Effect of Transcription in CGG Trinucleotide on the Fragile X Syndrome.  Dorianmarie Vargas FrancoRise ProgramBiol. 3019-Literature and Bibliography in Biology, University of Puerto Rico at Cayey

 Abstract)   Autism is presented as a disease categorized as a disability known as mental retardation. This condition is called Fragile X Syndrome which is heritable and is related to an alteration in FMR1 gene. [1] Fragile x syndrome can present some mental and physical impediments. The silencing of the CGG trinucleotide is the BEST known cause of the Fragile X syndrome. The Journal of Molecular Diagnostics published an article that states the real causes for Fragile X syndrome according to the demonstration of their results. As hypothesized, the substitution of G>C in the trinucleotide CGG is the cause of the mutation in DNA that causes mental retardation. The purpose of this investigation is to contrast and evaluate which information is correct to make proper conclusions.

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Introduction             Fragile X syndrome is the common form of mental retardation and is a heritable condition. This syndrome is also known as Martin-Bell syndrome in honor of J. Purdon Martin and Julia Bell (Ashley-Koch 2003). The Fragile X syndrome presents physical and  characteristics as intellectual disabilities, speech problems, attention deficit, long ears and chin, among others. The main cause for the disabilities is the damaging of the X chromosome specifically in the FMR1 gene. FMR1 or fragile X mental retardation 1 is the gene  that codes a protein called FMRP which is an RNA binding protein and is related to the control of brain and nerve cells, involved in the communication of the cells in the brain. Men have one X chromosome and women have two X chromosome, interestingly, Fragile X syndrome is more common in men than

women.  The process of silencing or deleting the CGG trinucleotide is known as

transcription which is better explained as the process by which the DNA transfers genetic information to RNA.  “The silencing process involves DNA methylation as well as modifications to the histone proteins around which the DNA is wrapped in vivo” (Biacsi et al. 2008 ). The complete deletion of the nucleotide is more unusual than a mosaic deletion. An alteration in the pattern the nucleotides can cause abnormalities as Fragile X syndrome because of a silencing in CGG nucleotide. To support this statement the scientist wrote: “This phenomenon is called X-inactivation or Lyonization. X-inactivation ensures that females, like males, have one functional copy of the X chromosome in each body cell. Because X-inactivation is random, in normal females the X chromosome inherited

from the mother is active in some cells, and the X chromosome inherited from the father is active in other cells.” from:Genetic Home Reference 2009. As known, there are contradicting results of the causes of Fragile X syndrome. The purpose is to analyze which one is valid.  Transcription: Deletion or expansion of CGG trinucleotide A mutation in FMR1 gene causes an elimination of FMR1 RNA which encode for the fragile mental retardation protein or FMRP. FMRP is the protein involved in transcription repressor. Transcription takes place in dendrites. Dendrites are terminals in the neurons and are responsible of the stimulus of nerves. Some receptor systems do not work correctly so interrupts the process transcription, silencing the FMR1 gene. The relationship between this repeat expansion and chromosome fragility is unknown (Kumari et al. 2009). The silencing of FMR1 gene is associated with the phenotype disorder caused by the Fragile X syndrome. Transcription in FMR1 gene, not responsible for Fragile X syndrome The Journal of Molecular Diagnosis published an article where scientist from Italy discovered, by experiments, that the silencing of FMR1 gene is not responsible of causing Fragile X syndrome. The substitution of CGG>CCG is said to be a non-coding region or polymorphism. Scientist demonstrates this hypothesis by examining a healthy family, mother, father and son. The mother has a familiar history of mental retardation. With the results were concluded that less than 1% of the population has the substitution in the nucleotide and the substitution of the

nucleotide does not affect the mRNA. Scientific suggest verifying the molecular composition which could be more involve with mRNA. Clarifies that healthy people has been described with the silencing of the gene that causes Fragile X syndrome. The results of the experiments made by the scientist demonstrate that a father and a son showed the variant allele and they were healthy. Finally cite: “Interestingly, a normal female individual carried a heterozygous G_T substitution (CGG_CTG) at the eighth repeat”, (Cecconi et al. 2008) 

               Figure 1.  Three different experiments with its result. A. Proband used for

the experiment were a permutated woman (PF), family individual

denominated as proband(P), proband’s father(F), proband’s son(S) and a

normal female as the control variable(NF). At 2.9kb, 2.8kb and 2.5kb is

presenting an x inactivation. The permutated female shows a weakness at

2.9kb. Both father and son show a loss at 2.8kb and appearance at 2.5kb. B.

The electropherogram shows the substitution of G>C. The change is

noticeable in the father and the son. The substitution nucleotide is marked by

arrows. C. This is an agarose gel experiment. Undigested normal allele

corresponds to 338bp and the variant allele corresponds to 198bp and 140bp.

At 338bp fluorescence the normal female. Finally the digested allele was

found in both, father and son. Journal of Molecular Diagnostics, Vol. 10, No.

3.

   

Figure 2. PCR amplification of the FMR1 CGG repeat of DNA isolated from the patient and a normal male B. Southern analysis of DNA isolated from this patient with atypical fragile X syndrome. column 1, normal female; column 2, the patient being tested; column 3, normal male; column 4, male with typical CGG expansion and methylation; column 5, molecular weight marker.

Materials and method: To work with a mental retardation patient is needed to make test to know the grade of retardation. The proband were healthy people to compare the results on people with fragile x syndrome. The mechanism used was a PCRs and mix with DNA in a reaction including a buffer called Invitrogen, primers and in room temperature making cycles to separate DNA from RNA. At the end was used a GC-Rich PCR System kit (Roche Applied Science, Indianapolis, IN) to analyze the results. (Cecconi et al. 2008). Another instrument used for PCR’s experiment is LA Taq enzyme kit from Takara Bio, Inc. (Otsu, Shiga, Japan CA). The analysis of in vivo protein/DNA interaction was other technique used for the experiment but finally was used PCR to separate DNA from RNA. A process used to analyze and evaluate the results was first of all analyzed the mosaic or expanded nucleotide. Then evaluate the extension and where is the deletion located. Furthermore determine which were the changes of the

pattern and finally compare with a healthy person.

Results In an experiment the healthy father and son reveal a variation in the alleles that is supposed to have the Fragile X syndrome. In the PCR the primer revealed a change from CGGCGG>CGGCCG noticing a change in the second repetition of CGG. Another discovery was that a healthy women present a substitution between G>T, CGG>CTG, but the scientist do not know what can cause this mutation. As another result, were discovered that a lesson or mutation in DNA can block the synthesis limiting replication stagnating the reproduction of bacteria and yeast.  To determine the result of the DNA is needed a Fluorescence Spectroscopy or also an agarose gel, so were discovered a presence of a fragment that determines the heterozygous deletion.  

Figure 3.  Detailed chart of tested people with mutation and no mutation

Discussion Is difficult to determine which of the articles is correct. There are many articles indicating that the silencing of CGG by the substitution  of G>C is the principal cause of Fragile X syndrome but there is only one article found that state that prove otherwise. In the article indicates the experiments and the results obtained but something is missing in every article that may unite the two versions of the problem. It makes sense to say that if occur a substitution in G>C there is a mutation that blocks the DNA. Moreover, has to beproven that the trinucleotide CGG is directly involved with the block of DNA and the synthesizing of RNA. The result of an experiment made with a woman with family history of mental retardation was positive to the fragile X syndrome, demonstrating the silencing of the gene.  Analyzing the data, the woman has a family history; probably she has the syndrome but not expressed, a recessive allele for the syndrome that expressed in the results.

References:

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