The fragile X mental retardation 1 gene, which codes for the fragile X mental retardation 1 protein, usually has 5 to 40 CGG repeats in the 5′ untranslated. Fragile X is the most common inherited cause of mental retardation with a prevalence of 1 in for males and 1 in to for females.
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Fragile X is the most common inherited cause of mental retardation with a prevalence of 1 in for males and 1 in to for females. Although medical and scientific professionals do not currently recommend screening nonsymptomatic populations, improvements in current treatment approaches and ongoing clinical trials have generated growing interest in screening for fragile X.
Here, we briefly review the relevant molecular basis of fragile X and fragile X testing and compare three different molecular technologies available for fragile X screening in both males and females. The first two-step method performed with high sensitivity and specificity. The second method provided agarose gel images that allow identification of males with expanded CGG repeats and females with expanded CGG-repeat bands which are sometimes faint.
The third melting curve analysis method would require controls in each run to correct for shifting optimal cutoff values. Reduction of the protein coded for by the fragile X mental retardation gene FMR1 causes fragile X syndrome, a genetic condition that causes a range of developmental problems, including learning disabilities, cognitive impairment, and behavioral abnormalities.
After the group 1 metabotropic glutamate receptor is stimulated, regulatory FMR1 protein FMRP production is believed to repress mRNA translation and protein synthesis and to control permanent physical changes that alter synaptic connections linked with the process of learning and memory. The CGG-repeat numbers of individual genes have been categorized, and the borders of these categories are approximate.
The normal range of CGG repeats is considered generally to be as high as 44 CGG repeats, and these repeats are interrupted typically every 9 or 10 repeats by an AGG triplet. The range of 45 to 54 CGG repeats is referred to as the gray zone or intermediate range; for alleles of this size, neither disease associations nor the rate of expansion are fully understood.
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Sendrkmu, this range is not associated with fragile X syndrome, and gray zone alleles expanding to a full mutation in one generation have not been observed. Alleles with approximately 55 to CGG repeats are considered premutations. These alleles are transmitted unstably from parent to child, and expansions from this range to the full-mutation range typically sndromu during maternal transmission. Because mutations of this size possibly can have somatic mosaicism that includes a full mutation, careful examination of the range of allele sizes is warranted.
Expansion of an allele into the premutation range perturbs gene expression, 7 and two conditions are associated with this range of expansions. Premutation alleles may shift transcription of the FMR1 mRNA to an upstream site, and this use of an alternative start site may correlate with increased transcription levels. Hypermethylation is present typically on most or all DNA copies except DNA extracted from chorionic villus sampling; hypermethylation results typically in lower or no production of FMRP.
Fragile X Syndrome
Relatively small studies have indicated that, when a full mutation on the single X chromosome in males is completely methylated, the most severe form of fragile X results.
Females have two X chromosomes; during normal development, one is methylated randomly and inactivated in different tissues. Therefore, females with full mutations may experience a range of symptoms, depending on whether a normal-X allele or the full-mutation allele is active in a fajil tissue. For full mutations, size mosaics of CGG repeats and methylation mosaics are observed.
Analysis of senddromu mutations typically indicates that multiple sizes of CGG repeats are present. Tissue-specific differences can be seen, and persons with size and methylation mosaicism may be higher functioning than persons with completely methylated full mutations. Almost all mutations are associated with fragile X and its related conditions, developmental delay, neurodevelopmental dysfunction, intellectual disability, or syndromic mental retardation.
These generally range from several hundred base pairs to millions of base pairs and frequently include the entire FMR1 gene. All but one include duplication of the entire FMR1 gene. Four point mutations include three missense mutations p.
The methods for measuring and estimating CGG repeats clinically include Southern blot analysis for size and methylation estimation. Widely used commercial reagents allow the following: The tight binding of CG-rich regions causes difficulty in sequencing.
Fragile X syndrome: A review of clinical management
The longer the CGG repeat, the more difficult it is to sequence, particularly in the full-mutation range. This means that expanded CGG repeats cannot be sequenced with conventional Sanger sequencing or commonly used next-generation sequencers. However, a new technology, single-molecule real-time sequencing, that may provide a means of better estimating the number of expanded CGG repeats in the future was reported recently.
Gross deletions and insertions or duplications account for 38 of the 65 currently known mutations in FMR1 Human Gene Mutation Database Professional The estimated prevalence of males with full mutations is approximately 1 in ; prevalence of females with full mutations is approximately 1 in to Although varying prevalences in different ethnic groups occur, few ethnicities have a higher prevalence than the white population.
Because of this relatively high prevalence in different ethnicities, improvements in current treatments, and several ongoing clinical trials that are evaluating agents to ameliorate or prevent the damage that occurs in fragile X Clinical Trials available at http: In addition to methods that use Southern blot analysis, researchers have taken different technical approaches to screening for fragile X.
Although commercial reagents are used for testing and screening in a clinical setting, in the newborn screening NBSpublic health setting NBS programs test every baby born in the United States and in other countries. The cost per test must be manageable for states with varying resources.
Various technical approaches to screening large populations target expanded CGG repeats, methylation of the promotor region, or FMRP itself. Some techniques that detect fragile X in males are not able to detect full mutations reliably in females.
These methods include an assessment of FMR1 methylation in DNA isolated from dried blood spots, developed by Warren frajli colleagues, 27 and a recent method that targets FMRP detection by using an immunoassay with a novel standard.
Several molecular techniques were published that attempt to detect both males and females with expanded CGG repeats. Fragile X controls were provided by Asuragen, and the World Health Organization standards were run to validate all assays. For this study, we used DNA from commercial sources; thus, the work did not meet the definition of human subjects as specified in 45 Crajil This method uses a two-step screening strategy and can be used with DNA extracted from blood spots.
In this procedure, a first round of PCR products are produced with sequence-based forward and reverse primers and are then analyzed by capillary electrophoresis Figure 2A. Normal samples yield one or two normal chromatographic peaks for females and one normal peak for males.
Samples that yield a single normal peak for females or no normal peak for frajjil are tested again in a second round sendrimu PCR by using one sequenced-based primer and a chimeric primer that targets the CGG repeats Figure 2B. Tassone fragile X screening method If there are less than two peaks for females or no peak for males, fraijl forward primer is run with a CGG-targeted reverse primer with capillary electrophoresis analysis of the resulting fragments.
The second approach, developed by Orpana and coworkers, uses heat pulses in the PCR extension step that destabilize the tight C-G bonds. Then the PCR products are analyzed with agarose gel zendromu, and length of the CGG repeat region is determined with standard molecular weight markers.
The Orpana fragile X screening method 29 uses multiple heat pulses in the PCR extension step to destabilize secondary structures and to sendormu the extension over the GC-rich sequence of the CGG repeat region. The method uses agarose gel electrophoresis to detect the resulting amplicons. The third approach, developed by Teo sendro,u al, srndromu uses melting curve analysis on a real-time PCR instrument to detect FMR1 expansions in males and females.
The amplicons are melted after the thermocycling program is completed, and then the first derivative melting curves are analyzed. This method ascertains whether the temperature at which the first derivative melting curve returns to baseline is above or below a cutoff to distinguish premutations and full mutations from normal alleles.
Gray zone mutations may fall in either category, and this method cannot identify them specifically. A male with no normal peak is likely swndromu have an expanded CGG repeat. For expanded samples of males and females, the standard and chimeric primers produce a series of peaks of diminishing amplitude, referred to as a stutter, which extend beyond the normal range and confirm the expanded repeat. When normal samples are tested with standard primers and the resulting fragments are analyzed by capillary electrophoresis, the Tassone method yields one or two peaks in the normal range for heterozygous females and frsjil peak in the normal range for males Figure 5A.
CGG repeats in the gray zone amplify and appear in the appropriate window Figure 5B. Usually, larger pre-mutations and full mutations do not amplify with the first step of this method Figure 5C.
Samples that have only one peak for females and no peak for males require analysis with Frajll primers Figure 5D ; this analysis does not distinguish between premutations and full mutations. A normal sample from a heterozygous female and a normal frajli from a male analyzed with sequence-based forward and reverse primers.
Samples from a female and male with a gray zone allele analyzed with sequence-based forward and reverse primers. A sample from a female with a normal 29 CGG-repeat allele and an expanded allele, and a sample from a male with an expanded CGG-repeat allele analyzed with sequence-based forward and reverse primers.
Samples from a homozygous female with two normal 30 CGG-repeat alleles, a sample from a female with a normal and an expanded CGG allele, and a sample from a male with an expanded CGG allele run with a sequence-based primer and a chimeric CGG-targeted primer.
The incorrect categorization of the one female sample with an expanded allele was a random error; a repeat analysis correctly identified this sample. Identification of the expanded alleles depends on the stutter that results sendromi the CGG-targeted PCR extending beyond the normal repeat range.
When fraajil alleles are present with the Orpana method, a distinct band is evident on the agarose gel Figure 6. This method works well for males with premutations and full mutations because no normal band is present Figure 6A even if an expanded allele is not evident. Therefore, for males we correctly identified all normal samples and all samples with senvromu and full mutations Table 1. Quantitating gray zone mutations is difficult for males and females because of the resolution of the gels.
We detected expanded bands for 10 of xendromu female premutation and full mutation samples. The allele sizes are indicated above the tracks. We obtained characteristic first-derivative melting curves for normal and expanded alleles with the Teo method Figure 7and the sensitivity and specificity of the allele characterization depended on the cutoffs used Figure 8. Only 81 normal, 3 gray zone samples, and 25 premutation and full mutation samples could be senfromu by this method because of the availability of the loaned instrument.
Under these conditions, we called one gray zone negative, one positive, and we could not call the third because it returned to baseline too close to the cutoff line. The Teo method does not claim to distinguish gray zone samples from fraajil or premutations and full mutations.
At this cutoff four of the normal samples tested positive, including three males and one female. Of these false positives, two males and sendeomu female had alleles with CGG repeats in the high normal range from 41 to 44, and the remaining normal male had 36 CGG repeats. With the use of a cutoff of The vertical reference lines represent cutoffs of Studies of population screening for fragile X conducted in a variety of settings have been systematically reviewed.
Studies were included when screening was offered to a general population and when psychosocial aspects of population screening of fragile X were eendromu. Of the 11 studies that offered population screening, all but one screened women of reproductive age to determine whether they carried an expanded CGG repeat allele.
Southern blot analysis is too labor intensive and z to be appropriate for NBS. Only one study offered NBS, and parents were only offered the option of testing their male newborns because the method used resulted in unreliable results in samples from females.
A recurrent theme across screening studies of women was the need for information and counseling because few participants had heard of fragile X before being offered screening and struggled sehdromu understand the clinical features of the disease.
Complex ethical and policy issues need to be resolved before mandated NBS is likely to be recommended. These issues include whether to screen only boys or both sexes, how to deal with incidental chromosomal findings, and whether to report only full mutations to parents or premutation expansions as well, which have adult-onset implications. The authors of the review by Hill et al 32 recommend research to address these issues, in addition to further clinical trials to establish the benefit of early interventions.
Of the three molecular screening methods potentially appropriate for NBS that we evaluated in samples of males and females, the Tassone method achieved high sensitivity and specificity for all samples tested. The Orpana method works particularly well for male samples. Expanded alleles in females can be faint and more difficult to detect and are best evaluated by an analyst experienced in reading gels.