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

Abstract

Background: Fragile X syndrome is the most common cause of inherited
\nX-linked mental retardation. It is due to a mutation in a gene on X
\nchromosome leading to hyper-expansion of a trinucleotide repeat
\nsequence. The two most common Fragile sites with clinical significance
\nare FRAXA at Xq27.3 comprising CGG repeat and a more distal FRAXE
\nassociated with amplification of a GCC repeat, located at Xq28. The
\nfrequency of occurrence of Fragile X syndrome is estimated to be 1/4000
\nmale births. Screening of referrals for the mutations associated with
\nthe Fragile X syndrome constitutes a significant workload in many
\ngenetic laboratories. Aims: The aim of the present study was to
\nestablish the use of PCR based simple and rapid method of initial
\nscreening of samples, so that only a minority of samples tested
\npositive with the above methods need to be screened by Southern
\nblotting which is more time consuming and involves use of radioactive
\nmaterial. Materials and Methods: Study includes 294 patients with
\nmental retardation. DNA extracted from blood was used for simultaneous
\namplification of the triplet repeat sequences at the FRAXA and FRAXE
\nloci. Secondly samples from females were analyzed for heterozygosity of
\nnormal FRAXA allele. For confirmation of the presence of an expanded
\nFRAXA allele in all the male positive cases, Southern blot
\nhybridization was carried out. PCR based assay was done to detect
\nmethylation of the CpG island upstream of the FMR-1 gene. Results: 
\nOut of the 294 cases 23 (7.8%) were found to be having full mutation
\n(FM) for FRAXA (21 males, 1 female & 1 male with mosaic FM/PM) and
\n13 females as having premutation (PM). All these 36 cases were
\nconfirmed by Southern blotting using appropriate probes. Among the
\nfemales the heterozygosity for FRAXA allele was found to be 46%. 
\nConclusion: Non-radioactive PCR methods are efficient and rapid test
\nfor intial screening of samples for the presence of FRAXA and FRAXE
\nmutations. Since a large majority of referrals do not have Fragile X,
\nthis economical and reliable method reduces the number of samples
\nneeding Southern blotting.