Editor—Turner syndrome is one of the most common chromosomal abnormality syndromes affecting 1 in 2500 liveborn females. The syndrome is characterised by short stature, gonadal dysgenesis, congenital heart disease, renal anomalies, and a variety of somatic features including neck webbing, cubitus valgus, short neck, and widely set nipples. Nearly half of the patients have a classical 45,X karyotype while others have structurally abnormal sex chromosomes (for example, 46,X,i(Xq)) or are mosaics with other cell lines with normal (46,XX) or abnormal sex chromosomes.1-4 Among these, patients with Y chromosomal material require specific attention since many of these 45,X/46,XY Turner syndrome patients develop gonadoblastoma or dysgerminoma later in life.5 6

Conventional chromosomal analysis indicates that 4-20% of patients with Turner syndrome have a Y chromosome or its derivatives.1-4 These figures could be even higher, since the more sensitive PCR based analysis has shown that 15-60% of cytogenetically 45,X females have Y chromosomal material.7-9 These findings mean that 10-50% of all Turner syndrome patients have Y chromosomal material and therefore are, to some extent, at risk of developing gonadoblastoma. A more precise understanding of the mechanism leading to generation of a 45,X/46,XY karyotype is therefore important for providing better care for these patients.

Karyotypes such as 45,X/46,XY are presumably caused by mitotic loss of the Y chromosome from the originally 46,XY fetus. It is not known, however, whether there is a predisposition towards the loss of the Y chromosome in these subjects or whether it is merely a random event caused by the inherent instability of XY chromosomal pairing. With regard to this question, Canto et al 10 recently reported the surprising finding that mutations in the SRY gene occurred in two out of three subjects with a 45,X/46,XY karyotype, one with a cytogenetically normal Y chromosome and the other with a Y derived marker chromosome. In these two patients, they identified an identical missense mutation (G2128A, Ser18Asn) upstream of the high mobility group (HMG) box of the gene. This not only implies that 45,X/46,XY Turner syndrome comprises a phenotypic spectrum with 46,XY females in which 10-15% have SRYmutations,11 12 but also suggests that single point mutations in the sex determining gene may cause gross structural chromosomal abnormalities, for example, formation of a marker chromosome or even the loss of the whole Y chromosome.

Methods

In order to determine the actual frequency ofSRY mutations in 45,X/46,XY patients, we systematically analysed the Y chromosome and theSRY gene in 90 consecutive Turner syndrome patients seen at one of our institutions and sequenced theSRY gene in 11 patients with Y chromosomal material. Blood samples were drawn after informed consent. Genomic DNA was then isolated from peripheral blood leucocytes using a QIAmp Blood Kit (QIAGEN, Germany) according to the supplier's instructions. A highly repetitive sequence in the Y centromere (DYZ3) was amplified by using a primer set 5′-TGAAAACTGCAC AGAAGCTG - 3′/5′ - ACACATCACAAAGAA CTATG-3′. The amplification procedure consisted of initial denaturation at 94°C for 10 minutes, then 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for two minutes in 25 μl reactions containing 50 ng of genomic DNA, 10 mmol/l Tris HCl (pH 8.3), 1.5 mmol/l MgCl₂, 50 mmol/l KCl, 0.2 mmol/l of dNTPs, 0.001% (w/v) gelatin, 25 pmol of each primer, and 1 unit ofTaqGold DNA polymerase (PE-Applied Biosystems, CA). The whole coding region of theSRY gene was amplified as two overlapping fragments using two primer sets (5′-TTGAGAATGAATACAT TGTCAGGG - 3′/5′ - GGGTCGCTTCACT CTATCCTG-3′ and 5′-CAGTGTGAAACG GGAGAAAACAGT - 3′/5′ - CTTTGCATGT TAATCGTGTTGACAC-3′) (fig 1). The amplification was performed under the same conditions as those for DYZ3. PCR products were purified with a Wizard PCR Preps DNA purification kit (Promega, WI) and directly sequenced with a BigDye Terminator Cycle Sequencing Kit (PE-Applied Biosystems) and an automatic sequencer (ABI-PRISM 310, PE-Applied Biosystems).

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Figure 1

Locations of the primers used to amplify and sequence the SRY gene (arrowheads). The rectangle represents the coding region of the gene.