Diagnosis of the fragile X syndrome in males using methylation-specific PCR of the FMRI locus

Abstract

We have developed a non-isotopic technique based on methylation-specific PCR (MSP) of the FMR1 promoter for the identification of fragile X full mutations among men. DNA samples were 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. We designed two primer pairs: one produced a 142-bp fragment from the bisulfite-treated methylated CpG island, while the other generated an 84-bp product from the treated non-methylated promoter. In normal males only the 84-bp fragment was seen, while the diagnosis of FRAXA was doubly indicated by the appearance of a 142-bp product together with absence or weak visualization of the 84-bp band. 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. Using the methylation-specific PCR we identified with 100% sensitivity and accuracy eight previously diagnosed FRAXA male patients mixed with 42 normal controls. Because of its simplicity and high efficiency, methylation-specific PCR may become the method of choice for the diagnosis of the fragile X syndrome in mentally retarded males.

Comparative Cytogenetic and PCR Studies in Fragile X Syndrome

“…Molecular study detects gene mutations by amplification of DNA fragments by Polymerase Chain Reaction (PCR) (Fu et al 1991, Heitz et al 1992, Haddad et al 1996, Pena and Sturzeneker 1999 and quickly identifies carriers of gene FMR1 full mutation. Haddad et al (1996) described a protocol in which FRAXA males, carriers of gene FMR1 full mutation, are identified by lack of amplification of mutated alleles.…”

Molecular Barr Bodies: Methylation‐Specific PCR of the Human X‐Linked Gene FMR‐1 for Diagnosis of Klinefelter Syndrome

“…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).…”