Test is found sensitive, specific for Klinefelter's

M-PCR may serve as an adjunct to karyotype and Y chromosome microdeletion assay, both of which are widely recommended forms of screening for men with low sperm production.

"This form of testing may advance our ability to detect genetic conditions in a more specific way than our current genetic testing methods," said lead investigator Peter N. Schlegel, MD, professor and chairman of urology at Cornell's Weill Medical College. "A gene-specific test should be more specific than a gross karyotype test, capable of detecting copy numbers of a gene rather than the presence or absence of an entire chromosome."

The researchers used M-PCR with primers for familial mental retardation 1 (FMR1) and X-chromosome inactivation transcript (CIST) genes to detect the presence of X-chromosome disomy in men. They extracted DNA from blood samples from four groups of subjects:

M-PCR with methylation-specific primers was then performed, and results were compared with known karyotype. The team also assessed X-chromosome inactivation patterns by measuring the net intensity of methylated and unmethylated XIST amplicons after gel electrophoresis.

"One of the theories of the medical problems related to Klinefelter syndrome is that X-chromosome inactivation, which gives normal females one acting chromosome and one 'inactivated' X chromosome, is defective in men with Klinefelter syndrome, so that a 'double dose' of genes may cause some of the abnormalities seen in these men," Dr. Schlegel said.

Indeed, the researchers observed that the methylation pattern of FMR1 and XIST genes in Klinefelter syndrome patients is the same as in females with one copy of the XIST gene methylated (inactive) and the other unmethylated (active). Dr. Schlegel and colleagues continue to evaluate their theory by testing a variety of other genes.

100% sensitive, specific Of equal importance, the M-PCR test using the FMR1 and XIST genes was found to be 100% sensitive and 100% specific for the diagnosis of Klinefelter syndrome. The investigators could detect a minimum of 1% of XX/XY mosaicism using methylated-to-unmethylated FMR1 PCR product ratio. Total material cost per test, excluding labor and overhead, was an inexpensive $5.49, with a turnaround time of 48 hours.

"We are continuing to research this test, using it to detect low-level mosaicism in men with infertility and further examining the diagnosis of Klinefelter syndrome in men who had this diagnosis based on karyotype alone," Dr. Schlegel said.