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      Cascade Screening for Fragile X Syndrome/CGG Repeat Expansions in Children Attending Special Education in Sri Lanka

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          Abstract

          Fragile X syndrome (FXS) is the commonest cause of inherited mental retardation and clinically presents with learning, emotional and behaviour problems. FXS is caused by expansion of cytosine-guanine-guanine (CGG) repeats present in the 5’ untranslated region of the FMR1 gene. The aim of this study was to screen children attending special education institutions in Sri Lanka to estimate the prevalence of CGG repeat expansions. The study population comprised a representative national sample of 850 children (540 males, 310 females) with 5 to 18 years of age from moderate to severe mental retardation of wide ranging aetiology. Screening for CGG repeat expansion was carried out on DNA extracted from buccal cells using 3’ direct triplet primed PCR followed by melting curve analysis. To identify the expanded status of screened positive samples, capillary electrophoresis, methylation specific PCR and Southern hybridization were carried out using venous blood samples. Prevalence of CGG repeat expansions was 2.2%. Further classification of the positive samples into FXS full mutation, pre-mutation and grey zone gave prevalence of 1.3%, 0.8% and 0.1% respectively. All positive cases were male. No females with FXS were detected in our study may have been due to the small sample size.

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          Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome.

          Fragile-X syndrome is a trinucleotide-repeat-expansion disorder in which the clinical phenotype is believed to result from transcriptional silencing of the fragile-X mental retardation 1 (FMR1) gene as the number of CGG repeats exceeds approximately 200. For premutation alleles ( approximately 55-200 repeats), no abnormalities in FMR1-gene expression have been described, despite growing evidence of clinical involvement in premutation carriers. To address this (apparent) paradox, we have determined, for 16 carrier males (55-192 repeats), the relative levels of leukocyte FMR1 mRNA, by use of automated fluorescence-detection reverse transcriptase-PCR, and the percent of lymphocytes that are immunoreactive for FMR1 protein (FMRP). For some alleles with>100 repeats, there was a reduction in the number of FMRP-positive cells. Unexpectedly, FMR1 mRNA levels were elevated at least fivefold within this same range. No significant increase in FMR1 mRNA stability was observed in a lymphoblastoid cell line (160 repeats) derived from one of the carrier males, suggesting that the increased message levels are due to an increased rate of transcription. Current results support a mechanism of involvement in premutation carriers, in which reduced translational efficiency is at least partially compensated through increased transcriptional activity. Thus, diminished translational efficiency may be important throughout much of the premutation range, with a mechanistic switch occurring in the full-mutation range as the FMR1 gene is silenced.
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            Prevalence of CGG expansions of the FMR1 gene in a US population-based sample.

            The primary goal of this study was to calculate the prevalence of the premutation of the FMR1 gene and of the "gray zone" using a population-based sample of older adults in Wisconsin (n = 6,747 samples screened). Compared with past research, prevalence was relatively high (1 in 151 females and 1 in 468 males for the premutation and 1 in 35 females and 1 in 42 males for the gray zone as defined by 45-54 CGG repeats). A secondary study goal was to describe characteristics of individuals found to have the premutation (n = 30, 7 males and 23 females). We found that premutation carriers had a significantly higher rate of divorce than controls, as well as higher rates of symptoms that might be indicative of fragile X-associated tremor ataxia syndrome (FXTAS; numbness, dizziness/faintness) and fragile X primary ovarian insufficiency (FXPOI; age at last menstrual period). Although not statistically significant, premutation carriers were twice as likely to have a child with disability. Copyright © 2012 Wiley Periodicals, Inc.
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              ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics.

              Molecular genetic testing of the FMR1 gene is commonly performed in clinical laboratories. Mutations in the FMR1 gene are associated with fragile X syndrome, fragile X tremor ataxia syndrome, and premature ovarian insufficiency. This document provides updated information regarding FMR1 gene mutations, including prevalence, genotype-phenotype correlation, and mutation nomenclature. Methodological considerations are provided for Southern blot analysis and polymerase chain reaction amplification of the FMR1 gene, including triplet repeat-primed and methylation-specific polymerase chain reaction. In addition to report elements, examples of laboratory reports for various genotypes are also included.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 December 2015
                2015
                : 10
                : 12
                : e0145537
                Affiliations
                [1 ]Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, Kynsey road, Colombo 08, Sri Lanka
                [2 ]Department of Psychological Medicine, Faculty of Medicine, University of Colombo, Kynsey road, Colombo 08, Sri Lanka
                [3 ]Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119074, Singapore
                [4 ]Khoo Teck Puat - National University Children’s Medical Institute, National University Health System, Singapore, 119228, Singapore
                [5 ]Department of Laboratory Medicine, National University Hospital, Singapore, 119074, Singapore
                Institute of Molecular Genetics IMG-CNR, ITALY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CHWMRBC WSSW HNP SSC ISRB. Performed the experiments: CHWMRBC ISRB. Analyzed the data: CHWMRBC ISRB. Contributed reagents/materials/analysis tools: CHWMRBC WSSW HNP SSC ISRB. Wrote the paper: CHWMRBC WSSW HNP.

                ‡ These authors also contributed equally to this work.

                Article
                PONE-D-15-37186
                10.1371/journal.pone.0145537
                4687912
                26694146
                c4837f08-84c3-4825-8625-9e2442ec2b20
                © 2015 Chandrasekara et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 6 September 2015
                : 4 December 2015
                Page count
                Figures: 3, Tables: 1, Pages: 10
                Funding
                This work was supported by the National Research Council Sri Lanka, 12-068, www.nrc.gov.lk/; and the University of Colombo Sri Lanka AP/3/2012/CG/11, www.cmb.ac.lk/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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