Use of methylation sensitive polymerase chain reaction for detection of fragile X full mutation & carrier state in males.

ABSTRACT

BACKGROUND & OBJECTIVE: Fragile X syndrome is the most common cause of inherited mental retardation. It is characterized by the progressive expansion of polymorphic (CGG) trinucleotide repeats located in the promoter region of the FMRI gene located at Xq27.3. The typical dysmorphic features that help in diagnosis are very often subtle or absent especially in pre-pubertal children. Confirmation is by molecular diagnosis based on repeat size and methylation analysis of the FMR1 gene. The present study was done to evaluate the utility of a methylation sensitive polymerase chain reaction (ms-PCR) method in the molecular diagnosis of fragile X syndrome in a select group of mentally retarded male children. METHODS: We used a methylation sensitive PCR technique, which initially modified DNA by bisulphite treatment. Two sets of PCR primers one each for methylated and unmethylated DNA sequences, were used. In full mutations, PCR specific for the methylated sequences was designed to amplify the CpG dinucleotide region upstream to the CGG repeats in clinically affected males. In healthy males and carriers, the second set of primers would amplify the unmethylated DNA sequences. The amplified PCR product size would help to differentiate between normal and premutation repeat size. RESULTS: In all, 25 blood samples collected from mentally retarded male children and five from normal controls were tested. Analysis of cases revealed one full blown mutation and one carrier state. These were further confirmed by southern blotting. INTERPRETATION & CONCLUSION: Unlike currently used methods, methylation sensitive PCR is a quick and accurate technique which could be used for the rapid screening of fragile X syndrome in mental retardation.