Original Communication Fragile X screening for FRAXA and FRAXE mutations using PCR based studies: Results of a five year study

BACKGROUND: Fragile X syndrome is the most common cause of inherited X-linked mental retardation. It is due to a mutation in a gene on X chromosome leading to hyper­ expansion of a trinucleotide repeat sequence. The two most common Fragile sites with clinical significance are FRAXA at Xq27.3 comprising CGG repeat and a more distal FRAXE associated with amplification of a GCC repeat, located at Xq28. The frequency of occurrence of Fragile X syndrome is estimated to be 1/4000 male births. Screening of refer­ rals for the mutations associated with the Fragile X syn­ drome constitutes a significant workload in many genetic laboratories. AIMS: The aim of the present study was to establish the use of PCR based simple and rapid method of initial screen­ ing of samples, so that only a minority of samples tested positive with the above methods need to be screened by Southern blotting which is more time consuming and in­ volves use of radioactive material. MATERIALS AND METHODS: Study includes 294 patients with mental retardation. DNA extracted from blood was used for simultaneous amplification of the triplet repeat se­ quences at the FRAXA and FRAXE loci. Secondly samples from females were analyzed for heterozygosity of normal FRAXA allele. For confirmation of the presence of an ex­ panded FRAXA allele in all the male positive cases, South­ ern blot hybridization was carried out. PCR based assay was done to detect methylation of the CpG island upstream of the FMR-1 gene. RESULTS: Out of the 294 cases 23 (7.8%) were found to be having full mutation (FM) for FRAXA (21 males, 1 fe­ male & 1 male with mosaic FM/PM) and 13 females as having premutation (PM). All these 36 cases were con­ firmed by Southern blotting using appropriate probes. Among the females the heterozygosity for FRAXA allele was found to be 46%. CONCLUSION: Non-radioactive PCR methods are efficient and rapid test for intial screening of samples for the pres­ ence of FRAXA and FRAXE mutations. Since a large ma­ jority of referrals do not have Fragile X, this economical and reliable method reduces the number of samples need­ ing Southern blotting.