genetics and Development, and Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.. Memorial Sloan-Kettering Cancer Center, New York, NY 10021. 4Departments of Pediatrics, New York Hospital-Cornell Medical Center, New York, NY 10021. © 1979 by the American Society of Human Genetics. 0002-9297n9/3104-0008$01.15
458
DETERMINANTS OF THE Y CHROMOSOME
459
the X chromosome, where extensive phenotypic-karyotypic correlations have been employed in the study of gonadal dysgenesis and Turner stigmata [1, 2]. Similar evaluation of the Y chromosome has been limited by the relative scarcity of morphological abnormalities unassociated with mosaicism, and by the inability to satisfactorily characterize Yp and proximal Yq by current banding techniques. Consequently, precise location of the gene(s) responsible for testicular differentiation remains controversial, and the presence of somatic determinants on the human Y chromosome is still speculative. Recent reports have established that a genetic locus on the Y chromosome controls expression of H-Y antigen [3, 4], a cell surface component responsible for rejection of male skin grafts by syngeneic females [5, 6]. H-Y antigen has been detected in the heterogametic sex (XY) of all vertebrates tested [7]. Accordingly, it has been proposed that H-Y antigen is the product of a phylogenetically conserved, Y-linked gene(s) that directs testicular differentiation of the primordial mammalian gonad [8-10]. Localization of the H-Y gene(s) thus becomes important to an understanding of the relationship between structural abnormalities of the Y chromosome and the various abnormalities of sexual development. A short arm deletion of the Y chromosome (46,XYp- ) in a phenotypic female with Turner stigmata provided us with an opportunity to localize genetic determinants of H-Y antigen and testicular differentiation. CASE REPORT
The patient was born at Stanford University Medical Center in 1963 to a 28-year-old primigravida. Pregnancy and delivery were normal. At birth, the infant weighed 3,500 grams, had marked lymphedema of the extremities, increased nuchal skin folds, and normal female external genitalia (figs. 1 and 2). Buccal smears were consistently negative, and initial nonbanded karyotypes from peripheral lymphocytes (orcein stain) were interpreted as 46,XY. Karyotypes of both parents revealed no abnormalities, and Xg(a) typing was consistent with maternal origin of the patient's X chromosome. Laparotomy performed at 3 months revealed a normal uterus and fallopian tubes, and bilateral 1.5 cm gonads, consisting of fibrous stroma with focal aggregations of undifferentiated cells arranged either in cords or circumscribed clusters scattered throughout the stroma (fig. 3). No distinct primordial follicles or seminiferous tubules could be identified. Karyotypic analysis of cells cultured from fascia and one gonad were again interpreted as 46,XY (orcein).
FIG. 1..-Lymphedema of the lower extremities at. birth
460
ROSENFELD ET AL.
FIG. 2. -Increased nuchal skin folds at birth
The patient was re-evaluated at 9 years (fig. 4). She was a phenotypic female with a height at the 25th percentile. External genitalia showed no evidence of masculinization. The neck was characterized by mild webbing and a low posterior hairline. Additional features included cubitus valgus, a broad chest, pigmented nevi, a high-arched palate, and clinodactyly of the fifth digits, with shortening of the middle phalanx but normal metacarpals. MATERIALS AND METHODS
Cytogenetic Studies Initial karyotypes performed in infancy employed conventional acetic orcein staining. Karyotypes of the patient, at 9 years, and her father were done with standard Q- and C-banding. Endocrine Studies Plasma testosterone and estradiol were measured by radioimmunoassay before and after stimulation with human chorionic gonadotropin (2,000 U I.M. q.o.d. x 6 doses) [11, 12]. Assay of plasma FSH and LH was performed by a modification of the radioimmunoassay described by Weinstein and Reitz [13].
FIG. 3. -Appearance of gonadal stroma at 3 months
DETERMINANTS OF THE Y CHROMOSOME
461
FIG. 4. -Patient at 9 years
Serological Studies Expression of H-Y antigen was determined by measuring the ability of patient and control cells to absorb H-Y antibodies from the serum of male-grafted female mice in three assays: (1) the sperm cytotoxicity test [14], (2) the mixed hemadsorption-hybrid antibody (MHA, HA) test [15, 16], and (3) the newly devised protein-A-sheep red blood cell (PA-SRBC) test [17], which uses antibodies of the IgG class only. For each assay, selected pools of H-Y antisera were subdivided into four parts: one part was not absorbed, and the other three were absorbed with cells from normal males, normal females, and the patient, respectively. Positive absorption, indicating the presence of H-Y antigen on absorbing cells, was manifested as a fall in cytotoxic titer of the antiserum (cytotoxicity test) or as a decrease in rosette formation (MHA, HA and PA-SRBC tests), when compared with values obtained after absorption with normal female cells.
FIG. 5. -Q-banding at 9 years, revealing a deletion of the short arm of the Y chromosome.
462
ROSENFELD ET AL.
FIG. 6.-Initial nonbanded chromosome studies at birth showing deletion of Yp. Father has a normal Y Chromosome. RESULTS
Cytogenetic Studies Initial nonbanded chromosome studies from lymphocyte cultures and gonadal biopsy were interpreted as 46,XY. Cytogenetic studies were repeated at 9 years employing Qand C-banding methods. Evaluation of metaphases from 30 peripheral lymphocytes and 50 cultured gonadal fibroblasts indicated a small acrocentric chromosome with bright fluorescence of the terminal long arms, a well-defined centromere, and a clear deletion of the short arms (fig. 5). A morphologically normal short arm could not be detected on any of the patient's Y chromosomes. The autosomes and X chromosome exhibited normal banding. A review of the initial nonbanded karyotypes confirmed the deletion of Yp (fig. 6), and the patient was assigned.a 46,XYp- karyotype. Chromosome analysis of the father revealed a 46,XY karyotype with a morphologically normal Y chromosome (figs. 5 and 6). Endocrine Studies The results of stimulation with chorionic gonadotropin are summarized in table 1. FSH and LH levels were elevated, compatible with values seen in patients with gonadal dysgenesis [18] or anorchia [19]. Baseline levels of testosterone and estradiol were low, consistent with the patient's prepuberal status, but failed to show the expected rise following a two-week course of HCG [20, 21]. Serological Studies (tables 1-3) Presence of H-Y antigen on peripheral leukocytes was determined by absorption in the sperm cytotoxicity test. The normative data in table 2 indicate a significant TABLE I HCG STIMULATION Normal male prepuberal
Normal prepuberal Honnone
FSH (mIU/ml) .................. LH (mIU/ml) ................... Testosterone (ng/dl) ............. Estradiol (pg/ml) ................
NOTE.-Method: HCG 2000U I.M. q.o.d.
Baseline
68 24 5
