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Departments of Human Genetics (B.K.J., M.J., E.V.), Pathology and Laboratory Medicine (S.N.), and Pediatrics (S.D.F., E.V.), University of California at Los Angeles, Los Angeles, California 90095-7088
Address all correspondence and requests for reprints to: Dr. Eric Vilain, Department of Human Genetics, University of California at Los Angeles, 695 Charles E. Young Drive South, Gonda Center, Room 6357A, Los Angeles, California 90095-7088. E-mail: . evilain{at}ucla.edu
Abstract
In humans, mutations in the testis-determining gene SRY result in XY sex reversal with pure gonadal dysgenesis (PGD). However, only about 10–15% of the cases of PGD can be explained by mutations within the SRY open reading frame, suggesting the existence of other sex-determining genes. Although SRY is known to bind and bend DNA, its target and mode of action remain elusive. Here, we describe a novel mutation in SRY at codon 127, resulting in a tyrosine (Y) to phenylalanine (F) substitution in the protein. This sequence variant was found not only in the XY female patient but also in her father, who is a phenotypically normal male. However, this Y127F variant was not found in the SRY sequences of 93 other randomly chosen males. This substitution affects a highly conserved tyrosine residue in the HMG box of SRY, in which two de novo mutations have been described previously in XY females with PGD. Furthermore, electromobility shift studies demonstrate that SRY protein harboring the Y127F variant is incapable of binding consensus SRY binding sites in vitro. Taken together, these data suggest that the Y127F variant is a novel mutation with functional consequences and not simply a polymorphism. The allelic variant of SRY transmitted in this family and shared by both a phenotypic female (proband) and a phenotypic male (proband’s father) emphasizes the importance of modifier genes in the sex determination pathway.
IN MAMMALIAN MALES, normal testicular development depends on the action of SRY, encoded by a gene located on the Y chromosome (1). The central motif of this 204 amino acid protein is an HMG box domain of 79 residues in length (Fig. 1
). Through its HMG box, SRY has been shown to bind and bend DNA in a sequence-specific manner (2). Like the other members of the SOX family of proteins of which it is the founding member, SRY is thought to function as a transcription factor.
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), shared between the father and one or more of his children (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). In four of these nine cases, paternal mosaicism for the mutant SRY provides an explanation for the familial inheritance. In four of the remaining five cases, paternal mosaicism was ruled out based on the transmission of the mutant allele to paternal relatives of the proband. In the last case, paternal mosaicism could not be determined because the proband’s father was deceased at the time of the study. In this report, we describe an XY female and her normal father who share one such familial mutation at a conserved tyrosine in the HMG box of SRY. Two XY females with PGD have been reported previously with mutations at this particular amino acid (14, 15). Although neither report describes a familial mutation, they emphasize the functional importance of this tyrosine residue. Subjects and Methods
Subjects
The proband, a 16-yr old XY female of Armenian descent, presented with primary amenorrhea, but was otherwise healthy and had no history of illness. The external genitalia and secondary sexual characteristics of this patient were like those of a normal female. Breasts were normally developed (Tanner stage IV). Pubic and axillary hair were also present (Tanner stage IV). The presence of secondary sexual characteristics is atypical of patients with SRY mutations and may indicate estrogen secretion from the dysgenetic gonads of this patient. Pelvic ultrasound revealed the presence of a hypoplastic uterus and small gonads. Endocrine function studies showed elevated LH (24.9 mIU/ml) and FSH (74.1 mIU/ml). Testosterone (25 ng/dl) and prolactin (17.7 ng/dl) fell within the normal range for an adult female. Estradiol levels (26 pg/ml) were below adult normal but above prepubertal levels. As a precaution against the possible development of dysgerminoma in the patient’s gonads, a bilateral gonadectomy was performed at age 16. In postsurgical measurements, estradiol levels fell to below 15 pg/ml (undetectable levels) before hormone therapy was begun. Microscopic examination of the left gonad shows discrete cellular aggregates composed of an admixture of germ cells and small epithelial cells of sex cord type. These nests are surrounded by a hyalinized basement membrane. Some of the nests show mulberry-like calcification. Sections of this gonad also reveal proliferation of atypical germ cells admixed with granulomatous chronic inflammation consistent with gonadoblastoma (Fig. 2) with focal malignant dysgerminoma (data not shown). The father of the proband was clinically unremarkable. Although no histological examination of the father’s gonads was performed, it can be inferred from his demonstrated fertility that his gonads are functional and probably normal.
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Puregene DNA Isolation kit (Gentra Systems, Minneapolis, MN) was used to extract genomic DNA from peripheral blood samples collected from the proband and her father after informed consent was obtained. Two overlapping sets of primers (Fig. 1) were used to amplify and sequence SRY (SRY1: 5'-gttgagggcggagaaatgcaag-3'; SRY2: 5'-acataggcaggctcacttctgg-3'; SRY3: 5'-cgcattcatcgtgtggtctcg-3'; SRY4: 5'-agctggtgctccattcttgag-3') from these two DNA samples. The PCRs were performed on 200 ng genomic DNA for 35 cycles with an annealing temperature of 58 C.
Allele-specific oligonucleotide (ASO) hybridization
With primers SRY3 and SRY4 described above, a 350-bp segment of SRY (including the Y127F mutation site) was amplified from the DNAs of the proband, her father, and 93 other randomly selected XY individuals. The DNAs from these 93 males were isolated from blood spots originally collected for standard neonatal screenings. The 350-bp SRY PCR products were then arrayed on two identical nylon membranes. Two oligonucleotides of 23 bases in length were designed and synthesized for use as probes in this study: one specific for wild-type SRY (5'-tacccgaattataagtatcgacc-3') and the other specific for Y127F SRY (5'- tacccgaattttaagtatcgacc-3'), differing only at the 11th nucleotide. Twenty-five nanograms of these oligonucleotides were end-labeled with 
-32P by T4 polynucleotide kinase (Amersham Pharmacia Biotech, Piscataway, NJ) and used separately to probe the duplicate membranes for 16 h at 65 C. The membranes were subsequently washed and exposed to film at -80 C.
EMSA
EMSAs were performed as described (16) with wild-type and Y127F proteins transcribed and translated from full-length clones with TnT Reticulocyte Lysate system (Promega Corp., Madison, WI). The wild-type and mutant proteins were incubated for 15 min at 22 C with a 29-bp double-stranded radiolabeled DNA target containing the consensus HMG box binding site (17). Control reactions were set up with 100-fold excess of unlabeled double-stranded target DNA (cold probe) to demonstrate binding specificity. Reaction products were separated on a 6% nondenaturing polyacrylamide gel run at 4 C for 75 min in 0.25x Tris-borate EDTA. The gels were dried and exposed to film at -80 C.
Histology
The gonads were fixed in formalin and embedded in paraffin before examination with routine hematoxylin and eosin stains.
Results
By direct sequencing of PCR products, we have identified a point mutation in the SRY gene of an XY female and her phenotypically normal and fertile father (Fig. 3a). This mutation at nucleotide 380 with respect to the initiation codon is an A
T transversion mutation that results in the replacement of a tyrosine residue with a phenylalanine residue at amino acid 127 within the HMG box. To determine whether or not the Y127F variant is a common polymorphism of SRY, we performed an ASO hybridization study on fragments of SRY amplified from the DNAs of the proband, her father, and 93 other males. Figure 4 shows that the Y127F variant was found only in the proband and her father, and not among any of the 93 other SRY alleles sampled. These data suggest that the Y127F variant is a mutation and not a common polymorphism. To study the functional consequences of the Y127F mutation, we performed EMSAs in which the binding of both wild-type and Y127F SRY proteins to the consensus HMG box target DNA was analyzed in vitro. In the presence of wild-type SRY produced by rabbit reticulocyte lysate, the migration of the HMG box target is retarded, showing binding of SRY (Fig. 5, lane 3). A faint band is observed in the presence of reticulocyte lysate alone, probably corresponding to the binding of unspecified HMG proteins present in the lysate (Fig. 5, lane 2). After chasing with a large excess of cold target probe, no band shift is observed, demonstrating the specific nature of SRY binding (Fig. 5, lane 4). Finally, when the mutated Y127F SRY protein is used in the assay, no specific binding is observed (Fig. 5, lane 5). Our results verify that wild-type SRY binds to its double-stranded consensus target, and they illustrate that the Y127F mutation in SRY abolishes this binding capacity.
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We have identified a mutation in the testis-determining gene SRY shared by an XY female and her phenotypically normal father. Our ASO hybridization studies suggest that this Y127F variant is a mutation and not a common polymorphism. Furthermore, our EMSA studies demonstrate that this mutation abolishes the binding capacity of SRY in vitro. Taken together, these data support the conclusion that the Y127F mutation creates a functional change in SRY that caused the XY sex reversal and PGD seen in the proband. However, although these data seem to explain adequately the phenotype of the proband, they stand in apparent conflict with the phenotype of her father, who shares the Y127F substitution.
Two independent cases describing de novo S18N mutations and two cases describing V60L and V60A mutations in SRY have suggested that human SRY may exhibit mutational hot spots at codons 18 and 60 (4, 7, 13, 18, 19). The mutation reported here describes the third novel mutation at tyrosine 127 and the second different mutation of the adenine at nucleotide 380. These reports suggest that there may be a mutational hot spot for human SRY at codon 127 as well. However, further study will be required to determine the validity and functional implications of this finding.
In most respects, the phenotype of the proband described here, XY sex reversal with PGD accompanied by gonadoblastoma and unambiguous female external genitalia, is typical of patients with mutations in SRY. One atypical aspect of the proband’s phenotype is the presence of secondary sexual characteristics, including normal breast development. Although most patients with mutations in SRY fail to develop secondary sexual characteristics at puberty, a small subset, similar to the proband, does develop these secondary sexual characteristics to varying degrees (20, 21, 22, 23). The origin of the hormones (estradiol in particular) driving the development of these secondary sexual characteristics is a source of much debate, but it seems likely that the gonadoblastoma associated with mutations in SRY could play a significant role in estradiol secretion in some cases. In one such case, an XY female with gonadoblastoma and normal breast development had increased aromatase activity and a subsequent increase in estradiol secretion in cells removed from the gonadoblastoma (24).
In the case of the proband, estradiol levels fell from 26 pg/ml before gonadectomy to undetectable levels after surgery, suggesting that her gonads were, in fact, the source of the estradiol. Although aromatase activity was not studied in this patient, the striking similarity in the phenotypes of this patient and the one described above by Wilson et al. (24) suggests a similar etiology of the breast development in these two cases.
Although it is widely recognized that dysgenetic gonads resulting from SRY mutations frequently develop gonadoblastoma, the molecular cause of these tumors remains elusive. The GBY (gonadoblastoma locus, Y chromosome) locus on the long arm of the Y chromosome describes a 1- to 2-Mb region thought to contain a proto-oncogene involved in the development of these gonadoblastomas (25). Of the five genes known to be located within the GBY locus, TSPY remains the best candidate based on its expression pattern and its homology to a family of oncogenes that encode cyclin B-binding proteins (26).
In addition, in at least one case, loss of heterozygosity at the RB1 (retinoblastoma 1) locus has been detected in DNA isolated from gonadal tumors removed from a patient with XY sex reversal with gonadoblastoma. This report suggests that there may be several different molecular defects secondary to the gonadal dysgenesis that result in the gonadoblastoma described in these patients (27). Further complicating the matter is the fact that "dysgerminoma," a term reserved for germ cell tumors, is occasionally, but not always associated with the gonadoblastoma. It is unclear whether the same molecular defect can cause both kinds of tumors directly or whether the dysgerminomas arise sporadically as a result of a separate mechanism (28).
The two previously identified XY female patients with mutations in codon 127 of SRY emphasize the functional importance of this Y residue (14, 15). However, in the case reported here, the normal and fertile father of the XY female proband shares the same Y127F substitution. If this mutation altered the DNA binding of SRY to the extent that it caused the proband’s sex reversal, then how could her father carry the same mutation and yet be asymptomatic? There are three reasonable hypotheses that could explain this apparent contradiction. First, it is possible that the Y127F substitution is a polymorphism subtle enough to conserve the normal function of SRY and that the sex reversal of the proband is caused by another mutation or an environmental influence during development. However, this explanation is unsatisfying in light of our EMSA data demonstrating the abolition of DNA binding by Y127F SRY. Furthermore, no functionally inconsequential polymorphisms have been reported within the HMG box of human SRY.
The second possibility is that the proband’s father is an unusual mosaic harboring lymphocytes and germ cells that carry the Y127F mutation in SRY and gonadal cells (other than germ cells) that carry wild-type SRY. Studies on DNA extracted from his lymphocytes suggest that there is no mosaicism in his peripheral blood cells (data not shown). However, because a gonadal tissue sample could not be obtained from the proband’s father, gonadal mosaicism cannot be completely excluded.
Third, it is possible that the influence of modifier genes that interact with SRY, for which the proband and her father carry different alleles, could explain the difference in their phenotypes (29). The Y127F mutation in SRY may not, by itself, cause an observable phenotype, but in the presence of a particular allele of a modifier gene, the phenotype may present itself. Several such potential modifier genes, including SIP-1, have been identified as binding partners of SRY in vitro, but further experimentation is required to validate and explore these interactions (30, 31).
Footnotes
This work was supported by grants from the National Institutes of Health (to E.V.) and from the Medical Investigation of Neurodevelopmental Disorders (MIND) Institute (to B.K.J.).
Abbreviations: ASO, Allele-specific oligonucleotide; PGD, pure gonadal dysgenesis.
Received December 19, 2001.
Accepted March 25, 2002.
References
This article has been cited by other articles:
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  B. Isidor, C. Capito, F. Paris, S. Baron, N. Corradini, B. Cabaret, M.-D. Leclair, M. Giraud, D. Martin-Coignard, A. David, et al. Familial Frameshift SRY Mutation Inherited from a Mosaic Father with Testicular Dysgenesis Syndrome J. Clin. Endocrinol. Metab., September 1, 2009; 94(9): 3467 - 3471. [Abstract] [Full Text] [PDF]
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 V. R. Harley, M. J. Clarkson, and A. Argentaro The Molecular Action and Regulation of the Testis-Determining Factors, SRY (Sex-Determining Region on the Y Chromosome) and SOX9 [SRY-Related High-Mobility Group (HMG) Box 9] Endocr. Rev., August 1, 2003; 24(4): 466 - 487. [Abstract] [Full Text] [PDF]
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