Identification of a new nonsense mutation (Tyr129Stop) of the SRY gene in a newborn infant with XY sex-reversal.код для вставкиСкачать
American Journal of Medical Genetics 128A:46 –47 (2004) Identification of a New Nonsense Mutation (Tyr129Stop) of the SRY Gene in a Newborn Infant With XY Sex-Reversal Mario Giuffrè,1* Piero Sammarco,2 Carmelo Fabiano,2 Fabio Giardina,2 Fabio Lunetta,2 and Giovanni Corsello1 1 Dipartimento Materno Infantile, Universitá degli Studi di Palermo, Italy Neonatologia e Terapia Intensiva Neonatale, Azienda Ospedaliera ‘‘Cervello,’’ Palermo, Italy 2 Point mutations and deletions of SRY gene have been described in several cases of XY gonadal dysgenesis. To date, most of these mutations affect the HMG domain of SRY which plays a central role in DNA binding activity of SRY. We report on a non-mosaic XY sex-reversed newborn girl (completely female external genitalia). The direct sequencing of SRY showed a new nonsense mutation in a codon of SRY gene flanking the 30 end of the HMG domain: a thymine is replaced by a guanine at position þ387 in codon 129, resulting in the replacement of the amino acid tyrosine (TAT) by a stop codon (TAG). The new mutation of this patient provides further evidence to support the functional importance of the putative DNA binding activity of the HMG-box domain. ß 2004 Wiley-Liss, Inc. KEY WORDS: gonadal dysgenesis; nonsense mutation; sex determining region Y; sex reversal INTRODUCTION In males, the sex determining region Y (SRY) gene encodes a protein (TDF, testis determining factor) that causes the undifferentiated gonads to develop into testes during embryogenesis. The SRY gene is located on the short arm of the Y chromosome and consists of an open reading frame (ORF) single exon within a region of 35 kb adjacent to the pseudoautosomal boundary. Analysis of the ORF has shown that the central third encodes a high-mobility-group (HMG) DNA binding domain. This protein binds to target DNA sequences and bends it. From this protein–DNA interaction and DNA flexing arises the main regulatory activity of the SRY gene. Point mutations and deletions of SRY gene have been described in several cases of XY gonadal dysgenesis. To date, most of these mutations affect the HMG domain of SRY. Here we report on a non-mosaic XY sex-reversed girl (completely female external genitalia) caused by a new nonsense mutation in a codon of SRY gene flanking the 30 end of the HMG domain. CLINICAL REPORT The present patient was the third child born to nonconsanguineous healthy parents; mother and father were, respectively, 40- and 39-years old. Family and pregnancy history were unremarkable. Because of the mother’s age, amniocentesis was performed and the karyotype was found to be 46,XY. Birth weight was 3,600 g, Apgar score was 10 at 10 and 50 , physical examination showed an apparently normal female with external female genitalia normal in shape and pigmentation. There were no palpable masses along the inguinal duct; all physical findings were normal. Cytogenetic analysis performed on T lymphocyte cultures from baby’s peripheral blood sample confirmed a normal 46,XY karyotype in all 50 cells examined. Abdominal ultrasound investigation documented the presence of a uterus; gonads were not visible. The levels of gonadotropins and gonadal steroids were normal with a slight increase of 17-OHprogesterone (Table I). GENETIC ANALYSIS Direct sequencing of the polymerase chain reaction (PCR) product of SRY was performed. A salting-out method [Miller et al., 1988] was used to prepare genomic DNA from peripheralblood leukocytes. The SRY gene was amplified using the upstream primer extF. 50 -ATGCAATCATATGCTTCTGCTA30 complementary to nucleotides þ1 bp to þ 22 bp and reverse primer extR. 50 -CTACAGCTTTGTCCAGTGGCTG-30 corresponding to nucleotides þ593 bp to þ615 bp relative to the ATG start codon. Using these primers, a 615 bp PCR product, encompassing the entire SRY gene, was amplified from genomic DNA. PCR product was purified, and both strands were sequenced directly using the ABI Prism 310 Capillary Sequencer and the following internal primers: intF. 50 ATCATATGCTTCTGCTATGTTA-30 and intR. 50 -GCTTTGTCCAGTGGCTGTAGCG-30 . A new nonsense mutation within the HMG-box of the SRY gene was discovered in the patient (Fig. 1): a thymine is replaced by a guanine at position þ387 in codon 129, resulting in the replacement of the amino acid tyrosine (TAT) by a stop codon (TAG). This point mutation has not been described previously. TABLE I. Serum Hormonal Levels Compared With Normal Control Females of the Same Age *Correspondence to: Dr. Mario Giuffrè, Dipartimento Materno Infantile, Universitá degli Studi di Palermo, Via Cardinale Rampolla, 1, 90142 Palermo, Italy. E-mail: email@example.com Received 8 July 2003; Accepted 13 October 2003 DOI 10.1002/ajmg.a.30075 ß 2004 Wiley-Liss, Inc. Hormone Patient Controls Total testosterone (ng/dl) Free testosterone (pg/ml) 17-OH-progesterone (ng/ml) D4-Androstenedione (ng/ml) Cortisol (mg/dl) FSH (mU/ml) LH (mU/ml) ACTH (pg/ml) 2.0 0.26 4.3 0.36 9.0 12.9 0.5 21.0 <10 0.1–1.5 <2 <0.5 1–24 <20 <9 <140 New Mutation (Tyr129Stop) in the SRY Gene 47 between species and the site of almost all mutations causing XY gonadal dysgenesis [Harley, 2002]. REFERENCES Affara NA, Chalmers IJ, Ferguson-Smith MA. 1993. Analysis of the SRY gene in 22 sex-reversed XY females identifies four new point mutations in the conserved DNA binding domain. Hum Mol Genet 2:785–789. Bilbao JR, Loridan L, Castaño L. 1996. A novel postzygotic nonsense mutation in SRY in familiar XY gonadal dysgenesis. Hum Genet 97: 537–539. Fig. 1. Sequence analysis of SRY DNA from the patient showing a novel nonsense point mutation (thymine to guanine) at position 387 determining a stop signal instead of incorporation of tyrosine at position 129 of the protein product. DISCUSSION Analysis of the SRY ORF demonstrated a T to G transversion at the third position of codon 129 (TAT, Tyr) giving rise to a stop codon (TAG). This point mutation may result in a truncated non-functional protein leading to complete 46,XY gonadal dysgenesis. The present patient underwent surgical removal of the gonadal streaks in the first year of life because of the high risk of developing a gonadoblastoma. Mutations in the SRY gene are among the known causes of XY sex reversal, and an increasing variety of mutations within this gene have been reported in XY sex reversal patients with gonadal dysgenesis [Hawkins et al., 1992; McElreavey et al., 1992a,b; Müller et al., 1992; Affara et al., 1993; Iida et al., 1994; Bilbao et al., 1996; Veitia et al., 1997; Brown et al., 1998]. Literature data suggest that there is an SRY mutation incidence ratio of about 10–15% in 46,XY females [Cameron and Sinclair, 1997], including point mutations, frame-shifts, and deletions. The novel point mutation, identified in this patient, creates a stop codon at the 30 end of the HMG-box and is very likely to produce an impairment of its DNA binding activity. The ability of the SRY protein to bind and bend the DNA helix appears to be critical to its function. 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