Hum Genet (1992) 90:308-310 human .. geneucs 9 Springer-Verlag 1992 Mutations in the conserved domain of SRY are uncommon in XY gonadal dysgenesis Enik6 K. Pivnick 1, Stephen Wachtel 2, Diane Woods 2, Joe Leigh Simpson 2, Colin E. Bishop 2 IDepartment of Pediatrics, University of Tennessee, Memphis, 711 Jefferson Avenue, Memphis TN 38105, USA 2Department of Obstetrics and Gynecology, University of Tennessee, Memphis, 71I Jefferson Avenue, Memphis TN 38105, USA Received: 12 May 1992 / Revised: 30 June 1992 Abstract. In order to evaluate the role of SRY in the de- termination of the testis, we sequenced the conserved domain of the SRY gene in 8 patients with 46,XY gonadal dysgenesis and 3 patients with related disorders, and compared our data with those obtained in 6 other similar studies. In our study, a 609-bp fragment of SRY was am- plified by the polymerase chain reaction and the internal conserved motif was sequenced. SRY sequences did not differ from those in normal males in any of our patients. Overall, 5 de novo mutations have been identified among 56 patients with sporadic XY gonadal dysgenesis (8.9%), and 2 de novo mutations have been identified among 18 patients with related conditions (11%). The unexpec- tedly low frequency of mutations within the SRY con- served domain in these patients could be caused by un- detected Y-linked mutations outside the conserved do- main in regions that control transcription during develop- ment (e.g., promotor/enhancer regions) or to downstream mutations in other sex-determining genes that need not map to the Y. Introduction Human sex differentiation is governed by a pathway in- volving autosomal and X-linked genes. The pathway is initiated by a dominant Y-situated "master gene" called TDF (testis-determining factor). In its presence, the bi- potential embryonic gonad becomes a testis and further development is male. In its absence, the gonad becomes an ovary and further development is female. Female development can occur in XY embryos when TDF or other genes in the testis-determining pathway are lost, mutated or otherwise compromised. In such cases, the gonad differentiates initially as an ovary, but in the absence of the second X chromosome, the germ cells undergo attrition at a rate greater than that in XX females. At birth, the ovary is usually represented by a "streak gonad", which is endocrinologically inert. The resulting condition is called 46,XY gonadal dysgenesis, or sometimes Swyer syndrome (Swyer 1955: review in Simpson 1989). Correspondence to: C. E. Bishop A prime candidate for TDF has now been cloned (Sinclair et al. 1990; see Gubbay et al. 1990). The new gene, called SRY (sex-determining region Y), is highly conserved in evolution. It is located in a 35-kb segment immediately proximal to the pseudoautosomal bound- ary. This segment is the smallest region of the Y chromo- some that is sex-determining in man. The insertion of a 14-kb segment containing the murine homolog, Sry, in- duces testicular development in at least some transgenic XX males of the mouse (Koopman et al. 1991). The SRY gene encodes a protein with a conserved 80 amino acid motif, similar to motifs found in the HMG (high mobility group) proteins that mediate DNA bind- ing (Nasrin et al. 1991). If SRY is the testis-determining gene, mutations within this motif would be expected in at least some cases of 46,XY gonadal dysgenesis. The nucleotide sequence of the conserved SRY motif accord- ingly has been analyzed in affected XY females in sev- eral studies. In our own study, we tested 8 subjects with sporadic 46,XY gonadal dysgenesis. Three subjects with related disorders were also studied. Here, we compare our results with those published by other groups. Materials and methods Patients" The criteria for selection of subjects with 46,XY gonadal dysgenesis were: (1) female external genitalia; (2) 46,XY karyotype without detectable mosaicism, as ascertained by karyotypic analysis; (3) elevated gonadotropins (FSH, LH) or streak gonads confirmed visually; (4) sporadic occurrence (absence of family history). Clin- ical data are summarized in Table 1. Three patients with related conditions were also studied: a 46,XY female with agonadia, a 46,XY true hermaphrodite, and a 46,XY/47,XXY female with mosaicism presumed secondary to mitotic nondysjunction. Clinical data for these patients are summarized in Table 2. Sequencing DNA was extracted from peripheral blood leukocytes from 9 of the patients and from cultured skin fibroblasts from 2 patients. A 609-bp fragment of SRY was then amplified by the polymerase chain reaction (PCR) with primers XES2 and XES7 (Berta ct al. 1990). The product was cloned into the Bluescript II KS+ Smal site and transformed into E. coli XLI Blue cells (Stratagene). The conserved motif was then sequenced from at least three indepen- dent clones by use of flanking primers (5'-3') CTGTGTAAG- ATCTTCAATC and GTGGTGAGAGGCACAAGT (Gubbay et