| Catalog Number: 40-3202-01 110m l Shipped at ambient temperature. Store at -20oC For research use only. Not for use in diagnostic procedures for clinical purposes. |
|
Background Fragile X syndrome is the most common form of inherited mental retardation. It affects approximately 1 in 1200 males and 1 in 2500 females. As suggested by the name, it is associated with a fragile site under specific cytogenetic laboratory conditions at position Xq27.3 (1). The inheritance pattern of fragile X puzzled geneticists as it did not follow a clear X linked pattern. Approximately 20% of males who are carriers based on pedigree analysis do not manifest any clinical symptoms and are thus termed as Normal Transmitting Males (NTM), mental retardation is rare among the daughters of male carriers. Approximately 35% of female carriers have some mental impairment. Based on the above it has been proposed that there are two states of the mutation, one mutation range in which there is no clinical expression (premutation), which could change to the disease causing state predominantly when transmitted by a female (full mutation)(2). The fragile X syndrome gene (FMR-1, fragile X mental retardation -1) was cloned in 1991 simultaneously by three groups (3-6). Soon the peculiar genetic mode of transmission was established and a new class of mutation came into existence- Triple repeat amplification. This explained the clinical state of ‘premutation’ and ‘full mutation’ as well as ‘anticipation’. The fragile X syndrome is caused by the amplification of CGG repeat which is located in the 5’ region of the cDNA. The most common allele in the normal population consists of 29 repeats, the range varying from 6 to 54 repeats. Premutations in fragile X families showing no phenotypic effect range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats are meiotically unstable with a mutation frequency of one. In general repeats up to 45 are considered normal, repeats above 50 to 200 are considered as premutation and above 200 as full mutation (3-7). The range between 40-55 is considered even by most experienced clinical geneticists and molecular geneticists very difficult to interpret and is considered as a ‘gray zone’ with interpretations made on a case by case basis (8). Genotyping Fragile X genotyping can be done by direct PCR amplification of the CGG triple repeat region or by southern analysis. In most cases both methods are used to complement the results, full mutations usually cannot be identified by PCR by most investigators and southern analysis is the preferred method to distinguish full mutations. The FMR-1 gene region containing the CGG triple repeat is flanked by Eco RI sites and a Eag I site in the region. Full mutation has been shown to methylate the active gene too and thus it prevents Eag I restriction of DNA. Hybridization of southern blots of Eco RI and Eag I double digested DNA clearly can distinguish between normal, premutation and full mutation genotypes (2). Southern analysis can not determine the exact number of repeats or the identification of genotypes corresponding to the ‘gray zone’. |
Fragile X southern blot. Lane 1 affected female. Non-radioactive detection, ~2hr. exposure. References
|
