Detection fragile X Using a little split out ?

Hello reader,

I just learning about the article about detection of Fragile X syndrome patients using methylation-specific PCR of the FMR1 gene. It has been reported that using this method could detect fragile X patient, especally in man with 100 % accuracy. I also have friend of mine in lab who did many samples with this method. All the samples were blood that was drawn from the samples. I am just wondering...We can develop the test like this USING a little split out..since it can be run in PCR......So in the future we can screen the people in large population, more cheapest than Southern, and in one single day....!!!!.....Hmmmmm...very interesting....!!!
-------------------------------------

here is the article :

INTRODUCTION

The fragile X syndrome (FRAXA) is the most common inherited form of mental retardation in man. The molecular pathogenesis of the disease generally involves expansion of a (CGG)_n trinucleotide repeat located on the 5' region of the FMR1 gene, leading to hypermethylation of the promoter and shutdown of gene expression (reviewed by Nelson, 1998; Kaufmann and Reiss, 1999). The molecular diagnosis of FRAXA has depended on simultaneous Southern blot analysis of (CGG)_n length and methylation status. However, this is a complex procedure and, to screen for FRAXA among mentally retarded children, we need simpler and cheaper PCR-based diagnostic tests.

The first PCR-based test that we developed was based on direct PCR amplification of the (CGG)_n trinucleotide repeat with primers flanking the microsatellite, with a product of 557 bp for the (CGG)₂₉ allele (Haddad et al., 1996). Conditions were established so that full mutations failed to amplify. To produce an internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 base pairs upstream the polymorphic region. In blind trials the PCR-based test showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. The test had two main disadvantages. Firstly, a normal (CGG)_n allele was preferentially amplified by PCR due to its smaller size and thus the PCR technique could not be used for the diagnosis of FRAXA in females, because they are heterozygous and would be scored as normal. For the same reason, mosaic patients with a normal sized allele might yield a false negative result. That is why the test was not 100% sensitive. The second drawback resulted from the "failure-to-amplify" characteristic of the test that thus could not provide a definitive diagnosis of fragile X syndrome. Although not quite suitable for medical diagnosis, the PCR test proved to be a useful tool for fragile X syndrome screening in populations of mentally retarded males (Haddad et al., 1999).

Recently Herman et al. (1996) developed an elegant PCR assay for methylation status of CpG islands. DNA samples are first treated with sodium bisulfite to convert unmethylated, but not methylated, cytosines to uracil, followed by PCR amplification with oligonucleotide primers specific for methylated versus unmethylated DNA. We wish to report the successful application of this methylation-specific PCR (MSP) for the study of the FMR1 promoter. This led to a much-improved method for PCR diagnosis of the fragile X syndrome in affected males.

MATERIAL AND METHODS

Patients

We used DNA from eight patients with fragile X syndrome, all confirmed by Southern blot analysis: five of these were ascertained in a screening study of mentally retarded boys in Brazil (Haddad et al., 1999), two were patients diagnosed at GENE and the other (NA06852) was obtained from the Coriell Mutant Cell Repository (Camden, NJ, USA). DNA samples from 42 normal controls were obtained from paternity testing cases at GENE.

PCR primers

For development of primers we used the data of Stöger et al. (1997) who described the pattern of cytosine methylation at the CpG island of the FMR1 gene. We designed two primer pairs: the first, 5'-AAATGGGCGTTTTGGTTTTCGC-3' and 5'-GCCAAAAATCATCGCGCATACG-3', produces a 142-bp fragment from the bisulfite-treated methylated CpG island, while the other, 5'-TGTTTTTTATTAAGTTTGTGTAT-3' and 5'-ACCAAAAATCATCACACATACA-3', generates an 84-bp product from the treated non-methylated promoter. The strategy behind the primer development is shown schematically in Figure 1.

Methylation-specific PCR

DNA samples were treated with sodium bisulfite as described by Herman et al. (1996). They were then submitted to PCR amplification in separate tubes with primers specific for the methylated (M) or non-methylated (N) versions of the CpG island of the FMR1 gene. In the reaction with the M primers we also included primers specific for the methylated version of the SNRPN gene (Kubota et al., 1997). PCR was performed in a final volume of 13 µl using 0.65 µ AmpliTaq Gold (Perkin Elmer, Foster City, CA, USA) in the manufacturer's recommended buffer, 200 µM of each dNTP, 0.4 µM of each primer and 100 ng of human genomic DNA. Thermal cycling conditions were: initial denaturation at 95^oC for 5 min, followed by 35 cycles of 1 min of annealing at 53^oC for the M reaction and 43^oC for the N reaction, 1 min of extension at 72^oC and denaturation at 95^oC for 1 min. Afterwards, the PCR reaction products were separated by electrophoresis in a 6% polyacrylamide gel and visualized by silver staining.

RESULTS AND DISCUSSION

In normal males only the 84-bp fragment was seen (Figure 2), while the diagnosis of FRAXA was doubly indicated by the appearance of a 142-bp product together with visualization of a much weaker 84-bp band (Figure 2). The probable reasons that the 84-bp product did not disappear as could be expected are that methylation is generally not complete (Stöger et al., 1997) and that somatic mosaicism occurs in the length of the (CGG)_n repeat in complete mutations. As an indispensable internal control for the efficiency of the sodium bisulfite treatment, we used a primer pair specific for the imprinted maternal methylated version of the CpG island of the SNRPN gene on human chromosome 15 (Kubota et al., 1997) generating a fragment of 174 bp (Figure 2).

Using the methylation-specific PCR we identified with 100% specificity, sensitivity and accuracy, eight previously diagnosed FRAXA male patients mixed with 42 normal controls. In theory the test should not be prone to producing false positive results and should also be very sensitive, permitting diagnosis even in mosaics with normal-sized alleles. Indeed, we have found that we can still obtain a clear methylated product even when DNA from FRAXA patients is diluted 20-fold with normal male DNA. If needed, sensitivity could be further increased by the use of fluorescently labeled primers and detection in an automatic DNA sequencer.

Apparently the pattern of methylation in the promoter region of the FMR1 is identical in full mutations of FRAXA and in X inactivation in normal females (Stöger et al., 1997). Thus, the MSP test cannot be used to diagnose the fragile X syndrome in affected females, since they already have, in virtue of X inactivation, a methylated FMR1 promoter region.

In summary, methylation-specific PCR emerges as a simple and efficient method for assessing methylation in the FMR1 CpG island. Indeed, it may become the method of choice for diagnosis of the fragile X syndrome in mentally retarded males.