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....!!!
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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.

New finding on Fragile X Syndrome gene

Scientists at The Scripps Research Institute have discovered a new gene involved in fragile X syndrome, a condition that often shares many symptoms of autism. The discovery may lead to new tests or treatments for several neurological disorders.

The new gene has been dubbed FMR4. "FMR4 is a novel gene that is located in the same chromosomal neighborhood as FMR1, a well established causative gene in fragile X syndrome," said Claes Wahlestedt, a professor at the Scripps Research campus in Jupiter, Florida. "Like FMR1, FMR4 is silenced in fragile X patients and up-regulated in FXTAS (fragile X-associated tremor/ataxia syndrome), a disease that resembles Parkinson's disease. Our discovery could lead to the development of new diagnostic tests or even to novel therapies for these defects."

Fragile X syndrome affects thousands of patients worldwide with severe learning disabilities, often accompanied by anxiety disorders, obsessive-compulsive behavior, and attention deficit hyperactivity disorder. There are currently no therapeutic treatments available for fragile X syndrome. Approximately one-third of all children diagnosed with fragile X syndrome also have some degree of autism, according to The National Fragile X Foundation, including such behaviors as social anxiety, poor eye contact, and hand biting.

More than 16 years ago, scientists linked fragile X syndrome to inactivation of FMR1 gene expression, leading to the lack of a protein known as the fragile X mental retardation protein, now considered to be critical for neuronal function. Until the current study, no other functional gene other than FMR1 had been shown to be inactivated in the disorder.

However, Wahlestedt knew the FMR1 gene locus-a specific point on a chromosome-was not well mapped. Wahlestedt and his colleagues hypothesized that unknown regulatory genes might be transcribed from the region.

The new study shows at least one other functional gene-FMR4-from this genetic region is linked to fragile X syndrome, although the gene's exact role in the intact brain remains uncharacterized..

"FMR4 is the new neighbor on the block and should not be ignored," Wahlestedt said. "While there is no direct relationship between these two genes [FMR1 and FMR4] that we know of, our study shows that FMR4 is not a conventional gene-it's a non-coding RNA transcript. It's not a dead piece of the genome, it has a pronounced functional effect in human cultured cells.."

The Role of Non-coding RNA

Non-coding RNA (ncRNA) transcripts or genes produce functional RNA molecules (ncRNAs) rather than encoding proteins. These ncRNAs are active in a number of different processes, including RNA modification, chromosome replication, and protein degradation.

A number of studies have suggested that at least 40 to 50 percent of the mammalian genome becomes transcribed, Wahlestedt pointed out, but only one to two percent of these transcripts are translated into proteins. "Several studies suggest that some ncRNA genes can be involved in various human diseases," he said. "FMR4 certainly falls into that category."

According to the study, FMR4 directly affects human cell proliferation in vitro-when the gene is silenced, changes in the cell cycle and a rise in apoptosis or programmed cell death occur. Overexpression, on the other hand, leads to increased cell proliferation.

The full meaning of this anti-apoptosis function is still unclear. "It could be critical for some cells to live or die at a certain stage in development, but we don't know what cells those might be," Wahlestedt added. "The fact that FMR4 is widely expressed in the human brain in both embryos and adults may possibly indicate a broad function."

The new study underscores the growing awareness among scientists of the complexity and unpredictability of the human genome.

"We know now that our genome is very busy and very complicated," Wahlestedt said "A great deal of this newly found complexity is about the regulation of other genes. As evolution has progressed, particularly in the higher organisms, there has been a corresponding increase in the need for regulatory mechanisms-to maintain more control over genome. Non-coding RNAs are at the center of these regulatory mechanisms."

The FMR4 discovery also highlights the mission of The Translational Research Institute at Scripps Florida, which is focused on translating basic research like the discovery of FMR4 into potential new therapeutics. The Translational Research Institute has a structure similar to a drug discovery company, and many of the researchers have pharmaceutical experience.

In addition to Wahlestedt, other authors of the study include Ahmad M. Khalil, Mohammad Ali Faghihi, Farzaneh Modarresi, and Shaun P. Brothers of The Scripps Research Institute in Jupiter, Florida.

The study, A Novel RNA Transcript with Antiapoptotic Function is Silenced in Fragile X Syndrome, was supported by Conquer Fragile X Foundation (now part of National Fragile X Foundation) and The Scripps Research Institute Florida. Upon publication, the study will be available at http://www.plosone.org/doi/pone.0001486.

Fragile X Syndrome

Today...

I am reading new post on fraxa foundation that effective treatments and a cure for all children and adults with Fragile X will be 'in front our eyes', by directly funding the most promising research.

Researchers are optimistic that the mGluR Theory of Fragile X will lead to treatments for Fragile X and for autism.
What is about mGluR theory ? OK lets me give you this references :

Development of the mGluR Theory :

• May 1997 FRAXA investigator and scientific advisor Dr. William Greenough reports that FMRP, the fragile X protein, is synthesized in dendrites in response to synaptic activity and stimulation of metabotropic glutamate receptors (mGluRs).
  
  
• Nov. 2000 In a FRAXA-funded research project started at Brown University, Drs. Mark Bear and Kim Huber make the important discovery that one mechanism of communication between neurons is defective in mice which have been bred to model Fragile X. This mechanism, called long-term depression (LTD), is a form of synaptic plasticity, the molecular basis of learning and memory. The team studied one specific form of LTD which occurs only if and when mGluRs are stimulated. They found that mGluR-LTD is excessive in the Fragile X knockout mouse.
  
  This discovery has enormous implications for our understanding of Fragile X and related autism spectrum disorders. It and follow-up experiments have led to the "mGluR Theory" of Fragile X: that exaggerated signaling in mGluR pathways underlies many cognitive, behavioral, and neurological symptoms of Fragile X (and probably autism, too.)
  
  
• May 2001 With FRAXA funding, of Columbia University tests the mGluR Theory by treating Fragile X mice with MPEP, a compound which blocks one kind of metabotropic glutamate receptor (mGluR5). According to the theory, this should reverse the major symptoms of Fragile X. In mice, the simplest symptoms to test are hyperactivity and sound-induced seizures. MPEP is found to reverse these symptoms with a single low dose in Fragile X mice.
  
  
• April 2002 The mGluR Theory is introduced at FRAXA's Banbury Conference on Fragile X at Cold Spring Harbor Laboratory, spurring other researchers to follow up on this discovery. Fragile X is now becoming accepted as the first known disease of "synaptic plasticity." Yearly Banbury meetings have since recruited some of the world's top neuroscientists to the study of Fragile X.
  
  
• 2003 A team led by Dr. Tom Jongens of the University of Pennsylvania demonstrate that fruit flies with a mutated Fragile X gene have learning deficits and that MPEP can rescue these abnormalities, even when given to adult flies. The team subsequently shows that the Fragile X flies have abnormal brain anatomy, which can also be corrected by treatment with MPEP during development. Further studies demonstrate that an available drug, lithium, which inhibits mGluR signaling pathways, also rescues Fragile X fly anatomy and cognition. FRAXA then commissions further studies at the Bauchwitz lab at Columbia, which confirm that lithium can treat seizures and hyperactivity in the Fragile X mouse model.
  
  
• 2004 FRAXA-funded researcher Dr. Robert Wong at SUNY Downstate demonstrates that isolated slices of Fragile X knockout mouse brain have more seizure activity than normal mouse brain. He shows that this seizure activity occurs only if mGluR5s are stimulated and shows also that MPEP blocks it.
  
  
• February 2005 Dr. Peter Vanderklish of Scripps Institute, a FRAXA-funded investigator, shows a distinct pattern of abnormal protein synthesis in Fragile X neurons. This pattern immediately normalizes with brief MPEP treatment.
  
  
• July 2005 FRAXA funds a clinical trial of lithium in Fragile X patients, run by Dr. Elizabeth Berry-Kravis at RUSH University, Chicago.
  
  
• July 2005 Researchers at Hoffman LaRoche report that fenobam, a compound used in Phase II/III human trials from 1978-82, is a selective mGluR5 antagonist. In these trials, fenobam showed efficacy for treatment of anxiety disorders, but it was never tested in patients with Fragile X. Its patent has now expired, so it can be tested at will.
  
  
• December 2005 FRAXA contracts with Scynexis to synthesize fenobam for experimental basic research and makes test batches available to qualified researchers free of charge.
  
  
• January 2006 FRAXA is now actively collaborating with several of the largest pharmaceutical companies in the world and several of the smallest startup companies to bring treatments for Fragile X into clinical trials. FRAXA is developing clinical trial sites, as well as improved biomarkers and outcome measures, which will make future trials more effective. FRAXA is also funding the testing of available medications like lithium, which may have been overlooked in the past as potential treatments for Fragile X. We will continue to develop the capacity necessary to advance potential therapies through clinical trials and into routine use.

Taken from Fraxa.org.

I also got newsletter form fraxa that phase II human trial of Fenobam is underway.
Here is the newsletter :

"In December we announced that an initial Phase 1 trial of fenobam in normal volunteers had started. That trial has been completed successfully, and a Phase II trial of fenobam in patients with Fragile X has begun.

Neuropharm and FRAXA Research Foundation are working together to explore the potential of the mGluR5 antagonist fenobam to treat Fragile X Syndrome. Neuropharm, a speciality pharmaceutical company focused on neurodevelopmental disorders, received Orphan Drug Designation in November 2006 for fenobam to treat Fragile X, after acquiring rights to relevant data on the compound from FRAXA. The Orphan Drug Act aims to speed up development of treatments for rare diseases, defined as affecting 200,000 or fewer U.S. residents, like Fragile X.

Fragile X Syndrome is the most common inherited cause of mental impairment and autism. Its symptoms include intellectual handicap, hyperactivity, attention problems, autistic features, emotional liability and epilepsy. There is currently no effective treatment for the condition.

This Phase II trial is being conducted in the US by Professor Randi Hagerman from the UC Davis MIND Institute (www.ucdmc.ucdavis.edu/mindinsti tute/) and Professor Elizabeth Berry-Kravis from Rush University Medical Center ( www.rush.edu/). Since mGluR5 antagonists have not been given to people with Fragile X before, this first study will use a single dose of fenobam in each patient. The outcome of this investigation will determine the feasibility of longer-term studies with fenobam.

Robert Mansfield, Neuropharm's CEO, commented: "We are delighted to announce the commencement of this study with Professors Hagerman and Berry-Kravis who are world leading experts in the clinical study of Fragile X Syndrome. Their trial of NPL-2009 (fenobam), a targeted treatment for Fragile X Syndrome, is a milestone for those affected by this condition. This study follows the work of FRAXA, and researchers such as Dr. Mark Bear, which has highlighted the potential of mGluR5 receptor antagonists for this patient group."

Lets pray together, may it will be success soon..