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Kamische et al (2003) should be congratulated for their thorough and informative article on Klinefelter syndrome, published in the January-February 2003 issue of the Journal of Andrology. However, we cannot agree with their favorable evaluation of Barr body analysis, because, in our experience, it is a test that does not have enough sensitivity to diagnose all cases of Klinefelter syndrome, especially in patients with mosaicism.
We wish to briefly describe a novel, simple, and highly sensitive molecular test based on methylation-specific PCR (MSP) of the human X-linked FMR-1 gene, which can replace with enormous advantage the morphological Barr body analysis. The MSP test is done exactly as we described elsewhere for the diagnosis of Fragile X syndrome (Pena and Sturzeneker, 1999). Accordingly, DNA samples are first treated with sodium bisulfite to convert unmethylated, but not methylated, cytosines to uracil, followed by PCR amplification with oligonucleotide primers specific for methylated versus unmethylated DNA (Herman et al, 1996). We designed two primer pairs: one produces a 142-bp fragment from the bisulfite-treated methylated CpG island, and the other generates an 84-bp product from the treated non-methylated promoter (Figure). In normal males, only the 84-bp fragment is seen, but the diagnosis of Klinefelter syndrome is indicated by the appearance of a 142-bp methylated product (Figure). As an indispensable internal control for the efficiency of the sodium bisulfite treatment, we used a primer pair specific for the imprinted maternal methylated version of the CpG island of the SNRPN gene on human chromosome 15 (Figure). Using MSP, we identified, with 100% sensitivity and accuracy, 15 previously diagnosed male patients with Klinefelter syndrome mixed in with 40 normal control subjects. The test is simple, fast (it can be done in less than 48 hours), and does not depend on the use of expensive equipment.
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The MSP test can detect the presence of the methylated X chromosome even when it is diluted 20-fold with normal male DNA (which does not contain the methylated X sequence). Thus, it should be sensitive enough to diagnose all patients with Klinefelter mosaicism. If needed, the test's sensitivity could be further increased by the use of fluorescently labeled primers and detection in an automatic DNA sequencer.
Because of its simplicity and high efficiency, MSP may become the method of choice for screening azoospermic males for Klinefelter syndrome. By its nature, the test can be aptly described as the "molecular Barr body test."
References
Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB.
Methylation-specific PCR: a novel PCR assay for methylation status of CpG
islands. Proc Natl Acad Sci USA. 1996; 93:9821-9826.
Kamischke A, Baumgardt A, Horst J, Nieschlag E. Clinical and
diagnostic features of patients with suspected Klinefelter syndrome.
J Androl. 2003; 24:41-48.
Kubota T, Das S, Christian SL, Baylin SB, Herman JG, Ledbetter DH. Methylation-specific PCR simplifies imprinting analysis. Nat Genet. 1997; 16:16-17.[Medline]
Pena SDJ, Sturzeneker R. Diagnosis of the fragile X syndrome in males using methylation-specific PCR of the FMR1 locus. Genet. Mol. Biol. 1999; 22:169-172.
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