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).

Southern analysis can not determine the exact number of repeats or the identification of genotypes corresponding to the ‘gray zone’ .

1. Nelson, D.L. (1993) Growth Genetics and Hormone. 9:1-4.
2. Rousseau, F. et al. (1991) NEJM 325:1673-1681.
3. Verkerk, A. et al. (1991) Cell 65:905-914
4. Fu, Y.H et al. (1991) Cell 67:1047-1058.
5. Oberle, I. et al. (1991) Science 252:1097-1102.
6. Yu, S. et al. (1991) Science 252: 1179-1181.
7. Nelson, D.L. (1996) Growth Gen. and Hormone. 12:1-4.
8. Richards, R and Sutherland, G.R (1992) TIG 8: 249-255.

One tube containing 500 ng of lyophilized Fragile X/GLFX1 probe. The DNA probe is stable in dried state for extended period at room temperature. Upon reconstitution it should be stored at -20 ^oC. The quantity supplied is sufficient for at least 5 random prime labeling reactions using 100ng for each reaction.

1. DNA digestion :

------------------------

2 Load samples to 0.8% agarose gel , run over night at 45mA for ~24 hours. (1.6 kb fragment on the bottom of the gel).

3. Depurination with 0.25N HCl (add 10 ml HCl to 500ml H₂O) for 10 minutes, denature the DNA with 0.4N NaOH/0.6M NaCl for 30 min. at RT, neutralize with 1.5M NaCl/0.5M Tris ( pH 7.5) for 30 min. at RT, transfer to the MagnaCharge Nylon membrane (MSI) by 10xSSC and 10 pieces of SIGMA QuickDraw blotting paper over night. Wash the membrane with 2x SSC, bake it at 80oC for 2 hours.

4. Perform prehybridization at 50^oC for 3 hours in 10 ml of hybrisol I buffer (Oncor) .

5. Label the probe as following: ( BM Random Primer DNA Labeling Kit)

GLFX1 GeneProbeä 25 -100 ng

Boil 10 minutes, and put on ice.

--------------

6. Wash the membrane in 2 x SSC/ 0.1% SDS at RT twice ( 5 min per wash)., then wash with 0.1 x SSC/ 0.1% SDS at 60oC twice (30 min. per wash). Wrap the membrane and put X-ray film on it, expose at -80oC over night. Develop the film next morning.

7. Strip the membrane by incubating in 0.5 N NaOH for 1 hour at RT with constant agitation. Change the solution and incubate over night if necessary. Rinse the membrane with 2x SSC, air dry.