Severe XIST hypomethylation clearly distinguishes (SRY+) 46,XX-maleness from Klinefelter syndrome.

OBJECTIVE: 46,XX-maleness affects 1 in 20 000 live male newborns resulting in infertility and hypergonadotrophic hypogonadism. Although the phenotypes of XX-males have been well described, the molecular nature of the X chromosomes remains elusive. We assessed the X inactivation status by DNA methylation analysis of four informative loci and compared those to Klinefelter syndrome (KS) and Turner syndrome.

DESIGN AND METHODS: Patient cohort consisted of ten sex-determining region of the Y (SRY+) XX-males, two (SRY-) XX-males, ten 47,XXY Klinefelter men, six 45,X Turner females and ten male and female control individuals each. Methylation analysis was carried out by bisulphite sequencing of DNA from peripheral blood lymphocytes analysing X-inactive-specific transcript (XIST), phosphoglycerate kinase 1 (PGK1), ferritin, heavy peptide-like 17 (FTHL17) and short stature homeobox (SHOX).

RESULTS: XIST methylation was 18% in (SRY+) XX-males, and thus they were severely hypomethylated compared to (SRY-) XX-males (48%; P<0.01), Klinefelter men (44%; P<0.01) and female controls (47%; P<0.01). Turner females and male controls displayed a high degree of XIST methylation of 98 and 94% respectively. Methylation of PGK1, undergoing X inactivation, was not significantly reduced in (SRY+) XX-males compared to female controls in spite of severe XIST hypomethylation (51 vs 69%; P>0.05). FTHL17, escaping X inactivation, but undergoing cell-type-specific inactivation was similarly methylated in XX-males (89%), KS patients (87%) and female controls (90%). SHOX, an X inactivation escapee located in the pseudoautosomal region, displays similarly low degrees of methylation for XX-males (7%), KS patients (7%) and female controls (9%).

CONCLUSIONS: XIST hypomethylation clearly distinguishes (SRY+) XX-males from Klinefelter men. It does not, however, impair appropriate epigenetic regulation of representative X-linked loci.