Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8590-8594, August 1996 Medical Sciences Loss of sequences 3' to the testis-determining gene, SRY, including the Y pseudoautosomal boundary associated with partial testicular determination K. MCELREAVEY*t, E. VILAIN*, S. BARBAUX*, J. S. FUQUAt, P. Y. FECHNERt, N. SOULEYREAU*, M. Doco-FENZY§, R. GABRIEL§, C. QUEREUX§, M. FELLOUS*, AND G. D. BERKOVITZt *Immunogenetique Humaine, Institut Pasteur, 75724 Paris, Cedex 15, France; tDivision of Pediatric Endocrinology, The Johns Hopkins University School of Medicine, 600 North Wolf Street, Baltimore, MD 21205-3311; and §H6pital Maison Blanche, 45 Rue Cognacq-Jay, 51092 Reims, France Communicated by Jean Dausset, Human Polymorphism Study Center, Paris, France, May 1, 1996 (received for review February 8, 1996) ABSTRACT The condition termed 46,XY complete go- In the first 5 weeks of gestation, the human fetus develops nadal dysgenesis is characterized by a completely female various bipotential and neutral sex structures, such as the phenotype and streak gonads. In contrast, subjects with 46,XY bipotential gonads, neutral external genitalia, and paired partial gonadal dysgenesis and those with embryonic testic- internal sex ducts. In the presence of SRY the bipotential ular regression sequence usually present ambiguous genitalia gonads become testes and male sex differentiation occurs. In and a mix of Mullerian and Wolffian structures. In 46,XY 46,XX individuals, in the absence of SRY, the fetal gonads partial gonadal dysgenesis gonadal histology shows evidence develop as ovaries. In rare individuals with a 46,XY karyotype, of incomplete testis determination. In 46,XY embryonic tes- there may be an abnormality in testis determination or in the ticular regression sequence there is lack of gonadal tissue on early steps of testis differentiation. The conditions that result both sides. Various lines of evidence suggest that embryonic are referred to as 46,XY gonadal dysgenesis and are further testicular regression sequence is a variant form of 46,XY classified as 46,XY complete (or pure) gonadal dysgenesis, gonadal dysgenesis. The sex-determining region Y chromo- 46,XY partial gonadal dysgenesis, and embryonic testicular some gene (SRY) encodes sequences for the testis-determining regression sequence (6-8). factor. To date germ-line mutations in SRYhave been reported The 46,XY complete gonadal dysgenesis is characterized by in "20%v of subjects with 46,XY complete gonadal dysgenesis. female external genitalia and well-developed Mullerian struc- However no germ-line mutations ofSRY have tures. The gonad consists of fibrous ovarian-like stroma with been reported in no evidence of testicular differentiation. The 46,XY partial subjects with the partial forms. We studied 20 subjects who gonadal dysgenesis is characterized by partial testis determi- presented either 46,XY partial gonadal dysgenesis or 46,XY nation in an individual with a normal XY karyotype. Partially embryonic testicular regression sequence. We examined the developed internal ducts usually consist of a mixture of Wolf- SRY gene and the minimum region of Y-specific DNA known fian (epididymus, vas deferens, and seminal vesicle) and to confer a male phenotype. The SRY-open reading frame Mullerian ducts (fallopian tube, uterus, and upper third of the (ORF) was normal in all subjects. However a de novo inter- vagina). Affected individuals exhibit varying degrees of mas- stitial deletion 3' to the SRY-ORF was found in one subject. culinization of the external genitalia. Embryonic testicular Although it is possible that the deletion was unrelated to the regression sequence can also be regarded as part of the clinical subject's phenotype, we propose that the deletion was respon- spectrum of 46,XY gonadal dysgenesis. Gonad tissue is absent sible for the abnormal gonadal development by diminishing on one or both sides. Affected individuals have a 46,XY expression ofSRY. We suggest that the deletion resulted either karyotype and usually present a mix of Wolffian and Mullerian in the loss of sequences necessary for normal SRY expression tissue and ambiguous genitalia. Various evidence supports the or in a position effect that altered SRY expression. This case claim that the embryonic gonads were functionally abnormal provides further evidence that deletions of the Y chromosome before their loss (8). outside the SRY-ORF can result in either complete or incom- Mutations in the SRY-ORF have been reported in -20% of plete sex reversal. subjects with 46,XY complete gonadal dysgenesis (9-14). The mutations are clustered in sequences encoding the HMG Sex determination in mammals is controlled by the presence or domain of the molecule. By contrast no germ-line mutations absence of a testis-determining factor encoded by a gene in the SRY-ORF have been reported among subjects with termed sex determining region Y (SRY) (1). In humans the partial forms of 46,XY gonadal dysgenesis. As SRY is respon- SRY gene resides in a 35-kb region on the short arm of the Y sible for the primary step in testis determination, mutations in chromosome, referred to as the testis-determining region. This the SRY-ORF could be expected to completely block testis is the smallest region of Y-specific DNA known to induce formation. However, it is conceivable that some cases of46,XY masculinization in XX males partial gonadal dysgenesis might result from mutations in the (2). The SRYgene itself has been SRY-ORF that modified SRY protein function in a subtle mapped to a region 5 kb proximal to the pseudoautosomal fashion. Similarly, a mutation in a putative regulatory element boundary. SRYconsists of a single exon of -700 bp (3, 4). The might result in reduced expression of SRY or an alteration in central one third of the open reading frame (ORF) encodes a the timing of SRY gene expression. To examine these possi- motif that has sequence homology to a conserved region of the bilities we studied 20 subjects who had either 46,XY partial high mobility group (HMG) proteins (5). This motif, termed gonadal dysgenesis or 46,XY embryonic testicular regression the HMG-box, has the capacity to bind to DNA, which is syndrome. We determined the presence of genetic abnormal- consistent with a cell-autonomous signal in the sex- ities in the testis-determining locus on the Y chromosome, determining pathway. including the SRY gene. In all cases the sequence of the SRY The publication costs of this article were defrayed in part by page charge Abbreviations: HMG, high mobility group; PABY, Y pseudoautoso- payment. This article must therefore be hereby marked "advertisement" in mal boundary; PABX, X pseudoautosomal boundary. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8590 Medical Sciences: McElreavey et aL Proc. Natl. Acad. Sci. USA 93 (1996) 8591 gene was found to be identical to that of a normal male. asymetrically amplified using the primers XES2 and XES7 (12) However one subject with 46,XY partial gonadal dysgenesis and sequenced directly using Sequenase (United States Bio- had a small de novo interstitial deletion 3' to the SRYgene. The chemical) using conditions described by the manufacturer. The results suggest that (i) human sex determination is sensitive to X and Y pseudoautosomal boundaries (PABX and PABY) variations in the activity of the testis-determining gene SRY were amplified using oligonucleotides A and C for the Y and (ii) small interstitial deletions in the Y chromosome may boundary and the oligonucleotides B and C for the X chro- be more common than previously supposed. mosomal boundary, as described (18). Amplification of Y- specific sequences 2 kb proximal to the pseudoautosomal boundary was performed using the primers Y2A, GCATTA- MATERIALS AND METHODS GTGAAGAATTAACT; and Y2B, TCTCATGAAGATTT- Subjects. Table 1 summarizes the clinical description of 20 TTACTC. These primers amplify a 343-bp Y-specific fragment subjects with a 46,XY karyotype and abnormalities of testis under standard conditions. Expression of SRY in peripheral determination. Subjects had 46,XY partial gonadal dysgenesis blood lymphocytes was determined as described elsewhere on the basis of (i) ambiguous external genitalia, (ii) mixture of (13). Paternity was determined by Southern analysis using both Wolffian and Mullerian ducts, and (iii) dysgenetic testis. minisatellite probes (19). Patients who have been described elsewhere are indicated. Blood karyotype analysis excluded mosaicism in all cases, and RESULTS none of the patients presented with Turner stigmata. DNA Analysis. Genomic DNA was prepared from periph- The SRY-ORF was sequenced in each case by using an eral blood lymphocytes. In Southern blot analysis, 15-,ug asymetrically amplified PCR product as a template. In all 20 samples were digested with restriction endonucleases accord- subjects the SRY-ORF sequence was identical to that of a ing to the manufacturer's recommended conditions. Agarose normal male control. To determine if mutations were present gel electrophoresis, transfer to nylon membranes (Hybond N; in the testis-determining region of the Y short arm, Southern Amersham), prehybridization, hybridization to 32P-labeled analysis was performed by using the probes, pY53.3 (SRY), probes, and autoradiography were performed essentially as pO.9, pDP1226, and HfO.2. Southern analysis using the probes described (13, 15). Blots were hybridized with the Y-DNA- pDP1226 and pO.9 failed to detect in all patients any change specific probes pY53.3 (SRY), pO.9, pDP1226, and with the in fragment size from that of a normal male control (data not pseudoautosomal probe HFO.2 (1, 16-18). Hybridization was shown). Southern analysis was performed on StuI digested also performed using probes from 10 kb and 4 kb distal to the DNA using the probe pY53.3 which contains the SRY gene. pseudoautosomal boundary. Primer pairs KPR1OA (GAGT- Hybridization of StuI digested DNA from a normal male CTGAGACTTTTAGAGAG) and KPR1OB (GGGCGGG- control with the probe pY53.3 results in two Y-specific frag- GATATGCCTGTCCTAAGC) amplify a 738-bp fragment ments of 9 and 6 kb (Fig.