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Kul E, Santos M, Stork O. Nigrostriatal Degeneration Underpins Sensorimotor Dysfunction in an Inducible Mouse Model of Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS). Int J Mol Sci 2025; 26:1511. [PMID: 40003975 PMCID: PMC11855849 DOI: 10.3390/ijms26041511] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2024] [Revised: 02/07/2025] [Accepted: 02/10/2025] [Indexed: 02/27/2025] Open
Abstract
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by moderately expanded CGG trinucleotide repeats in the 5' untranslated region (UTR) of the FMR1 gene. Characterized by motor deficits such as action tremor and cerebellar gait ataxia, FXTAS is further distinguished by ubiquitin-positive intranuclear inclusions in neurons and glia. However, its clinical spectrum often overlaps with other neurodegenerative conditions such as Parkinson's disease (PD). Sensorimotor gating deficits, commonly associated with disorders affecting the nigrostriatal pathway such as PD, have been reported in FXTAS, but the underlying connection between these two phenotypes remains undetermined. In this study, we used the P90CGG mouse model of FXTAS, which expresses 90 CGG repeats upon doxycycline induction, to investigate sensorimotor gating deficits and their relationship to nigrostriatal degeneration. After induction, the P90CGG model exhibited late-onset impairments in prepulse inhibition (PPI), a cross-species measure of sensorimotor gating. These deficits coincided with pronounced nigrostriatal degeneration but occurred without evidence of inclusion formation in the substantia nigra. Our findings highlight nigrostriatal degeneration, which has not previously been reported in animal models of FXTAS, and suggest a potential link to sensorimotor gating dysfunction within the context of the disorder.
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Affiliation(s)
- Emre Kul
- Department of Genetics & Molecular Neurobiology, Institute of Biology, Otto-von-Guericke University Magdeburg, 39106 Magdeburg, Germany; (E.K.); (M.S.)
| | - Mónica Santos
- Department of Genetics & Molecular Neurobiology, Institute of Biology, Otto-von-Guericke University Magdeburg, 39106 Magdeburg, Germany; (E.K.); (M.S.)
| | - Oliver Stork
- Department of Genetics & Molecular Neurobiology, Institute of Biology, Otto-von-Guericke University Magdeburg, 39106 Magdeburg, Germany; (E.K.); (M.S.)
- Center for Behavioral Brain Sciences, 39106 Magdeburg, Germany
- Center for Intervention and Research on Adaptive and Maladaptive Brain Circuits Underlying Mental Health (C-I-R-C), Jena-Magdeburg-Halle, 07743 Jena, Germany
- German Center for Mental Health (DZPG), Site Jena-Magdeburg-Halle, 07745 Jena, Germany
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Kul E, Stork O. Trehalose consumption ameliorates pathogenesis in an inducible mouse model of the Fragile X-associated tremor/ataxia syndrome. Nutr Neurosci 2024; 27:826-835. [PMID: 37776526 DOI: 10.1080/1028415x.2023.2261682] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/02/2023]
Abstract
Trehalose is a naturally occurring sugar found in various food and pharmaceutical preparations with the ability to enhance cellular proteostasis and reduce the formation of toxic intracellular protein aggregates, making it a promising therapeutic candidate for various neurodegenerative disorders. OBJECTIVES Here, we explored the effectiveness of nutritional trehalose supplementation in ameliorating symptoms in a mouse model of Fragile X-associated tremor/ataxia syndrome (FXTAS), an incurable late onset manifestation of moderately expanded trinucleotide CGG repeat expansion mutations in the 5' untranslated region of the fragile X messenger ribonucleoprotein 1 gene (FMR1). METHODS An inducible mouse model of FXTAS expressing 90 CGG repeats in the brain had been previously developed, which faithfully captures hallmarks of the disorder, the formation of intracellular inclusions, and the disturbance of motor function. Taking advantage of the inducible nature of the model, we investigated the therapeutic potential of orally administered trehalose under two regimens, modelling disease prevention and disease treatment. RESULTS AND DISCUSSION Trehalose's effectiveness in combating protein aggregation is frequently attributed to its ability to induce autophagy. Accordingly, trehalose supplementation under the prevention regimen ameliorated the formation of intranuclear inclusions and improved the motor deficiencies resulting from the induced expression of 90 CGG repeats, but it failed to reverse the existing nuclear pathology as a treatment strategy. Given the favorable safety profile of trehalose, it is promising to further explore the potential of this agent for early stage FXTAS.
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Affiliation(s)
- Emre Kul
- Department of Genetics & Molecular Neurobiology, Institute of Biology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
| | - Oliver Stork
- Department of Genetics & Molecular Neurobiology, Institute of Biology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
- Center for Behavioral Brain Sciences, Magdeburg, Germany
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Jiraanont P, Zafarullah M, Sulaiman N, Espinal GM, Randol JL, Durbin-Johnson B, Schneider A, Hagerman RJ, Hagerman PJ, Tassone F. FMR1 Protein Expression Correlates with Intelligence Quotient in Both Peripheral Blood Mononuclear Cells and Fibroblasts from Individuals with an FMR1 Mutation. J Mol Diagn 2024; 26:498-509. [PMID: 38522837 PMCID: PMC11983694 DOI: 10.1016/j.jmoldx.2024.02.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2023] [Revised: 02/15/2024] [Accepted: 02/23/2024] [Indexed: 03/26/2024] Open
Abstract
Fragile X syndrome (FXS) is the most common heritable form of intellectual disability and is caused by CGG repeat expansions exceeding 200 (full mutation). Such expansions lead to hypermethylation and transcriptional silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene. As a consequence, little or no FMR1 protein (FMRP) is produced; absence of the protein, which normally is responsible for neuronal development and maintenance, causes the syndrome. Previous studies have demonstrated the causal relationship between FMRP levels and cognitive abilities in peripheral blood mononuclear cells (PBMCs) and dermal fibroblast cell lines of patients with FXS. However, it is arguable whether PBMCs or fibroblasts would be the preferred surrogate for measuring molecular markers, particularly FMRP, to represent the cognitive impairment, a core symptom of FXS. To address this concern, CGG repeats, methylation status, FMR1 mRNA, and FMRP levels were measured in both PBMCs and fibroblasts derived from 66 individuals. The findings indicated a strong association between FMR1 mRNA expression levels and CGG repeat numbers in PBMCs of premutation males after correcting for methylation status. Moreover, FMRP expression levels from both PBMCs and fibroblasts of male participants with a hypermethylated full mutation and with mosaicism demonstrated significant association between the intelligence quotient levels and FMRP levels, suggesting that PBMCs may be preferable for FXS clinical studies, because of their greater accessibility.
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Affiliation(s)
- Poonnada Jiraanont
- Division of Molecular and Cellular Medicine, Faculty of Medicine, King Mongkut's Institute of Technology Ladkrabang, Bangkok, Thailand
| | - Marwa Zafarullah
- Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California
| | - Noor Sulaiman
- Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California
| | - Glenda M Espinal
- Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California
| | - Jamie L Randol
- Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California
| | - Blythe Durbin-Johnson
- Division of Biostatistics, University of California, Davis, School of Medicine, Davis, California
| | - Andrea Schneider
- Department of Pediatrics, University of California, Davis, School of Medicine, Davis, California; UC Davis MIND Institute, University of California, Davis, Sacramento, California
| | - Randi J Hagerman
- Department of Pediatrics, University of California, Davis, School of Medicine, Davis, California; UC Davis MIND Institute, University of California, Davis, Sacramento, California
| | - Paul J Hagerman
- Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California; UC Davis MIND Institute, University of California, Davis, Sacramento, California
| | - Flora Tassone
- Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California; UC Davis MIND Institute, University of California, Davis, Sacramento, California.
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da Silva CP, Camuzi D, Reis AHDO, Gonçalves AP, Dos Santos JM, Machado FB, Medina-Acosta E, Soares-Lima SC, Santos-Rebouças CB. Identification of a novel epigenetic marker for typical and mosaic presentations of Fragile X syndrome. Expert Rev Mol Diagn 2023; 23:1273-1281. [PMID: 37970883 DOI: 10.1080/14737159.2023.2284782] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2023] [Accepted: 11/07/2023] [Indexed: 11/19/2023]
Abstract
BACKGROUND Fragile X syndrome (FXS) is primarily due to CGG repeat expansions in the FMR1 gene. FMR1 alleles are classified as normal (N), intermediate (I), premutation (PM), and full mutation (FM). FXS patients often carry an FM, but size mosaicism can occur. Additionally, loss of methylation boundary upstream of repeats results in de novo methylation spreading to FMR1 promoter in FXS patients. RESEARCH DESIGN AND METHODS This pilot study investigated the methylation boundary and adjacent regions in 66 males with typical and atypical FXS aged 1 to 30 years (10.86 ± 6.48 years). AmplideX FMR1 mPCR kit was used to discriminate allele profiles and methylation levels. CpG sites were assessed by pyrosequencing. RESULTS 40 out of 66 FXS patients (60.6%) showed an exclusive FM (n = 40), whereas the remaining (n = 26) exhibited size mosaicism [10 PM_FM (15.15%); 10 N_FM (15.15%); 2 N_PM_FM (3%)]. Four patients (6.1%) had deletions near repeats. Noteworthy, a CpG within FMR1 intron 2 displayed hypomethylation in FXS patients and hypermethylation in controls, demonstrating remarkable specificity, sensitivity, and accuracy when a methylation threshold of 69.5% was applied. CONCLUSIONS Since intragenic methylation is pivotal in gene regulation, the intronic CpG might be a novel epigenetic biomarker for FXS diagnosis.
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Affiliation(s)
- Camilla Pereira da Silva
- Department of Genetics, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Diego Camuzi
- Molecular Carcinogenesis Program, Brazilian National Cancer Institute, Rio de Janeiro, Brazil
| | - Adriana Helena de Oliveira Reis
- Department of Genetics, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Andressa Pereira Gonçalves
- Department of Genetics, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Jussara Mendonça Dos Santos
- Department of Genetics, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Filipe Brum Machado
- Department of Biological Sciences, Minas Gerais State University, Minas Gerais, Brazil
| | - Enrique Medina-Acosta
- Biotechnology Laboratory, Molecular Diagnostic, and Research Center, State University of the North Fluminense Darcy Ribeiro, Campos dos Goytacazes, Brazil
| | | | - Cíntia Barros Santos-Rebouças
- Department of Genetics, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
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Lee HG, Imaichi S, Kraeutler E, Aguilar R, Lee YW, Sheridan SD, Lee JT. Site-specific R-loops induce CGG repeat contraction and fragile X gene reactivation. Cell 2023; 186:2593-2609.e18. [PMID: 37209683 PMCID: PMC11505655 DOI: 10.1016/j.cell.2023.04.035] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2021] [Revised: 01/15/2023] [Accepted: 04/26/2023] [Indexed: 05/22/2023]
Abstract
Here, we describe an approach to correct the genetic defect in fragile X syndrome (FXS) via recruitment of endogenous repair mechanisms. A leading cause of autism spectrum disorders, FXS results from epigenetic silencing of FMR1 due to a congenital trinucleotide (CGG) repeat expansion. By investigating conditions favorable to FMR1 reactivation, we find MEK and BRAF inhibitors that induce a strong repeat contraction and full FMR1 reactivation in cellular models. We trace the mechanism to DNA demethylation and site-specific R-loops, which are necessary and sufficient for repeat contraction. A positive feedback cycle comprising demethylation, de novo FMR1 transcription, and R-loop formation results in the recruitment of endogenous DNA repair mechanisms that then drive excision of the long CGG repeat. Repeat contraction is specific to FMR1 and restores the production of FMRP protein. Our study therefore identifies a potential method of treating FXS in the future.
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Affiliation(s)
- Hun-Goo Lee
- Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
| | - Sachiko Imaichi
- Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
| | - Elizabeth Kraeutler
- Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
| | - Rodrigo Aguilar
- Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
| | - Yong-Woo Lee
- Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
| | - Steven D Sheridan
- Center for Quantitative Health Center for Genomic Medicine and Department of Psychiatry, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Psychiatry, Harvard Medical School, Boston, MA 02114, USA
| | - Jeannie T Lee
- Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
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Shieh M, Amkraut K, Spiridigliozzi GA, Adayev T, Nicholson K, McConkie‐Rosell A, McDonald M, Pennington M, Sebastian S, Lachiewicz AM. High performing male with fragile X syndrome with an unmethylated FMR1 full mutation: The relevance of clinical and genetic correlations. Clin Case Rep 2023; 11:e7371. [PMID: 37361657 PMCID: PMC10290186 DOI: 10.1002/ccr3.7371] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Revised: 04/28/2023] [Accepted: 05/08/2023] [Indexed: 06/28/2023] Open
Abstract
A high performing male with an unmethylated full mutation in the fragile X messenger ribonucleoprotein 1 (FMR1) gene surpassed our expectations into young adulthood. Although initial genetic findings helped make a correct fragile X syndrome (FXS) determination, the report was insufficient. Ten years later, we repeated and conducted additional genetic and clinical studies to determine whether more information could assist with treatment and counseling. The genetic findings were very consistent with his high functioning and would have enabled us to be more confident about a good developmental outcome had they been available previously. As FXS enters the mainstream of well-understood genetic disorders and technological advancements improve genetic tests, it should be clearer to clinical providers what a full FXS assessment could include to provide high quality information for care. For individuals with FXS who are high functioning, their families and clinical professionals would benefit from knowing more genetic findings, including, most importantly, methylation status, but also the FMR1 protein (FMRP) level and mRNA level. While we now know that obtaining only the CGG repeat number is not always adequate to inform accurate clinical care, future studies are likely to show the benefit of studying other biomarkers, such as mRNA levels.
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Affiliation(s)
- Meg Shieh
- Department of ChemistryBrown UniversityProvidenceRhode IslandUSA
- Department of PediatricsDuke University Health SystemDurhamNorth CarolinaUSA
| | - Keren Amkraut
- Department of PediatricsDuke University Health SystemDurhamNorth CarolinaUSA
| | - Gail A. Spiridigliozzi
- Department of PediatricsDuke University Health SystemDurhamNorth CarolinaUSA
- Department of Psychiatry and Behavioral SciencesDuke University Medical CenterDurhamNorth CarolinaUSA
| | - Tatyana Adayev
- Department of Human GeneticsNew York State Institute for Basic Research in Developmental DisabilitiesNew YorkNew YorkUSA
| | - Kaylea Nicholson
- Department of Communication SciencesDuke University Health CenterDurhamNorth CarolinaUSA
| | | | - Marie McDonald
- Department of PediatricsDuke University Health SystemDurhamNorth CarolinaUSA
| | | | - Siby Sebastian
- Department of PathologyDuke University Health SystemDurhamNorth CarolinaUSA
| | - Ave M. Lachiewicz
- Department of PediatricsDuke University Health SystemDurhamNorth CarolinaUSA
- Department of Psychiatry and Behavioral SciencesDuke University Medical CenterDurhamNorth CarolinaUSA
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7
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Burger P, Colin F, Strehle A, Mazzucotelli T, Collot N, Coutelle R, Durand B, Bouman A, Landau Prat D, Kleefstra T, Parrend P, Piton A, Koolen DA, Mandel JL. GenIDA: an international participatory database to gain knowledge on health issues related to genetic forms of neurodevelopmental disorders. J Neural Transm (Vienna) 2023; 130:459-471. [PMID: 36436153 PMCID: PMC9702708 DOI: 10.1007/s00702-022-02569-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2022] [Accepted: 11/15/2022] [Indexed: 11/28/2022]
Abstract
Intellectual disability with or without manifestations of autism and/or epilepsy affects 1-2% of the population, and it is estimated that more than 30-50% of these cases have a single genetic cause. More than 1000 genes and recurrent chromosomal abnormalities are involved in these genetic forms of neurodevelopmental disorders, which often remain insufficiently described in terms of clinical spectrum, associated medical problems, etc., due to their rarity and the often-limited number of patients' phenotypes reported. GenIDA is an international online participatory database that aims to better characterise the clinical manifestations and natural histories of these rare diseases. Clinical information is reported by parents of affected individuals using a structured questionnaire exploring physical parameters, cognitive and behavioural aspects, the presence or absence of neurological disorders or problems affecting major physiological functions, as well as autonomy and quality of life. This strengthens the implication in research of the concerned families. GenIDA aims to construct international cohorts of significant size of individuals affected by a given condition. As of July 2022, GenIDA counts some 1545 documented patient records from over 60 nationalities and collaborates with clinicians and researchers around the world who have access to the anonymized data collected to generate new, medically meaningful information to improve patient care. We present the GenIDA database here, together with an overview of the possibilities it offers to affected individuals, their families, and professionals in charge of the management of genetic forms of neurodevelopmental disorders. Finally, case studies of cohorts will illustrate the usefulness of GenIDA.
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Affiliation(s)
- Pauline Burger
- Department of Neurogenetics and Translational Medicine, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France.
- Institut National de la Santé et de la Recherche Médicale, U 1258, Illkirch, France.
- Centre National de la Recherche Scientifique, UMR 7104, Illkirch, France.
- Université de Strasbourg, Strasbourg, France.
| | - Florent Colin
- Department of Neurogenetics and Translational Medicine, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France
- Institut National de la Santé et de la Recherche Médicale, U 1258, Illkirch, France
- Centre National de la Recherche Scientifique, UMR 7104, Illkirch, France
- Université de Strasbourg, Strasbourg, France
- INSERM UMR S1109, Tumor Biomechanics Lab, Fédération de Médecine Translationnelle de Strasbourg (FMTS), University of Strasbourg, Strasbourg, France
| | - Axelle Strehle
- Department of Neurogenetics and Translational Medicine, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France
- Institut National de la Santé et de la Recherche Médicale, U 1258, Illkirch, France
- Centre National de la Recherche Scientifique, UMR 7104, Illkirch, France
- Université de Strasbourg, Strasbourg, France
| | - Timothée Mazzucotelli
- Department of Neurogenetics and Translational Medicine, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France
- Institut National de la Santé et de la Recherche Médicale, U 1258, Illkirch, France
- Centre National de la Recherche Scientifique, UMR 7104, Illkirch, France
- Université de Strasbourg, Strasbourg, France
| | - Nicole Collot
- Department of Neurogenetics and Translational Medicine, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France
- Institut National de la Santé et de la Recherche Médicale, U 1258, Illkirch, France
- Centre National de la Recherche Scientifique, UMR 7104, Illkirch, France
- Université de Strasbourg, Strasbourg, France
| | - Romain Coutelle
- Service de Psychiatrie de l'enfant et de l'adolescent, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
- INSERM U 1114, Clinique Psychiatrique, Strasbourg, France
| | - Benjamin Durand
- Service de Génétique Médicale, Institut de Génétique Médicale d'Alsace, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
| | - Arianne Bouman
- Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Daphna Landau Prat
- Division of Ophthalmology, The Goldschleger Eye Institute, Sheba Medical Center, Tel Hashomer, Israel
- Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
- The Sheba Talpiot Medical Leadership Program, Tel Hashomer, Israel
| | - Tjitske Kleefstra
- Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
- Centre of Excellence for Neuropsychiatry, Vincent Van Gogh Institute for Psychiatry, Venray, The Netherlands
| | - Pierre Parrend
- ICube Laboratory (Laboratoire Des Sciences de l'ingénieur, de l'informatique et de l'imagerie), UMR 7357, Université de Strasbourg, CNRS, Strasbourg, France
- EPITA, Strasbourg, France
| | - Amélie Piton
- Department of Neurogenetics and Translational Medicine, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France
- Institut National de la Santé et de la Recherche Médicale, U 1258, Illkirch, France
- Centre National de la Recherche Scientifique, UMR 7104, Illkirch, France
- Université de Strasbourg, Strasbourg, France
- Laboratoire de Diagnostic Génétique, IGMA, Hôpitaux Universitaire de Strasbourg, Strasbourg, France
- Institut Universitaire de France, Paris, France
| | - David A Koolen
- Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Jean-Louis Mandel
- Department of Neurogenetics and Translational Medicine, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France
- Institut National de la Santé et de la Recherche Médicale, U 1258, Illkirch, France
- Centre National de la Recherche Scientifique, UMR 7104, Illkirch, France
- Université de Strasbourg, Strasbourg, France
- University of Strasgourg Institute for Advanced Studies (USIAS), University of Strasbourg, Strasbourg, France
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Baker EK, Arpone M, Bui M, Kraan CM, Ling L, Francis D, Hunter MF, Rogers C, Field MJ, Santa María L, Faundes V, Curotto B, Morales P, Trigo C, Salas I, Alliende AM, Amor DJ, Godler DE. Tissue mosaicism, FMR1 expression and intellectual functioning in males with fragile X syndrome. Am J Med Genet A 2023; 191:357-369. [PMID: 36349505 PMCID: PMC10952635 DOI: 10.1002/ajmg.a.63027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 09/13/2022] [Accepted: 10/13/2022] [Indexed: 11/10/2022]
Abstract
Fragile X syndrome (FXS) is caused by hypermethylation of the FMR1 promoter due to the full mutation expansion (full mutation [FM]: CGG ≥ 200 repeats) and silencing of FMR1. Assessment of mosaicism for active-unmethylated alleles has prognostic utility. This study examined relationships between FMR1 methylation in different tissues with FMR1 messenger ribonucleic acid (mRNA) and intellectual functioning in 87 males with FXS (1.89-43.17 years of age). Methylation sensitive Southern blot (mSB) and Methylation Specific-Quantitative Melt Aanalysis (MS-QMA) were used to examine FMR1 methylation. FMR1 mRNA levels in blood showed strong relationships with FMR1 methylation assessed using MS-QMA in blood (n = 68; R2 = 0.597; p = 1.4 × 10-10 ) and buccal epithelial cells (BEC) (n = 62; R2 = 0.24; p = 0.003), with these measures also showing relationships with intellectual functioning scores (p < 0.01). However, these relationships were not as strong for mSB, with ~40% of males with only FM alleles that were 100% methylated and non-mosaic by mSB, showing methylation mosaicism by MS-QMA. This was confirmed through presence of detectable levels of FMR1 mRNA in blood. In summary, FMR1 methylation levels in blood and BEC examined by MS-QMA were significantly associated with FMR1 mRNA levels and intellectual functioning in males with FXS. These relationships were not as strong for mSB, which underestimated prevalence of mosaicism.
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Affiliation(s)
- Emma K. Baker
- Diagnosis and Development, Murdoch Children's Research InstituteRoyal Children's HospitalMelbourneVictoriaAustralia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health SciencesUniversity of MelbourneParkvilleVictoriaAustralia
- School of Psychology and Public HealthLa Trobe UniversityBundooraVictoriaAustralia
| | - Marta Arpone
- Diagnosis and Development, Murdoch Children's Research InstituteRoyal Children's HospitalMelbourneVictoriaAustralia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health SciencesUniversity of MelbourneParkvilleVictoriaAustralia
- Brain and Mind, Murdoch Children's Research InstituteRoyal Children's HospitalParkvilleVictoriaAustralia
| | - Minh Bui
- Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global HealthUniversity of MelbourneMelbourneVictoriaAustralia
| | - Claudine M. Kraan
- Diagnosis and Development, Murdoch Children's Research InstituteRoyal Children's HospitalMelbourneVictoriaAustralia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health SciencesUniversity of MelbourneParkvilleVictoriaAustralia
| | - Ling Ling
- Diagnosis and Development, Murdoch Children's Research InstituteRoyal Children's HospitalMelbourneVictoriaAustralia
| | - David Francis
- Victorian Clinical Genetics Services and Murdoch Children's Research InstituteThe Royal Children's HospitalMelbourneVictoriaAustralia
| | - Mathew F. Hunter
- Monash GeneticsMonash HealthClaytonVictoriaAustralia
- Department of PaediatricsMonash UniversityClaytonVictoriaAustralia
| | - Carolyn Rogers
- Genetics of Learning Disability ServiceHunter GeneticsWaratahNew South WalesAustralia
| | - Michael J. Field
- Genetics of Learning Disability ServiceHunter GeneticsWaratahNew South WalesAustralia
| | - Lorena Santa María
- Molecular and Cytogenetics LaboratoryINTA University of ChileSantiagoChile
| | - Víctor Faundes
- Molecular and Cytogenetics LaboratoryINTA University of ChileSantiagoChile
| | - Bianca Curotto
- Molecular and Cytogenetics LaboratoryINTA University of ChileSantiagoChile
| | - Paulina Morales
- Molecular and Cytogenetics LaboratoryINTA University of ChileSantiagoChile
| | - Cesar Trigo
- Molecular and Cytogenetics LaboratoryINTA University of ChileSantiagoChile
| | - Isabel Salas
- Molecular and Cytogenetics LaboratoryINTA University of ChileSantiagoChile
| | | | - David J. Amor
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health SciencesUniversity of MelbourneParkvilleVictoriaAustralia
- Neurodisability and Rehabilitation, Murdoch Children's Research InstituteRoyal Children's HospitalMelbourneVictoriaAustralia
| | - David E. Godler
- Diagnosis and Development, Murdoch Children's Research InstituteRoyal Children's HospitalMelbourneVictoriaAustralia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health SciencesUniversity of MelbourneParkvilleVictoriaAustralia
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Mirceta M, Shum N, Schmidt MHM, Pearson CE. Fragile sites, chromosomal lesions, tandem repeats, and disease. Front Genet 2022; 13:985975. [PMID: 36468036 PMCID: PMC9714581 DOI: 10.3389/fgene.2022.985975] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2022] [Accepted: 09/02/2022] [Indexed: 09/16/2023] Open
Abstract
Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites ( ≳ 40 known), ≤ 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.
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Affiliation(s)
- Mila Mirceta
- Program of Genetics and Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON, Canada
- Program of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Natalie Shum
- Program of Genetics and Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON, Canada
- Program of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Monika H. M. Schmidt
- Program of Genetics and Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON, Canada
- Program of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Christopher E. Pearson
- Program of Genetics and Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON, Canada
- Program of Molecular Genetics, University of Toronto, Toronto, ON, Canada
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10
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Flavell J, Franklin C, Nestor PJ. A Systematic Review of Fragile X-Associated Neuropsychiatric Disorders. J Neuropsychiatry Clin Neurosci 2022; 35:110-120. [PMID: 36172690 DOI: 10.1176/appi.neuropsych.21110282] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
OBJECTIVE Fragile X premutation carriers are reported to have increased neuropsychiatric problems, and thus the term fragile X-associated neuropsychiatric disorders (FXAND) has been proposed. Unfortunately, published prevalence estimates of these phenomena are inconsistent. This systematic review clarified this issue by reviewing both fragile X premutation prevalence in patients with neurodevelopmental disorders and psychiatric disorder prevalence in premutation carriers without fragile X-associated tremor/ataxia syndrome (FXTAS). Average prevalence was derived from studies that used semistructured clinical interviews, diagnostic criteria, and validated rating scales. METHODS Forty-six studies were reviewed. The rate of fragile X premutation in neurodevelopmental disorders was assessed from five studies. Probands with neurodevelopmental disorders were more likely than those in the general population to be premutation carriers. The rate of psychiatric disorders in premutation carriers was assessed from five studies for neurodevelopmental, 13 studies for mood, 12 studies for anxiety, and two studies for psychotic disorders. The phenotype and sex distribution among premutation carriers were similar to those with fragile X syndrome. RESULTS Compared to control group and general population estimates, the most prevalent psychiatric disorders were neurodevelopmental disorders, anxiety disorders, and bipolar II disorder. Psychiatric disorders were also more common in males. Most studies relied only on past medical history to define the prevalence of psychiatric disorders, yielding variability in results. CONCLUSIONS Future studies are needed to avoid bias by identifying cohorts from population-based sampling, to describe cohort demographic characteristics to elucidate differences in age and sex, and to prioritize the use of validated psychiatric assessment methods.
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Affiliation(s)
- Joshua Flavell
- Mater Intellectual Disability and Autism Service (Flavell, Franklin) and Mater Centre for Neurosciences (Flavell, Nestor), Mater Hospital, Brisbane, Australia; Metro North Hospital and Health Service, Brisbane (Flavell); Queensland Brain Institute (Flavell, Nestor) and Mater Research Institute (Franklin), University of Queensland, Brisbane
| | - Catherine Franklin
- Mater Intellectual Disability and Autism Service (Flavell, Franklin) and Mater Centre for Neurosciences (Flavell, Nestor), Mater Hospital, Brisbane, Australia; Metro North Hospital and Health Service, Brisbane (Flavell); Queensland Brain Institute (Flavell, Nestor) and Mater Research Institute (Franklin), University of Queensland, Brisbane
| | - Peter J Nestor
- Mater Intellectual Disability and Autism Service (Flavell, Franklin) and Mater Centre for Neurosciences (Flavell, Nestor), Mater Hospital, Brisbane, Australia; Metro North Hospital and Health Service, Brisbane (Flavell); Queensland Brain Institute (Flavell, Nestor) and Mater Research Institute (Franklin), University of Queensland, Brisbane
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11
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Neklyudova A, Smirnov K, Rebreikina A, Martynova O, Sysoeva O. Electrophysiological and Behavioral Evidence for Hyper- and Hyposensitivity in Rare Genetic Syndromes Associated with Autism. Genes (Basel) 2022; 13:671. [PMID: 35456477 PMCID: PMC9027402 DOI: 10.3390/genes13040671] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2022] [Revised: 03/29/2022] [Accepted: 04/05/2022] [Indexed: 01/27/2023] Open
Abstract
Our study reviewed abnormalities in spontaneous, as well as event-related, brain activity in syndromes with a known genetic underpinning that are associated with autistic symptomatology. Based on behavioral and neurophysiological evidence, we tentatively subdivided the syndromes on primarily hyper-sensitive (Fragile X, Angelman) and hypo-sensitive (Phelan-McDermid, Rett, Tuberous Sclerosis, Neurofibromatosis 1), pointing to the way of segregation of heterogeneous idiopathic ASD, that includes both hyper-sensitive and hypo-sensitive individuals. This segmentation links abnormalities in different genes, such as FMR1, UBE3A, GABRB3, GABRA5, GABRG3, SHANK3, MECP2, TSC1, TSC2, and NF1, that are causative to the above-mentioned syndromes and associated with synaptic transmission and cell growth, as well as with translational and transcriptional regulation and with sensory sensitivity. Excitation/inhibition imbalance related to GABAergic signaling, and the interplay of tonic and phasic inhibition in different brain regions might underlie this relationship. However, more research is needed. As most genetic syndromes are very rare, future investigations in this field will benefit from multi-site collaboration with a common protocol for electrophysiological and event-related potential (EEG/ERP) research that should include an investigation into all modalities and stages of sensory processing, as well as potential biomarkers of GABAergic signaling (such as 40-Hz ASSR).
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Affiliation(s)
- Anastasia Neklyudova
- Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Science, 117485 Moscow, Russia; (A.N.); (K.S.); (A.R.); (O.M.)
| | - Kirill Smirnov
- Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Science, 117485 Moscow, Russia; (A.N.); (K.S.); (A.R.); (O.M.)
| | - Anna Rebreikina
- Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Science, 117485 Moscow, Russia; (A.N.); (K.S.); (A.R.); (O.M.)
- Sirius Center for Cognitive Research, Sirius University of Science and Technology, 354340 Sochi, Russia
| | - Olga Martynova
- Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Science, 117485 Moscow, Russia; (A.N.); (K.S.); (A.R.); (O.M.)
| | - Olga Sysoeva
- Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Science, 117485 Moscow, Russia; (A.N.); (K.S.); (A.R.); (O.M.)
- Sirius Center for Cognitive Research, Sirius University of Science and Technology, 354340 Sochi, Russia
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12
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Adegbenro A, Coleman S, Nesterova IV. Stoichiometric approach to quantitative analysis of biomolecules: the case of nucleic acids. Anal Bioanal Chem 2022; 414:1587-1594. [PMID: 34800148 PMCID: PMC8766926 DOI: 10.1007/s00216-021-03781-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Revised: 11/02/2021] [Accepted: 11/09/2021] [Indexed: 02/03/2023]
Abstract
Majority of protocols for quantitative analysis of biomarkers (including nucleic acids) require calibrations and target standards. In this work, we developed a principle for quantitative analysis that eliminates the need for a standard of a target molecule. The approach is based on stoichiometric reporting. While stoichiometry is a simple and robust analytical platform, its utility toward the analysis of biomolecules is very limited due to the lack of general methodologies for detecting the equivalence point. In this work, we engineer a new target/probe-binding model that enables detecting the equivalence point while maintaining an appropriate level of specificity. We establish the probe design principles through theoretical simulations and experimental confirmation. Further, we demonstrate the utility of the stoichiometric analysis via a proof-of-concept system based on oligonucleotide hybridization. Overall, the approach that requires neither standard nor calibration yields quantitative results with an adequate accuracy (> 90-110%) and a high specificity. The principles established in our work are very general and can extend beyond oligonucleotide targets toward quantitative analysis of many other biomolecules such as antibodies and proteins.
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Affiliation(s)
- Adeyinka Adegbenro
- Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, 60115, USA
| | - Seth Coleman
- Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, 60115, USA
| | - Irina V Nesterova
- Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, 60115, USA.
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13
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FMR1 allele frequencies in 51,000 newborns: a large-scale population study in China. World J Pediatr 2021; 17:653-658. [PMID: 34738199 DOI: 10.1007/s12519-021-00473-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Accepted: 10/05/2021] [Indexed: 10/19/2022]
Abstract
BACKGROUND Fragile X syndrome (FXS), caused by CGG-repeat expansion in FMR1 promoter, is one of the most common causes of mental retardation. Individuals with full mutation and premutation alleles have a high risk of psychophysiological disorder and of having affected offspring. Frequencies of FMR1 alleles in general newborns have been reported in Caucasians but have not been investigated in the large-scale population in the mainland of China. METHODS The sizes of FMR1 CGG-repeats were analyzed in 51,661 newborns (28,114 males and 23,547 females) and also in a cohort of 33 children diagnosed with developmental delay using GC-rich polymerase chain reaction (PCR) and triple repeat primed PCR. RESULTS The frequency of CGG repeats > 100 was 1/9371 in males and 1/5887 in females, and the frequency of CGG repeats > 54 was 1/1561 in males and 1/1624 in females. FMR1 full mutation and premutation were identified in 27.27% of children who had Ages and Stages Questionnaire scores less than two standard deviations from the cutoff value. CONCLUSIONS Our study revealed the prevalence of FXS in China and improved the sample databases of FXS, suggesting that the prevalence of FXS in Chinese is higher than estimated previously and that FXS screening can be advised to high-risk families.
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14
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Movaghar A, Page D, Scholze D, Hong J, DaWalt LS, Kuusisto F, Stewart R, Brilliant M, Mailick M. Artificial intelligence-assisted phenotype discovery of fragile X syndrome in a population-based sample. Genet Med 2021; 23:1273-1280. [PMID: 33772223 PMCID: PMC8257481 DOI: 10.1038/s41436-021-01144-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Revised: 03/01/2021] [Accepted: 03/02/2021] [Indexed: 11/09/2022] Open
Abstract
PURPOSE Fragile X syndrome (FXS), the most prevalent inherited cause of intellectual disability, remains underdiagnosed in the general population. Clinical studies have shown that individuals with FXS have a complex health profile leading to unique clinical needs. However, the full impact of this X-linked disorder on the health of affected individuals is unclear and the prevalence of co-occurring conditions is unknown. METHODS We mined the longitudinal electronic health records from more than one million individuals to investigate the health characteristics of patients who have been clinically diagnosed with FXS. Additionally, using machine-learning approaches, we created predictive models to identify individuals with FXS in the general population. RESULTS Our discovery-oriented approach identified the associations of FXS with a wide range of medical conditions including circulatory, endocrine, digestive, and genitourinary, in addition to mental and neurological disorders. We successfully created predictive models to identify cases five years prior to clinical diagnosis of FXS without relying on any genetic or familial data. CONCLUSION Although FXS is often thought of primarily as a neurological disorder, it is in fact a multisystem syndrome involving many co-occurring conditions, some primary and some secondary, and they are associated with a considerable burden on patients and their families.
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Affiliation(s)
- Arezoo Movaghar
- Waisman Center, University of Wisconsin-Madison, Madison, WI, USA
| | - David Page
- Department of Biostatistics and Bioinformatics, Duke University, Durham, NC, USA
| | - Danielle Scholze
- Waisman Center, University of Wisconsin-Madison, Madison, WI, USA
- Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA
| | - Jinkuk Hong
- Waisman Center, University of Wisconsin-Madison, Madison, WI, USA
| | | | | | - Ron Stewart
- Morgridge Institute for Research, Madison, WI, USA
| | - Murray Brilliant
- Waisman Center, University of Wisconsin-Madison, Madison, WI, USA
- Marshfield Clinic Research Institute, Marshfield, WI, USA
| | - Marsha Mailick
- Waisman Center, University of Wisconsin-Madison, Madison, WI, USA.
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15
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Erbs E, Fenger-Grøn J, Jacobsen CM, Lildballe DL, Rasmussen M. Spontaneous rescue of a FMR1 repeat expansion and review of deletions in the FMR1 non-coding region. Eur J Med Genet 2021; 64:104244. [PMID: 34022415 DOI: 10.1016/j.ejmg.2021.104244] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2020] [Revised: 04/16/2021] [Accepted: 05/02/2021] [Indexed: 12/07/2022]
Abstract
Fragile X syndrome (FXS) is caused by CGG-repeat expansion in the 5' UTR of FMR1 of >200 repeats. Rarely, FXS is caused by deletions; however, it is not clear whether deletions including only the non-coding region of FMR1 are pathogenic. We report a deletion in the 5' UTR of FMR1 in an unaffected male infant and review 12 reported deletions involving only the non-coding region of FMR1. Genetic testing was requested in a male infant born to a mother harbouring a FMR1 full mutation. The maternal grandmother carried a FMR1 premutation. FMR1 CGG repeats were analysed using repeat-primed PCR. Only a short PCR fragment was amplified and subsequent Sanger sequencing detected an 88 bp deletion in hemizygous form. The deletion included all CGG repeats and flanking sequences but no FMR1 exons. Linkage analysis using STR markers revealed that the deletion had occurred on the allele, which was expanded in the mother and the maternal grandmother. Reverse transcription and quantitative PCR showed normal FMR1 mRNA levels. Grønskov et al. reported a 157 bp deletion of all CGG repeats and flanking sequences in a female without FXS hemizygous for the FMR1 gene due to a deletion on the other X chromosome. Protein expression was unaffected by the deletion. The reported deletion comprises the deletion detected in the male infant. At almost 2 years of age he is unaffected. Based on these observations and the normal FMR1 mRNA level, we conclude that a spontaneous rescue of an FMR1 repeat expansion has occurred.
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Affiliation(s)
- Emilie Erbs
- Department of Clinical Genetics, Lillebaelt Hospital, University Hospital of Southern Denmark, Vejle, Denmark.
| | - Jesper Fenger-Grøn
- Department of Paediatrics, Lillebaelt Hospital, University Hospital of Southern Denmark, Kolding, Denmark
| | - Cecilie Mondrup Jacobsen
- Department of Clinical Genetics, Lillebaelt Hospital, University Hospital of Southern Denmark, Vejle, Denmark
| | - Dorte Launholt Lildballe
- Department of Clinical Genetics, Lillebaelt Hospital, University Hospital of Southern Denmark, Vejle, Denmark
| | - Maria Rasmussen
- Department of Clinical Genetics, Lillebaelt Hospital, University Hospital of Southern Denmark, Vejle, Denmark; Department of Regional Health Research, University of Southern Denmark, Odense M, Denmark
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16
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Laboratory testing for fragile X, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med 2021; 23:799-812. [PMID: 33795824 DOI: 10.1038/s41436-021-01115-y] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Revised: 02/01/2021] [Accepted: 02/02/2021] [Indexed: 11/08/2022] Open
Abstract
Molecular genetic testing of the FMR1 gene is commonly performed in clinical laboratories. Pathogenic variants in the FMR1 gene are associated with fragile X syndrome, fragile X-associated tremor ataxia syndrome (FXTAS), and fragile X-associated primary ovarian insufficiency (FXPOI). This document provides updated information regarding FMR1 pathogenic variants, including prevalence, genotype-phenotype correlations, and variant nomenclature. Methodological considerations are provided for Southern blot analysis and polymerase chain reaction (PCR) amplification of FMR1, including triplet repeat-primed and methylation-specific PCR.The American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee has the mission of maintaining high technical standards for the performance and interpretation of genetic tests. In part, this is accomplished by the publication of the document ACMG Technical Standards for Clinical Genetics Laboratories, which is now maintained online ( http://www.acmg.net ). This subcommittee also reviews the outcome of national proficiency testing in the genetics area and may choose to focus on specific diseases or methodologies in response to those results. Accordingly, the subcommittee selected fragile X syndrome to be the first topic in a series of supplemental sections, recognizing that it is one of the most frequently ordered genetic tests and that it has many alternative methods with different strengths and weaknesses. This document is the fourth update to the original standards and guidelines for fragile X testing that were published in 2001, with revisions in 2005 and 2013, respectively.This versionClarifies the clinical features associated with different FMRI variants (Section 2.3)Discusses important reporting considerations (Section 3.3.1.3)Provides updates on technology (Section 4.1).
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17
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Baker EK, Arpone M, Vera SA, Bretherton L, Ure A, Kraan CM, Bui M, Ling L, Francis D, Hunter MF, Elliott J, Rogers C, Field MJ, Cohen J, Maria LS, Faundes V, Curotto B, Morales P, Trigo C, Salas I, Alliende AM, Amor DJ, Godler DE. Intellectual functioning and behavioural features associated with mosaicism in fragile X syndrome. J Neurodev Disord 2019; 11:41. [PMID: 31878865 PMCID: PMC6933737 DOI: 10.1186/s11689-019-9288-7] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2019] [Accepted: 11/07/2019] [Indexed: 02/06/2023] Open
Abstract
Background Fragile X syndrome (FXS) is a common cause of intellectual disability and autism spectrum disorder (ASD) usually associated with a CGG expansion, termed full mutation (FM: CGG ≥ 200), increased DNA methylation of the FMR1 promoter and silencing of the gene. Mosaicism for presence of cells with either methylated FM or smaller unmethylated pre-mutation (PM: CGG 55–199) alleles in the same individual have been associated with better cognitive functioning. This study compares age- and sex-matched FM-only and PM/FM mosaic individuals on intellectual functioning, ASD features and maladaptive behaviours. Methods This study comprised a large international cohort of 126 male and female participants with FXS (aged 1.15 to 43.17 years) separated into FM-only and PM/FM mosaic groups (90 males, 77.8% FM-only; 36 females, 77.8% FM-only). Intellectual functioning was assessed with age appropriate developmental or intelligence tests. The Autism Diagnostic Observation Schedule-2nd Edition was used to examine ASD features while the Aberrant Behavior Checklist-Community assessed maladaptive behaviours. Results Comparing males and females (FM-only + PM/FM mosaic), males had poorer intellectual functioning on all domains (p < 0.0001). Although females had less ASD features and less parent-reported maladaptive behaviours, these differences were no longer significant after controlling for intellectual functioning. Participants with PM/FM mosaicism, regardless of sex, presented with better intellectual functioning and less maladaptive behaviours compared with their age- and sex-matched FM-only counterparts (p < 0.05). ASD features were similar between FM-only and PM/FM mosaics within each sex, after controlling for overall intellectual functioning. Conclusions Males with FXS had significantly lower intellectual functioning than females with FXS. However, there were no significant differences in ASD features and maladaptive behaviours, after controlling for intellectual functioning, independent of the presence or absence of mosaicism. This suggests that interventions that primarily target cognitive abilities may in turn reduce the severity of maladaptive behaviours including ASD features in FXS.
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Affiliation(s)
- Emma K Baker
- Diagnosis and Development, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia. .,Faculty of Medicine, Dentistry and Health Sciences, Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia. .,School of Psychology and Public Health, La Trobe University, Bundoora, VIC, Australia.
| | - Marta Arpone
- Diagnosis and Development, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia.,Faculty of Medicine, Dentistry and Health Sciences, Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia.,Brain and Mind, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia
| | - Solange Aliaga Vera
- Diagnosis and Development, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia
| | - Lesley Bretherton
- Brain and Mind, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia
| | - Alexandra Ure
- Faculty of Medicine, Dentistry and Health Sciences, Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia.,Neurodisability and Rehabilitation, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia.,Royal Children's Hospital, Melbourne, VIC, Australia.,Department of Pediatrics, Monash University, Clayton, VIC, Australia
| | - Claudine M Kraan
- Diagnosis and Development, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia.,Faculty of Medicine, Dentistry and Health Sciences, Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia
| | - Minh Bui
- Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, University of Melbourne, Carlton, VIC, Australia
| | - Ling Ling
- Diagnosis and Development, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia
| | - David Francis
- Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia
| | - Matthew F Hunter
- Department of Pediatrics, Monash University, Clayton, VIC, Australia.,Monash Genetics, Monash Health, Melbourne, VIC, Australia
| | - Justine Elliott
- Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia
| | - Carolyn Rogers
- Genetics of Learning Disability Service, Hunter Genetics, Waratah, NSW, Australia
| | - Michael J Field
- Genetics of Learning Disability Service, Hunter Genetics, Waratah, NSW, Australia
| | - Jonathan Cohen
- Fragile X Alliance Inc, Centre for Developmental Disability Health Victoria, Monash University, North Caulfield, Clayton, VIC, Australia
| | - Lorena Santa Maria
- Laboratory of Molecular Cytogenetics, Department of Genetics and Metabolic Diseases, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile
| | - Victor Faundes
- Laboratory of Molecular Cytogenetics, Department of Genetics and Metabolic Diseases, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile
| | - Bianca Curotto
- Laboratory of Molecular Cytogenetics, Department of Genetics and Metabolic Diseases, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile
| | - Paulina Morales
- Laboratory of Molecular Cytogenetics, Department of Genetics and Metabolic Diseases, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile
| | - Cesar Trigo
- Laboratory of Molecular Cytogenetics, Department of Genetics and Metabolic Diseases, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile
| | - Isabel Salas
- Laboratory of Molecular Cytogenetics, Department of Genetics and Metabolic Diseases, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile
| | - Angelica M Alliende
- Laboratory of Molecular Cytogenetics, Department of Genetics and Metabolic Diseases, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile
| | - David J Amor
- Faculty of Medicine, Dentistry and Health Sciences, Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia.,Neurodisability and Rehabilitation, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia
| | - David E Godler
- Diagnosis and Development, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia.,Faculty of Medicine, Dentistry and Health Sciences, Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia
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18
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Significantly Elevated FMR1 mRNA and Mosaicism for Methylated Premutation and Full Mutation Alleles in Two Brothers with Autism Features Referred for Fragile X Testing. Int J Mol Sci 2019; 20:ijms20163907. [PMID: 31405222 PMCID: PMC6721168 DOI: 10.3390/ijms20163907] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Revised: 08/04/2019] [Accepted: 08/07/2019] [Indexed: 11/26/2022] Open
Abstract
Although fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥200 cytosine-guanine-guanine (CGG) repeats, and a decrease in FMR1 mRNA and its protein (FMRP), incomplete silencing has been associated with more severe autism features in FXS males. This study reports on brothers (B1 and B2), aged 5 and 2 years, with autistic features and language delay, but a higher non-verbal IQ in comparison to typical FXS. CGG sizing using AmplideX PCR only identified premutation (PM: 55–199 CGGs) alleles in blood. Similarly, follow-up in B1 only revealed PM alleles in saliva and skin fibroblasts; whereas, an FM expansion was detected in both saliva and buccal DNA of B2. While Southern blot analysis of blood detected an unmethylated FM, methylation analysis with a more sensitive methodology showed that B1 had partially methylated PM alleles in blood and fibroblasts, which were completely unmethylated in buccal and saliva cells. In contrast, B2 was partially methylated in all tested tissues. Moreover, both brothers had FMR1 mRNA ~5 fold higher values than those of controls, FXS and PM cohorts. In conclusion, the presence of unmethylated FM and/or PM in both brothers may lead to an overexpression of toxic expanded mRNA in some cells, which may contribute to neurodevelopmental problems, including elevated autism features.
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Baker EK, Arpone M, Aliaga SM, Bretherton L, Kraan CM, Bui M, Slater HR, Ling L, Francis D, Hunter MF, Elliott J, Rogers C, Field M, Cohen J, Cornish K, Santa Maria L, Faundes V, Curotto B, Morales P, Trigo C, Salas I, Alliende AM, Amor DJ, Godler DE. Incomplete silencing of full mutation alleles in males with fragile X syndrome is associated with autistic features. Mol Autism 2019; 10:21. [PMID: 31073396 PMCID: PMC6499941 DOI: 10.1186/s13229-019-0271-7] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2019] [Accepted: 04/03/2019] [Indexed: 11/10/2022] Open
Abstract
Background Fragile X syndrome (FXS) is a common monogenic cause of intellectual disability with autism features. While it is caused by loss of the FMR1 product (FMRP), mosaicism for active and inactive FMR1 alleles, including alleles termed premutation (PM: 55-199 CGGs), is not uncommon. Importantly, both PM and active full mutation (FM: ≥ 200 CGGs) alleles often express elevated levels of mRNA that are thought to be toxic. This study determined if complete FMR1 mRNA silencing from FM alleles and/or levels of FMR1 mRNA (if present) in blood are associated with intellectual functioning and autism features in FXS. Methods The study cohort included 98 participants (70.4% male) with FXS (FM-only and PM/FM mosaic) aged 1-43 years. A control group of 14 females were used to establish control FMR1 mRNA reference range. Intellectual functioning and autism features were assessed using the Mullen Scales of Early Learning or an age-appropriate Wechsler Scale and the Autism Diagnostic Observation Schedule-2nd Edition (ADOS-2), respectively. FMR1 mRNA was analysed in venous blood collected at the time of assessments, using the real-time PCR relative standard curve method. Results Females with FXS had significantly higher levels of FMR1 mRNA (p < 0.001) than males. FMR1 mRNA levels were positively associated with age (p < 0.001), but not with intellectual functioning and autistic features in females. FM-only males (aged < 19 years) expressing FM FMR1 mRNA had significantly higher ADOS calibrated severity scores compared to FM-only males with completely silenced FMR1 (p = 0.011). However, there were no significant differences between these subgroups on intellectual functioning. In contrast, decreased levels of FMR1 mRNA were associated with decreased intellectual functioning in FXS males (p = 0.029), but not autism features, when combined with the PM/FM mosaic group. Conclusion Incomplete silencing of toxic FM RNA may be associated with autistic features, but not intellectual functioning in FXS males. While decreased levels of mRNA may be more predictive of intellectual functioning than autism features. If confirmed in future studies, these findings may have implications for patient stratification, outcome measure development, and design of clinical and pre-clinical trials in FXS.
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Affiliation(s)
- Emma K. Baker
- Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, 50 Flemington Rd, Parkville, VIC 3052 Australia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia
| | - Marta Arpone
- Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, 50 Flemington Rd, Parkville, VIC 3052 Australia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia
- Brain and Mind, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Australia
| | - Solange M. Aliaga
- Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, 50 Flemington Rd, Parkville, VIC 3052 Australia
| | - Lesley Bretherton
- Brain and Mind, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Australia
| | - Claudine M. Kraan
- Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, 50 Flemington Rd, Parkville, VIC 3052 Australia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia
| | - Minh Bui
- Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, University of Melbourne, Carlton, Australia
| | - Howard R. Slater
- Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, 50 Flemington Rd, Parkville, VIC 3052 Australia
| | - Ling Ling
- Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, 50 Flemington Rd, Parkville, VIC 3052 Australia
| | - David Francis
- Victorian Clinical Genetics Services and Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC Australia
| | - Matthew F. Hunter
- Monash Genetics, Monash Health, Melbourne, VIC Australia
- Department of Paediatrics, Monash University, Clayton, VIC Australia
| | - Justine Elliott
- Victorian Clinical Genetics Services and Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC Australia
| | - Carolyn Rogers
- Genetics of Learning Disability Service, Hunter Genetics, Waratah, NSW Australia
| | - Michael Field
- Genetics of Learning Disability Service, Hunter Genetics, Waratah, NSW Australia
| | - Jonathan Cohen
- Fragile X Alliance Inc, North Caulfield, VIC and Center for Developmental Disability Health Victoria, Monash University, Clayton, Australia
| | - Kim Cornish
- Monash Institute of Cognitive and Clinical Neurosciences, Monash University, Clayton, VIC Australia
| | - Lorena Santa Maria
- Molecular and Cytogenetics Laboratory, INTA, University of Chile, Santiago, Chile
| | - Victor Faundes
- Molecular and Cytogenetics Laboratory, INTA, University of Chile, Santiago, Chile
| | - Bianca Curotto
- Molecular and Cytogenetics Laboratory, INTA, University of Chile, Santiago, Chile
| | - Paulina Morales
- Molecular and Cytogenetics Laboratory, INTA, University of Chile, Santiago, Chile
| | - Cesar Trigo
- Molecular and Cytogenetics Laboratory, INTA, University of Chile, Santiago, Chile
| | - Isabel Salas
- Molecular and Cytogenetics Laboratory, INTA, University of Chile, Santiago, Chile
| | - Angelica M. Alliende
- Molecular and Cytogenetics Laboratory, INTA, University of Chile, Santiago, Chile
| | - David J. Amor
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia
- Neurodisability and Rehabilitation, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Australia
| | - David E. Godler
- Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, 50 Flemington Rd, Parkville, VIC 3052 Australia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia
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Abu Diab M, Eiges R. The Contribution of Pluripotent Stem Cell (PSC)-Based Models to the Study of Fragile X Syndrome (FXS). Brain Sci 2019; 9:brainsci9020042. [PMID: 30769941 PMCID: PMC6406836 DOI: 10.3390/brainsci9020042] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2019] [Revised: 02/11/2019] [Accepted: 02/13/2019] [Indexed: 02/06/2023] Open
Abstract
Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from a deficiency in the fragile X mental retardation protein (FMRP) due to a CGG repeat expansion in the 5′-UTR of the X-linked FMR1 gene. When CGGs expand beyond 200 copies, they lead to epigenetic gene silencing of the gene. In addition, the greater the allele size, the more likely it will become unstable and exhibit mosaicism for expansion size between and within tissues in affected individuals. The timing and mechanisms of FMR1 epigenetic gene silencing and repeat instability are far from being understood given the lack of appropriate cellular and animal models that can fully recapitulate the molecular features characteristic of the disease pathogenesis in humans. This review summarizes the data collected to date from mutant human embryonic stem cells, induced pluripotent stem cells, and hybrid fusions, and discusses their contribution to the investigation of FXS, their key limitations, and future prospects.
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Affiliation(s)
- Manar Abu Diab
- Stem Cell Research Laboratory, Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem 91031, Israel.
- School of Medicine, Hebrew University of Jerusalem, Jerusalem 9112102, Israel.
| | - Rachel Eiges
- Stem Cell Research Laboratory, Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem 91031, Israel.
- School of Medicine, Hebrew University of Jerusalem, Jerusalem 9112102, Israel.
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21
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Yau SY, Bettio L, Chiu J, Chiu C, Christie BR. Fragile-X Syndrome Is Associated With NMDA Receptor Hypofunction and Reduced Dendritic Complexity in Mature Dentate Granule Cells. Front Mol Neurosci 2019; 11:495. [PMID: 30705620 PMCID: PMC6344420 DOI: 10.3389/fnmol.2018.00495] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2018] [Accepted: 12/27/2018] [Indexed: 12/11/2022] Open
Abstract
Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. It is caused by the overexpansion of cytosine-guanine-guanine (CGG) trinucleotide in Fmr1 gene, resulting in complete loss of the fragile X mental retardation protein (FMRP). Previous studies using Fmr1 knockout (Fmr1 KO) mice have suggested that a N-methyl-D-aspartate receptors (NMDAR) hypofunction in the hippocampal dentate gyrus may partly contribute to cognitive impairments in FXS. Since activation of NMDAR plays an important role in dendritic arborization during neuronal development, we examined whether deficits in NMDAR function are associated with alterations in dendritic complexity in the hippocampal dentate region. The dentate granule cell layer (GCL) presents active postnatal neurogenesis, and consists of a heterogenous neuronal population with gradient ages from the superficial to its deep layer. Here, we show that neurons with multiple primary dendrites that reside in the outer GCL of Fmr1 KO mice display significantly smaller NMDAR excitatory post-synaptic currents (EPSCs) and a higher α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to NMDA ratio in comparison to their wild-type counterparts. These deficits were associated with a significant decrease in dendritic complexity, with both dendritic length and number of intersections being significantly reduced. In contrast, although neurons with a single primary dendrite resided in the inner GCL of Fmr1 KO mice had a trend toward a reduction in NMDAR EPSCs and a higher AMPA/NMDA ratio, no alterations were found in dendritic complexity at this developmental stage. Our data indicate that the loss of FMRP causes NMDAR deficits and reduced dendritic complexity in granule neurons with multiple primary dendrites which are thought to be more mature in the GCL.
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Affiliation(s)
- Suk-Yu Yau
- Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, BC, Canada
| | - Luis Bettio
- Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, BC, Canada
| | - Jason Chiu
- Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, BC, Canada
| | - Christine Chiu
- Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, BC, Canada
| | - Brian R Christie
- Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, BC, Canada
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22
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Elizur SE, Friedman Gohas M, Dratviman-Storobinsky O, Cohen Y. Pathophysiology Mechanisms in Fragile-X Primary Ovarian Insufficiency. Methods Mol Biol 2019; 1942:165-171. [PMID: 30900184 DOI: 10.1007/978-1-4939-9080-1_14] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
Women who carry the FMR1 premutation may suffer from ongoing deterioration of ovarian function. The lucidity of the molecular mechanism of FXTAS is emerging and findings from research in the field of FXTAS could elucidate the pathogenesis of FXPOI. To date there are three possible mechanisms for ovarian dysfunction in FMR1 permutation carriers. The first is the RNA toxic gain-of-function mechanism initiating loss of function of over 30 specific RNA-binding proteins. The second is associated to the formation of an abnormal polyglycine-containing protein (FMRpolyG), and the third is related to novel lncRNAs, named FMR4 and FMR6. Herein we describe our laboratory methodology, focusing on the culturing and manipulation of granulosa cells from human female premutation carriers, trying to reveal the actual possible mechanisms liable to FXPOI. Detecting the precise pathways in premutation carrier might facilitate in offering these women the opportunity to make an informed decision regarding their reproductive and family planning.
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Affiliation(s)
- Shai E Elizur
- IVF unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center (Tel Hashomer), Ramat Gan, Israel
- The Fertility Research Laboratory, Chaim Sheba Medical Center (Tel Hashomer), Ramat Gan, Israel
| | - Moran Friedman Gohas
- The Fertility Research Laboratory, Chaim Sheba Medical Center (Tel Hashomer), Ramat Gan, Israel
| | - Olga Dratviman-Storobinsky
- IVF unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center (Tel Hashomer), Ramat Gan, Israel
| | - Yoram Cohen
- IVF unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center (Tel Hashomer), Ramat Gan, Israel.
- The Fertility Research Laboratory, Chaim Sheba Medical Center (Tel Hashomer), Ramat Gan, Israel.
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23
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Abstract
Fragile X syndrome (FXS) is one of the most common reasons for intellectual disability (ID). First described in the 1940s, it took many years to understand the disease. The awe-inspiring breakthroughs in both science and technology facilitated the recognition of the unique inheritance pattern and the genetic mechanism of fragile X. In this chapter we describe the history and evolution of our understanding of FXS as mirrored by advances in genetics.
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Affiliation(s)
- Adi Reches
- Genetic Institute and Racine IVF Unit at Lis Maternity Hospital Tel Aviv, Sackler Faculty of Medicine, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel.
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24
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Cai X, Arif M, Wan H, Kornreich R, Edelmann LJ. Clinical Genetic Testing for Fragile X Syndrome by Polymerase Chain Reaction Amplification and Southern Blot Analyses. Methods Mol Biol 2019; 1942:11-27. [PMID: 30900172 DOI: 10.1007/978-1-4939-9080-1_2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
Fragile X syndrome (FXS) is characterized by mental retardation and in the vast majority of cases it is caused by expansion of CGG trinucleotide repeats in the 5' untranslated region (or UTR) in the FMR1 gene on the X chromosome. The size and methylation status of CGG repeats are correlated with the clinical phenotype of FMR1-related disorders. The methods used for clinical genetic testing of FXS include polymerase chain reaction (PCR) amplification and Southern blot analyses, which effectively detect alleles with repeats in the normal, intermediate, premutation, and full mutation size ranges, as well as the methylation status of FMR1 promoter region.
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Affiliation(s)
- Xiaoqiang Cai
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Sema4, a Mount Sinai Venture, Stamford, CT, USA
- WuXi AppTec Group, Shanghai, China
| | - Mohammad Arif
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Haolei Wan
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Sema4, a Mount Sinai Venture, Stamford, CT, USA
| | - Ruth Kornreich
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Sema4, a Mount Sinai Venture, Stamford, CT, USA
| | - Lisa J Edelmann
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
- Sema4, a Mount Sinai Venture, Stamford, CT, USA.
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25
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Mailick MR, Movaghar A, Hong J, Greenberg JS, DaWalt LS, Zhou L, Jackson J, Rathouz PJ, Baker MW, Brilliant M, Page D, Berry-Kravis E. Health Profiles of Mosaic Versus Non-mosaic FMR1 Premutation Carrier Mothers of Children With Fragile X Syndrome. Front Genet 2018; 9:173. [PMID: 29868121 PMCID: PMC5964198 DOI: 10.3389/fgene.2018.00173] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Accepted: 04/27/2018] [Indexed: 11/19/2022] Open
Abstract
The FMR1 premutation is of increasing interest to the FXS community, as questions about a primary premutation phenotype warrant research attention. 100 FMR1 premutation carrier mothers (mean age = 58; 67–138 CGG repeats) of adults with fragile X syndrome were studied with respect to their physical and mental health, motor, and neurocognitive characteristics. We explored the correlates of CGG repeat mosaicism in women with expanded alleles. Mothers provided buccal swabs from which DNA was extracted and the FMR1 CGG genotyping was performed (Amplidex Kit, Asuragen). Mothers were categorized into three groups: Group 1: premutation non-mosaic (n = 45); Group 2: premutation mosaic (n = 41), and Group 3: premutation/full mutation mosaic (n = 14). Group 2 mothers had at least two populations of cells with different allele sizes in the premutation range besides their major expanded allele. Group 3 mothers had a very small population of cells in the full mutation range (>200 CGGs) in addition to one or multiple populations of cells with different allele sizes in the premutation range. Machine learning (random forest) was used to identify symptoms and conditions that correctly classified mothers with respect to mosaicism; follow-up comparisons were made to characterize the three groups. In categorizing mosaicism, the random forest yielded significantly better classification than random classification, with overall area under the receiver operating characteristic curve (AUROC) of 0.737. Among the most important symptoms and conditions that contributed to the classification were anxiety, menopause symptoms, executive functioning limitations, and difficulty walking several blocks, with the women who had full mutation mosaicism (Group 3) unexpectedly having better health. Although only 14 premutation carrier mothers in the present sample also had a small population of full mutation cells, their profile of comparatively better health, mental health, and executive functioning was unexpected. This preliminary finding should prompt additional research on larger numbers of participants with more extensive phenotyping to confirm the clinical correlates of low-level full mutation mosaicism in premutation carriers and to probe possible mechanisms.
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Affiliation(s)
- Marsha R Mailick
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States
| | - Arezoo Movaghar
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States.,Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, United States
| | - Jinkuk Hong
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States
| | - Jan S Greenberg
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States
| | - Leann S DaWalt
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States
| | - Lili Zhou
- Department of Pediatrics, Rush University Medical Center, Chicago, IL, United States.,Department of Pathology, Rush University Medical Center, Chicago, IL, United States
| | - Jonathan Jackson
- Department of Pediatrics, Rush University Medical Center, Chicago, IL, United States
| | - Paul J Rathouz
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States.,Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI, United States
| | - Mei W Baker
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States.,Wisconsin State Laboratory of Hygiene, Madison, WI, United States
| | - Murray Brilliant
- Waisman Center, University of Wisconsin-Madison, Madison, WI, United States.,Marshfield Clinic Research Institute, Marshfield, WI, United States
| | - David Page
- Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI, United States
| | - Elizabeth Berry-Kravis
- Department of Pediatrics, Rush University Medical Center, Chicago, IL, United States.,Department of Neurological Sciences and Biochemistry, Rush University Medical Center, Chicago, IL, United States
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26
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Zeidman Kalman T, Khalandovsky R, Tenenbaum Gonikman E, Bercovici M. Monitoring Dissociation Kinetics during Electrophoretic Focusing to Enable High-Specificity Nucleic Acid Detection. Angew Chem Int Ed Engl 2018. [DOI: 10.1002/ange.201711673] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Tal Zeidman Kalman
- Russell Berrie Nanotechnology Institute; Technion-Israel Institute of Technology; Haifa 3200003 Israel
| | - Rebecca Khalandovsky
- Faculty of Mechanical Engineering; Technion-Israel Institute of Technology; Haifa 3200003 Israel
| | - Elena Tenenbaum Gonikman
- Faculty of Mechanical Engineering; Technion-Israel Institute of Technology; Haifa 3200003 Israel
| | - Moran Bercovici
- Faculty of Mechanical Engineering; Technion-Israel Institute of Technology; Haifa 3200003 Israel
- Russell Berrie Nanotechnology Institute; Technion-Israel Institute of Technology; Haifa 3200003 Israel
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27
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Zeidman Kalman T, Khalandovsky R, Tenenbaum Gonikman E, Bercovici M. Monitoring Dissociation Kinetics during Electrophoretic Focusing to Enable High-Specificity Nucleic Acid Detection. Angew Chem Int Ed Engl 2018; 57:3343-3348. [DOI: 10.1002/anie.201711673] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2017] [Revised: 12/31/2017] [Indexed: 12/12/2022]
Affiliation(s)
- Tal Zeidman Kalman
- Russell Berrie Nanotechnology Institute; Technion-Israel Institute of Technology; Haifa 3200003 Israel
| | - Rebecca Khalandovsky
- Faculty of Mechanical Engineering; Technion-Israel Institute of Technology; Haifa 3200003 Israel
| | - Elena Tenenbaum Gonikman
- Faculty of Mechanical Engineering; Technion-Israel Institute of Technology; Haifa 3200003 Israel
| | - Moran Bercovici
- Faculty of Mechanical Engineering; Technion-Israel Institute of Technology; Haifa 3200003 Israel
- Russell Berrie Nanotechnology Institute; Technion-Israel Institute of Technology; Haifa 3200003 Israel
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28
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Esanov R, Andrade NS, Bennison S, Wahlestedt C, Zeier Z. The FMR1 promoter is selectively hydroxymethylated in primary neurons of fragile X syndrome patients. Hum Mol Genet 2018; 25:4870-4880. [PMID: 28173181 DOI: 10.1093/hmg/ddw311] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2016] [Revised: 08/17/2016] [Accepted: 09/02/2016] [Indexed: 12/13/2022] Open
Abstract
Fragile X syndrome (FXS) results from a repeat expansion mutation near the FMR1 gene promoter and is the most common form of heritable intellectual disability and autism. Full mutations larger than 200 CGG repeats trigger FMR1 heterochromatinization and loss of gene expression, which is primarily responsible for the pathological features of FXS . In contrast, smaller pre-mutations of 55–200 CGG are associated with FMR1 overexpression and Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative condition. While the role of 5-methylcytosine (5mC) in FMR1 gene silencing has been studied extensively, the role of 5-hydroxymethylation (5hmC), a newly discovered epigenetic mark produced through active DNA demethylation, has not been previously investigated in FXS neurons. Here, we used two complementary epigenetic assays, 5hmC sensitive restriction digest and ten-eleven translocation-assisted bisulfite pyrosequencing, to quantify FMR1 5mC and 5hmC levels. We observed increased levels of 5hmC at the FMR1 promoter in FXS patient brains with full-mutations relative to pre-mutation carriers and unaffected controls. In addition, we found that 5hmC enrichment at the FMR1 locus in FXS cells is specific to neurons by utilizing a nuclei sorting technique to separate neuronal and glial DNA fractions from post-mortem brain tissues. This FMR1 5hmC enrichment was not present in cellular models of FXS including fibroblasts, lymphocytes and reprogrammed neurons, indicating they do not fully recapitulate this epigenetic feature of disease. Future studies could investigate the potential to leverage this epigenetic pathway to restore FMR1 expression and discern whether levels of 5hmC correlate with phenotypic severity.
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Affiliation(s)
- Rustam Esanov
- Department of Psychiatry & Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Nadja S Andrade
- Department of Psychiatry & Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Sarah Bennison
- Department of Psychiatry & Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Claes Wahlestedt
- Department of Psychiatry & Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Zane Zeier
- Department of Psychiatry & Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, Miami, FL 33136, USA
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29
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Mor-Shaked H, Eiges R. Reevaluation of FMR1 Hypermethylation Timing in Fragile X Syndrome. Front Mol Neurosci 2018; 11:31. [PMID: 29467618 PMCID: PMC5808132 DOI: 10.3389/fnmol.2018.00031] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2017] [Accepted: 01/24/2018] [Indexed: 12/27/2022] Open
Abstract
Fragile X syndrome (FXS) is one of the most common heritable forms of cognitive impairment. It results from a fragile X mental retardation protein (FMRP) protein deficiency caused by a CGG repeat expansion in the 5'-UTR of the X-linked FMR1 gene. Whereas in most individuals the number of CGGs is steady and ranges between 5 and 44 units, in patients it becomes extensively unstable and expands to a length exceeding 200 repeats (full mutation). Interestingly, this disease is exclusively transmitted by mothers who carry a premutation allele (55-200 CGG repeats). When the CGGs reach the FM range, they trigger the spread of abnormal DNA methylation, which coincides with a switch from active to repressive histone modifications. This results in epigenetic gene silencing of FMR1 presumably by a multi-stage, developmentally regulated process. The timing of FMR1 hypermethylation and transcription silencing is still hotly debated. There is evidence that hypermethylation varies considerably between and within the tissues of patients as well as during fetal development, thus supporting the view that FMR1 silencing is a post-zygotic event that is developmentally structured. On the other hand, it may be established in the female germ line and transmitted to the fetus as an integral part of the mutation. This short review summarizes the data collected to date concerning the timing of FMR1 epigenetic gene silencing and reassess the evidence in favor of the theory that gene inactivation takes place by a developmentally regulated process around the 10th week of gestation.
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Affiliation(s)
- Hagar Mor-Shaked
- Stem Cell Research Laboratory, Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.,Hebrew University Medical School, Jerusalem, Israel
| | - Rachel Eiges
- Stem Cell Research Laboratory, Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel.,Hebrew University Medical School, Jerusalem, Israel
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30
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Bearden CE, Glahn DC. Cognitive genomics: Searching for the genetic roots of neuropsychological functioning. Neuropsychology 2017; 31:1003-1019. [PMID: 29376674 PMCID: PMC5791763 DOI: 10.1037/neu0000412] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
OBJECTIVE Human cognition has long been known to be under substantial genetic control. With the complete mapping of the human genome, genome-wide association studies for many complex traits have proliferated; however, the highly polygenic nature of intelligence has made the identification of the precise genes that influence both global and specific cognitive abilities more difficult than anticipated. METHOD Here, we review the latest developments in the genomics of cognition, including a discussion of methodological advances in the genetic analysis of complex traits, and shared genetic contributions to cognitive abilities and neuropsychiatric disorders. RESULTS A wealth of twin and family studies have provided compelling evidence for a strong heritable component of both global and specific cognitive abilities, and for the existence of "generalist genes" responsible for a large portion of the variance in diverse cognitive abilities. Increasingly sophisticated analytic tools and ever-larger sample sizes are now facilitating the identification of specific genetic and molecular underpinnings of cognitive abilities, leading to optimism regarding possibilities for novel treatments for illnesses related to cognitive function. CONCLUSIONS We conclude with a set of future directions for the field, which will further accelerate discoveries regarding the biological pathways relevant to cognitive abilities. These, in turn, may be further interrogated in order to link biological mechanisms to behavior. (PsycINFO Database Record
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Affiliation(s)
- Carrie E Bearden
- Department of Psychiatry, University of California at Los Angeles
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Hayward BE, Kumari D, Usdin K. Recent advances in assays for the fragile X-related disorders. Hum Genet 2017; 136:1313-1327. [PMID: 28866801 DOI: 10.1007/s00439-017-1840-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Accepted: 08/24/2017] [Indexed: 12/17/2022]
Abstract
The fragile X-related disorders are a group of three clinical conditions resulting from the instability of a CGG-repeat tract at the 5' end of the FMR1 transcript. Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI) are disorders seen in carriers of FMR1 alleles with 55-200 repeats. Female carriers of these premutation (PM) alleles are also at risk of having a child who has an FMR1 allele with >200 repeats. Most of these full mutation (FM) alleles are epigenetically silenced resulting in a deficit of the FMR1 gene product, FMRP. This results in fragile X Syndrome (FXS), the most common heritable cause of intellectual disability and autism. The diagnosis and study of these disorders is challenging, in part because the detection of alleles with large repeat numbers has, until recently, been either time-consuming or unreliable. This problem is compounded by the mosaicism for repeat length and/or DNA methylation that is frequently seen in PM and FM carriers. Furthermore, since AGG interruptions in the repeat tract affect the risk that a FM allele will be maternally transmitted, the ability to accurately detect these interruptions in female PM carriers is an additional challenge that must be met. This review will discuss some of the pros and cons of some recently described assays for these disorders, including those that detect FMRP levels directly, as well as emerging technologies that promise to improve the diagnosis of these conditions and to be useful in both basic and translational research settings.
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Affiliation(s)
- Bruce E Hayward
- Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Building 8, Room 2A19, National Institutes of Health, 8 Center Drive MSC 0830, Bethesda, MD, 20892, USA
| | - Daman Kumari
- Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Building 8, Room 2A19, National Institutes of Health, 8 Center Drive MSC 0830, Bethesda, MD, 20892, USA
| | - Karen Usdin
- Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Building 8, Room 2A19, National Institutes of Health, 8 Center Drive MSC 0830, Bethesda, MD, 20892, USA.
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Ciaccio C, Fontana L, Milani D, Tabano S, Miozzo M, Esposito S. Fragile X syndrome: a review of clinical and molecular diagnoses. Ital J Pediatr 2017; 43:39. [PMID: 28420439 PMCID: PMC5395755 DOI: 10.1186/s13052-017-0355-y] [Citation(s) in RCA: 90] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2016] [Accepted: 04/07/2017] [Indexed: 12/24/2022] Open
Abstract
Background Fragile X Syndrome (FXS) is the second cause of intellectual disability after Down syndrome and the most prevalent cause of intellectual disability in males, affecting 1:5000–7000 men and 1:4000–6000 women. It is caused by an alteration of the FMR1 gene, which maps at the Xq27.3 band: more than 99% of individuals have a CGG expansion (>200 triplets) in the 5′ UTR of the gene, and FMR1 mutations and duplication/deletion are responsible for the remaining (<1%) molecular diagnoses of FXS. The aim of this review was to gather the current clinical and molecular knowledge about FXS to provide clinicians with a tool to guide the initial assessment and follow-up of FXS and to offer to laboratory workers and researchers an update about the current diagnostic procedures. Discussion FXS is a well-known condition; however, most of the studies thus far have focused on neuropsychiatric features. Unfortunately, some of the available studies have limitations, such as the paucity of patients enrolled or bias due to the collection of the data in a single-country population, which may be not representative of the average global FXS population. In recent years, insight into the adult presentation of the disease has progressively increased. Pharmacological treatment of FXS is essentially symptom based, but the growing understanding of the molecular and biological mechanisms of the disease are paving the way to targeted therapy, which may reverse the effects of FMRP deficiency and be a real cure for the disease itself, not just its symptoms. Conclusions The clinical spectrum of FXS is wide, presenting not only as an isolated intellectual disability but as a multi-systemic condition, involving predominantly the central nervous system but potentially affecting any apparatus. Given the relative high frequency of the condition and its complex clinical management, FXS appears to have an important economic and social burden.
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Affiliation(s)
- Claudia Ciaccio
- Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation, University of Milan, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via Commenda 9, 20122, Milan, Italy
| | - Laura Fontana
- Division of Pathology, Department of Pathophysiology and Transplantation, University of Milan, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Donatella Milani
- Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation, University of Milan, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via Commenda 9, 20122, Milan, Italy
| | - Silvia Tabano
- Division of Pathology, Department of Pathophysiology and Transplantation, University of Milan, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Monica Miozzo
- Division of Pathology, Department of Pathophysiology and Transplantation, University of Milan, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Susanna Esposito
- Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, Piazza Lucio Severi 1, Loc. S. Andrea delle Fratte, 06132, Perugia, Italy.
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Martindale JE. Diagnosis of Spinocerebellar Ataxias Caused by Trinucleotide Repeat Expansions. CURRENT PROTOCOLS IN HUMAN GENETICS 2017; 92:9.30.1-9.30.22. [PMID: 28075481 DOI: 10.1002/cphg.30] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
Spinocerebellar ataxias (SCAs) are a group of disorders that are both clinically and genetically heterogeneous. They usually demonstrate onset in adulthood, but some forms may have juvenile or infantile onset. There are many different types of SCA, demonstrating different modes of inheritance and types of mutation. The most common forms are due to dominantly inherited expansions in trinucleotide repeat sequences located within the coding region of the relevant genes, and these are readily identifiable by molecular genetic testing. In general, it is possible to test for these disorders using PCR-based assays, amplifying across the trinucleotide repeat regions and sizing the PCR products to determine the number of repeats. Larger expansions are generally associated with a more severe presentation of the disorder, and alternative methods may be necessary to detect these alleles. This protocol describes methods for detecting normal and expanded triplet repeat alleles in the most common SCA genes. © 2017 by John Wiley & Sons, Inc.
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Affiliation(s)
- Joanne E Martindale
- Sheffield Diagnostic Genetics Service, Sheffield Children's NHS Foundation Trust, Sheffield, United Kingdom
- Wellington Regional Genetics Laboratory, Wellington Hospital, Newtown, Wellington, New Zealand
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Hadd AG, Filipovic-Sadic S, Zhou L, Williams A, Latham GJ, Berry-Kravis E, Hall DA. A methylation PCR method determines FMR1 activation ratios and differentiates premutation allele mosaicism in carrier siblings. Clin Epigenetics 2016; 8:130. [PMID: 27980694 PMCID: PMC5131543 DOI: 10.1186/s13148-016-0280-8] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2016] [Accepted: 10/23/2016] [Indexed: 01/02/2023] Open
Abstract
Background Epigenetic modifications of the fragile X mental retardation 1 (FMR1) gene locus may impact the risk for reproductive and neurological disorders associated with expanded trinucleotide repeats and methylation status in the 5′ untranslated region. FMR1 methylation is commonly assessed by Southern blot (SB) analysis, yet this method suffers a cumbersome workflow and relatively poor sizing resolution especially for premutation allele characteristic for fragile X-associated disorders. In this study, a methylation PCR (mPCR) assay was used to evaluate correlations among genotype, epitype, and phenotype in fragile X premutation (PM) carrier women in order to advance the understanding of the association between molecular determinants and the presence of fragile X-associated tremor and ataxia (FXTAS). Results Activation ratios (ARs) in 39 PM women were determined by mPCR and compared with SB analysis. ARs were distributed across a range of values including five samples with <20% AR and six with >80% AR. The two methods were correlated (R2 of 0.87 and F test of <0.001), indicating that mPCR can measure AR in agreement with established assays. However, mPCR was unique in identifying novel and distinct patterns of methylation mosaicism in premutation carrier women, including seven sibling pairs that were assessed using FXTAS clinical rating scales. Of note, four of six pairs with defined age of onset for neurological signs showed ARs consistent with skewed activation of the pathogenic expanded allele. One subject with severe FXTAS had a mosaic full mutation allele identified using mPCR but not detected by SB analysis. Conclusions We utilized a repeatable and streamlined methodology to characterize FMR1 inactivation in premutation carrier women. The method was concordant with SB analysis and provided higher resolution information on allele and methylation mosaicism. This approach can facilitate the characterization of epigenetic factors influencing fragile X phenotypes in larger cohort studies that can advance understanding and treatment of fragile X-associated disorders. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0280-8) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Andrew G Hadd
- Research and Technology Development, Asuragen, Inc., Austin, TX 78744 USA
| | | | - Lili Zhou
- Department of Neurological Sciences, Rush University Medical Center, Chicago, IL USA ; Departments of Pediatrics and Biochemistry, Rush University Medical Center, Chicago, IL USA
| | - Arianna Williams
- Department of Neurological Sciences, Rush University Medical Center, Chicago, IL USA
| | - Gary J Latham
- Research and Technology Development, Asuragen, Inc., Austin, TX 78744 USA
| | - Elizabeth Berry-Kravis
- Department of Neurological Sciences, Rush University Medical Center, Chicago, IL USA ; Departments of Pediatrics and Biochemistry, Rush University Medical Center, Chicago, IL USA
| | - Deborah A Hall
- Department of Neurological Sciences, Rush University Medical Center, Chicago, IL USA
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Yau S, Bostrom C, Chiu J, Fontaine C, Sawchuk S, Meconi A, Wortman R, Truesdell E, Truesdell A, Chiu C, Hryciw B, Eadie B, Ghilan M, Christie B. Impaired bidirectional NMDA receptor dependent synaptic plasticity in the dentate gyrus of adult female Fmr1 heterozygous knockout mice. Neurobiol Dis 2016; 96:261-270. [DOI: 10.1016/j.nbd.2016.09.012] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2016] [Revised: 09/07/2016] [Accepted: 09/17/2016] [Indexed: 11/29/2022] Open
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Macpherson JN, Murray A. Development of Genetic Testing for Fragile X Syndrome and Associated Disorders, and Estimates of the Prevalence of FMR1 Expansion Mutations. Genes (Basel) 2016; 7:genes7120110. [PMID: 27916885 PMCID: PMC5192486 DOI: 10.3390/genes7120110] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2016] [Revised: 11/10/2016] [Accepted: 11/24/2016] [Indexed: 12/15/2022] Open
Abstract
The identification of a trinucleotide (CGG) expansion as the chief mechanism of mutation in Fragile X syndrome in 1991 heralded a new chapter in molecular diagnostic genetics and generated a new perspective on mutational mechanisms in human genetic disease, which rapidly became a central paradigm (“dynamic mutation”) as more and more of the common hereditary neurodevelopmental disorders were ascribed to this novel class of mutation. The progressive expansion of a CGG repeat in the FMR1 gene from “premutation” to “full mutation” provided an explanation for the “Sherman paradox,” just as similar expansion mechanisms in other genes explained the phenomenon of “anticipation” in their pathogenesis. Later, FMR1 premutations were unexpectedly found associated with two other distinct phenotypes: primary ovarian insufficiency and tremor-ataxia syndrome. This review will provide a historical perspective on procedures for testing and reporting of Fragile X syndrome and associated disorders, and the population genetics of FMR1 expansions, including estimates of prevalence and the influence of AGG interspersions on the rate and probability of expansion.
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Affiliation(s)
- James N Macpherson
- Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury District Hospital, Salisbury SP2 8BJ, UK.
| | - Anna Murray
- Medical School, University of Exeter, RILD Level 3, Royal Devon & Exeter Hospital, Barrack Road, Exeter EX2 5DW, UK.
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Zwemer LM, Nolin SL, Okamoto PM, Eisenberg M, Wick HC, Bianchi DW. Global transcriptome dysregulation in second trimester fetuses with FMR1 expansions. Prenat Diagn 2016; 37:43-52. [PMID: 27646161 DOI: 10.1002/pd.4928] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2016] [Revised: 08/30/2016] [Accepted: 09/14/2016] [Indexed: 02/06/2023]
Abstract
OBJECTIVE We tested the hypothesis that FMR1 expansions would result in global gene dysregulation as early as the second trimester of human fetal development. METHOD Using cell-free fetal RNA obtained from amniotic fluid supernatant and expression microarrays, we compared RNA levels in samples from fetuses with premutation or full mutation allele expansions with control samples. RESULTS We found clear signals of differential gene expression relating to a variety of cellular functions, including ubiquitination, mitochondrial function, and neuronal/synaptic architecture. Additionally, among the genes showing differential gene expression, we saw links to related diseases of intellectual disability and motor function. Finally, within the unique molecular phenotypes established for each mutation set, we saw clear signatures of mitochondrial dysfunction and disrupted neurological function. Patterns of differential gene expression were very different in male and female fetuses with premutation alleles. CONCLUSION These results support a model for which genetic misregulation during fetal development may set the stage for late clinical manifestations of FMR1-related disorders. © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Lillian M Zwemer
- Mother Infant Research Institute, Tufts Medical Center, Boston, MA, USA
| | - Sarah L Nolin
- New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA
| | - Patricia M Okamoto
- Integrated Genetics/Laboratory Corporation of America® Holdings, Westborough, MA, USA
| | - Marcia Eisenberg
- Laboratory Corporation of America® Holdings, Research Triangle Park, NC, USA
| | - Heather C Wick
- Department of Computer Science, Tufts University, Medford, MA, USA
| | - Diana W Bianchi
- Mother Infant Research Institute, Tufts Medical Center, Boston, MA, USA
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Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders. Genes (Basel) 2016; 7:genes7100087. [PMID: 27754417 PMCID: PMC5083926 DOI: 10.3390/genes7100087] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2016] [Revised: 10/06/2016] [Accepted: 10/08/2016] [Indexed: 12/12/2022] Open
Abstract
Fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability and autism. Molecular diagnostic testing of FXS and related disorders (fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS)) relies on a combination of polymerase chain reaction (PCR) and Southern blot (SB) for the fragile X mental retardation 1 (FMR1) CGG-repeat expansion and methylation analyses. Recent advancements in PCR-based technologies have enabled the characterization of the complete spectrum of CGG-repeat mutation, with or without methylation assessment, and, as a result, have reduced our reliance on the labor- and time-intensive SB, which is the gold standard FXS diagnostic test. The newer and more robust triplet-primed PCR or TP-PCR assays allow the mapping of AGG interruptions and enable the predictive analysis of the risks of unstable CGG expansion during mother-to-child transmission. In this review, we have summarized the correlation between several molecular elements, including CGG-repeat size, methylation, mosaicism and skewed X-chromosome inactivation, and the extent of clinical involvement in patients with FMR1-related disorders, and reviewed key developments in PCR-based methodologies for the molecular diagnosis of FXS, FXTAS and FXPOI, and large-scale (CGG)n expansion screening in newborns, women of reproductive age and high-risk populations.
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Mor-Shaked H, Eiges R. Modeling Fragile X Syndrome Using Human Pluripotent Stem Cells. Genes (Basel) 2016; 7:genes7100077. [PMID: 27690107 PMCID: PMC5083916 DOI: 10.3390/genes7100077] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2016] [Revised: 09/02/2016] [Accepted: 09/12/2016] [Indexed: 02/06/2023] Open
Abstract
Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from a loss-of-function mutation by a CGG repeat expansion at the 5′ untranslated region of the X-linked fragile X mental retardation 1 (FMR1) gene. Expansion of the CGG repeats beyond 200 copies results in protein deficiency by leading to aberrant methylation of the FMR1 promoter and the switch from active to repressive histone modifications. Additionally, the CGGs become increasingly unstable, resulting in high degree of variation in expansion size between and within tissues of affected individuals. It is still unclear how the FMR1 protein (FMRP) deficiency leads to disease pathology in neurons. Nor do we know the mechanisms by which the CGG expansion results in aberrant DNA methylation, or becomes unstable in somatic cells of patients, at least in part due to the lack of appropriate animal or cellular models. This review summarizes the current contribution of pluripotent stem cells, mutant human embryonic stem cells, and patient-derived induced pluripotent stem cells to disease modeling of FXS for basic and applied research, including the development of new therapeutic approaches.
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Affiliation(s)
- Hagar Mor-Shaked
- Stem Cell Research Laboratory, Medical Genetics Institute, Shaare Zedek Medical Center Affiliated with the Hebrew University School of Medicine, Jerusalem 91031, Israel.
| | - Rachel Eiges
- Stem Cell Research Laboratory, Medical Genetics Institute, Shaare Zedek Medical Center Affiliated with the Hebrew University School of Medicine, Jerusalem 91031, Israel.
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Bostrom C, Yau SY, Majaess N, Vetrici M, Gil-Mohapel J, Christie BR. Hippocampal dysfunction and cognitive impairment in Fragile-X Syndrome. Neurosci Biobehav Rev 2016; 68:563-574. [DOI: 10.1016/j.neubiorev.2016.06.033] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Revised: 06/21/2016] [Accepted: 06/22/2016] [Indexed: 01/03/2023]
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Schenkel LC, Schwartz C, Skinner C, Rodenhiser DI, Ainsworth PJ, Pare G, Sadikovic B. Clinical Validation of Fragile X Syndrome Screening by DNA Methylation Array. J Mol Diagn 2016; 18:834-841. [PMID: 27585064 DOI: 10.1016/j.jmoldx.2016.06.005] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2016] [Revised: 06/14/2016] [Accepted: 06/21/2016] [Indexed: 01/11/2023] Open
Abstract
Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. It is most frequently caused by an abnormal expansion of the CGG trinucleotide repeat (>200 repeats) located in the promoter of the fragile X mental retardation gene (FMR1), resulting in promoter DNA hypermethylation and gene silencing. Current clinical tests for FXS are technically challenging and labor intensive, and may involve use of hazardous chemicals or radioisotopes. We clinically validated the Illumina Infinium HumanMethylation450 DNA methylation array for FXS screening. We assessed genome-wide and FMR1-specific DNA methylation in 32 males previously diagnosed with FXS, including nine with mosaicism, as well as five females with full mutation, and premutation carrier males (n = 11) and females (n = 11), who were compared to 300 normal control DNA samples. Our findings demonstrate 100% sensitivity and specificity for detection of FXS in male patients, as well as the ability to differentiate patients with mosaic methylation defects. Full mutation and premutation carrier females did not show FMR1 methylation changes. We have clinically validated this genome-wide DNA methylation assay as a cost- and labor-effective alternative for sensitive and specific screening for FXS, while ruling out the most common differential diagnoses of FXS, Prader-Willi syndrome, and Sotos syndrome in the same assay.
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Affiliation(s)
- Laila C Schenkel
- Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada
| | - Charles Schwartz
- Center for Molecular Studies, J.C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, South Carolina
| | - Cindy Skinner
- Center for Molecular Studies, J.C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, South Carolina
| | - David I Rodenhiser
- Department of Biochemistry, Oncology and Paediatrics, Western University, London, Ontario, Canada; London Regional Cancer Program, London Health Sciences Center, London, Ontario, Canada; Children's Health Research Institute, London Health Sciences Center, London, Ontario, Canada
| | - Peter J Ainsworth
- Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada; Department of Biochemistry, Oncology and Paediatrics, Western University, London, Ontario, Canada; London Regional Cancer Program, London Health Sciences Center, London, Ontario, Canada; Children's Health Research Institute, London Health Sciences Center, London, Ontario, Canada; Molecular Genetics Laboratory, London Health Sciences Center, London, Ontario, Canada
| | - Guillaume Pare
- Departments of Pathology and Molecular Medicine and Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Ontario, Canada
| | - Bekim Sadikovic
- Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada; London Regional Cancer Program, London Health Sciences Center, London, Ontario, Canada; Children's Health Research Institute, London Health Sciences Center, London, Ontario, Canada; Molecular Genetics Laboratory, London Health Sciences Center, London, Ontario, Canada.
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Grigsby J. The fragile X mental retardation 1 gene (FMR1): historical perspective, phenotypes, mechanism, pathology, and epidemiology. Clin Neuropsychol 2016; 30:815-33. [PMID: 27356167 DOI: 10.1080/13854046.2016.1184652] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
OBJECTIVES To provide an historical perspective and overview of the phenotypes, mechanism, pathology, and epidemiology of the fragile X-associated tremor/ataxia syndrome (FXTAS) for neuropsychologists. METHODS Selective review of the literature on FXTAS. RESULTS FXTAS is an X-linked neurodegenerative disorder of late onset. One of several phenotypes associated with different mutations of the fragile X mental retardation 1 gene (FMR1), FXTAS involves progressive action tremor, gait ataxia, and impaired executive functioning, among other features. It affects carriers of the FMR1 premutation, which may expand when passed from a mother to her children, in which case it is likely to cause fragile X syndrome (FXS), the most common inherited developmental disability. CONCLUSION This review briefly summarizes current knowledge of the mechanisms, epidemiology, and mode of transmission of FXTAS and FXS, as well as the neuropsychological, neurologic, neuropsychiatric, neuropathologic, and neuroradiologic phenotypes of FXTAS. Because it was only recently identified, FXTAS is not well known to most practitioners, and it remains largely misdiagnosed, despite the fact that its prevalence may be relatively high.
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Affiliation(s)
- Jim Grigsby
- a Departments of Psychology and Medicine , University of Colorado Denver , Denver , CO , USA
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Hall DA, Robertson-Dick EE, O'Keefe JA, Hadd AG, Zhou L, Berry-Kravis E. X-inactivation in the clinical phenotype of fragile X premutation carrier sisters. NEUROLOGY-GENETICS 2016; 2:e45. [PMID: 27066582 PMCID: PMC4817899 DOI: 10.1212/nxg.0000000000000045] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/12/2015] [Accepted: 11/17/2015] [Indexed: 12/28/2022]
Abstract
Objective: The purpose of this study is to describe a case series of 4 sisters with discordant clinical phenotypes associated with fragile X–associated tremor/ataxia syndrome (FXTAS) that may be explained by varying CGG repeat sizes and activation ratios (ARs) (the ratio of cells carrying the normal fragile X mental retardation 1 [FMR1] allele on the active X chromosome). Methods: Four sisters with premutation size FMR1 gene repeats underwent detailed clinical characterization. CGG repeat length was determined by PCR, and AR was determined using a newly developed commercial methylation PCR assay and was compared with the results from Southern blot with densitometric image analysis. Results: Sister 1 had the largest CGG expansion (82) and the lowest AR (12%), with the most severe clinical presentation. Sister 2 had a lower CGG expansion (70) and an AR of 10% but had a milder clinical presentation.Sister 3 had a similar CGG expansion (79) but a slightly higher AR of 15% and less neurologic involvement. Sister 4 had a similar CGG expansion size of 80 but had the largest AR (40%) and was the only sister not to be affected by FXTAS or have any neurologic signs on examination. Conclusions: These results suggest that premutation carrier women who have higher ARs may be less likely to show manifestations of FXTAS. If larger studies show similar patterns, AR data could potentially be beneficial to supplement CGG repeat size when counseling premutation carrier women in the clinic.
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Affiliation(s)
- Deborah A Hall
- Department of Neurological Sciences (D.A.H., E.B.-K.), Department of Anatomy and Cell Biology (E.E.R.-D., J.A.O.), Department of Biochemistry (L.Z., E.B.-K.), and Department of Pediatrics (E.B.-K.), Rush University, Chicago, IL; and Asuragen, Inc. (A.G.H.), Austin, TX
| | - Erin E Robertson-Dick
- Department of Neurological Sciences (D.A.H., E.B.-K.), Department of Anatomy and Cell Biology (E.E.R.-D., J.A.O.), Department of Biochemistry (L.Z., E.B.-K.), and Department of Pediatrics (E.B.-K.), Rush University, Chicago, IL; and Asuragen, Inc. (A.G.H.), Austin, TX
| | - Joan A O'Keefe
- Department of Neurological Sciences (D.A.H., E.B.-K.), Department of Anatomy and Cell Biology (E.E.R.-D., J.A.O.), Department of Biochemistry (L.Z., E.B.-K.), and Department of Pediatrics (E.B.-K.), Rush University, Chicago, IL; and Asuragen, Inc. (A.G.H.), Austin, TX
| | - Andrew G Hadd
- Department of Neurological Sciences (D.A.H., E.B.-K.), Department of Anatomy and Cell Biology (E.E.R.-D., J.A.O.), Department of Biochemistry (L.Z., E.B.-K.), and Department of Pediatrics (E.B.-K.), Rush University, Chicago, IL; and Asuragen, Inc. (A.G.H.), Austin, TX
| | - Lili Zhou
- Department of Neurological Sciences (D.A.H., E.B.-K.), Department of Anatomy and Cell Biology (E.E.R.-D., J.A.O.), Department of Biochemistry (L.Z., E.B.-K.), and Department of Pediatrics (E.B.-K.), Rush University, Chicago, IL; and Asuragen, Inc. (A.G.H.), Austin, TX
| | - Elizabeth Berry-Kravis
- Department of Neurological Sciences (D.A.H., E.B.-K.), Department of Anatomy and Cell Biology (E.E.R.-D., J.A.O.), Department of Biochemistry (L.Z., E.B.-K.), and Department of Pediatrics (E.B.-K.), Rush University, Chicago, IL; and Asuragen, Inc. (A.G.H.), Austin, TX
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Aliaga SM, Slater HR, Francis D, Du Sart D, Li X, Amor DJ, Alliende AM, Santa Maria L, Faundes V, Morales P, Trigo C, Salas I, Curotto B, Godler DE. Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis. Clin Chem 2016; 62:343-52. [DOI: 10.1373/clinchem.2015.244681] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2015] [Accepted: 12/02/2015] [Indexed: 01/13/2023]
Abstract
Abstract
BACKGROUND
FMR1 full mutations (FMs) (CGG expansion >200) in males mosaic for a normal (<45 CGG) or gray-zone (GZ) (45–54 CGG) allele can be missed with the standard 2-step fragile X syndrome (FXS) testing protocols, largely because the first-line PCR tests showing a normal or GZ allele are not reflexed to the second-line test that can detect FM.
METHODS
We used methylation-specific quantitative melt analysis (MS-QMA) to determine the prevalence of cryptic FM alleles in 2 independent cohorts of male patients (994 from Chile and 2392 from Australia) referred for FXS testing from 2006 to 2013. All MS-QMA–positive cases were retested with commercial triplet primed PCR, methylation-sensitive Southern blot, and a methylation-specific EpiTYPER-based test.
RESULTS
All 38 FMs detected with the standard 2-step protocol were detected with MS-QMA. However, MS-QMA identified methylation mosaicism in an additional 15% and 11% of patients in the Chilean and Australian cohorts, respectively, suggesting the presence of a cryptic FM. Of these additional patients, 57% were confirmed to carry cryptic expanded alleles in blood, buccal mucosa, or saliva samples. Further confirmation was provided by identifying premutation (CGG 55–199) alleles in mothers of probands with methylation-sensitive Southern blot. Neurocognitive assessments showed that low-level mosaicism for cryptic FM alleles was associated with cognitive impairment or autism.
CONCLUSIONS
A substantial number of mosaic FM males who have cognitive impairment or autism are not diagnosed with the currently recommended 2-step testing protocol and can be identified with MS-QMA as a first-line test.
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Affiliation(s)
- Solange M Aliaga
- Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia
- Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia
- Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile
| | - Howard R Slater
- Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia
- Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia
| | - David Francis
- Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia
| | - Desiree Du Sart
- Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia
| | - Xin Li
- Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia
| | - David J Amor
- Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia
- Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia
| | - Angelica M Alliende
- Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile
| | - Lorena Santa Maria
- Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile
- Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile
| | - Víctor Faundes
- Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile
- Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile
| | - Paulina Morales
- Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile
- Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile
| | - Cesar Trigo
- Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile
| | - Isabel Salas
- Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile
| | - Bianca Curotto
- Cytogenetics and Molecular Laboratory, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile
- Centre for Diagnosis and Treatment of Fragile X Syndrome, INTA University of Chile, Santiago, Chile
| | - David E Godler
- Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia
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Cascade Screening for Fragile X Syndrome/CGG Repeat Expansions in Children Attending Special Education in Sri Lanka. PLoS One 2015; 10:e0145537. [PMID: 26694146 PMCID: PMC4687912 DOI: 10.1371/journal.pone.0145537] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2015] [Accepted: 12/04/2015] [Indexed: 01/08/2023] Open
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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A Pilot Study on Assessment of Triplet Repeat Primed PCR for Fragile X Syndrome Diagnosis. ACTA ACUST UNITED AC 2015. [DOI: 10.1007/978-981-287-670-6_10] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/23/2023]
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Gerhardt J. Epigenetic modifications in human fragile X pluripotent stem cells; Implications in fragile X syndrome modeling. Brain Res 2015; 1656:55-62. [PMID: 26475977 DOI: 10.1016/j.brainres.2015.10.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Revised: 08/18/2015] [Accepted: 10/02/2015] [Indexed: 12/18/2022]
Abstract
Patients with fragile X syndrome (FXS) exhibit moderate to severe intellectual disabilities. In addition, one-third of FXS patients show characteristics of autism spectrum disorder. FXS is caused by a trinucleotide repeat expansion, which leads to silencing of the fragile X mental retardation (FMR1) gene. The absence of the FMR1 gene product, FMRP, is the reason for the disease symptoms. It has been suggested that repeat instability and transcription of the FMR1 gene occur during early embryonic development, while after cell differentiation repeats become stable and the FMR1 gene is silent. Epigenetic marks, such as DNA methylation, are associated with gene silencing and repeat stability at the FMR1 locus. However, the mechanisms leading to gene silencing and repeat expansion are still ambiguous, because studies at the human genomic locus were limited until now. The FXS pluripotent stem cells, recently derived from FXS adult cells and FXS blastocysts, are new useful tools to examine these mechanisms at the human endogenous FMR1 locus. This review summarizes the epigenetic features and experimental studies of FXS human embryonic and FXS induced pluripotent stem cells, generated so far. This article is part of a Special Issue entitled SI: Exploiting human neurons.
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Affiliation(s)
- Jeannine Gerhardt
- Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx 10461, USA.
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Chen X, Wang J, Xie H, Zhou W, Wu Y, Wang J, Qin J, Guo J, Gu Q, Zhang X, Ji T, Zhang Y, Xiong Z, Wang L, Wu X, Latham GJ, Jiang Y. Fragile X syndrome screening in Chinese children with unknown intellectual developmental disorder. BMC Pediatr 2015; 15:77. [PMID: 26174701 PMCID: PMC4502947 DOI: 10.1186/s12887-015-0394-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/09/2014] [Accepted: 06/25/2015] [Indexed: 11/18/2022] Open
Abstract
Background Fragile X syndrome is the most common genetic disorder of intellectual developmental disorder/mental retardation (IDD/MR). The prevalence of FXS in a Chinese IDD children seeking diagnosis/treatment in mainland China is unknown. Methods Patients with unknown moderate to severe IDD were recruited from two children’s hospitals. Informed consent was obtained from the children's parents. The size of the CGG repeat was identified using a commercial TP-PCR assay. The influence of AGG interruptions on the CGG expansion during maternal transmission was analyzed in 24 mother-son pairs (10 pairs with 1 AGG and 14 pairs with 2 AGGs). Results 553 unrelated patients between six months and eighteen years of age were recruited. Specimens from 540 patients (male:female = 5.2:1) produced high-quality TP-PCR data, resulting in the determination of the FMR1 CGG repeat number for each. The most common repeat numbers were 29 and 30, and the most frequent interruption pattern was 2 or 3 AGGs. Five full mutations were identified (1 familial and 4 sporadic IDD patients), and size mosaicism was apparent in 4 of these FXS patients (4/5 = 80 %). The overall yield of FXS in the IDD cohort was 0.93 % (5/540). Neither the mean size of CGG expansion (0.20 vs. 0.79, p > 0.05) nor the frequency of CGG expansion (2/10 vs. 9/14, p > 0.05) was significantly different between the 1 and 2 AGG groups following maternal transmission. Conclusions The FMR1 TP-PCR assay generates reliable and sensitive results across a large number of patient specimens, and is suitable for clinical genetic diagnosis. Using this assay, the prevalence of FXS was 0.93 % in Chinese children with unknown IDD. Electronic supplementary material The online version of this article (doi:10.1186/s12887-015-0394-8) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Xiaoli Chen
- Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China.
| | - Jingmin Wang
- Department of Pediatrics, Peking University First Hospital, Beijing, China.
| | - Hua Xie
- Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China.
| | - Wenjuan Zhou
- Department of Pediatrics, Peking University First Hospital, Beijing, China.
| | - Ye Wu
- Department of Pediatrics, Peking University First Hospital, Beijing, China.
| | - Jun Wang
- Department of Neurology, Affiliated Children's Hospital of Capital Institute of Pediatrics, Beijing, China.
| | - Jian Qin
- Beijing Microread Genetech Co., Ltd, Beijing, China.
| | - Jin Guo
- Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China.
| | - Qiang Gu
- Department of Pediatrics, Peking University First Hospital, Beijing, China.
| | - Xiaozhen Zhang
- Department of Genetics, Jiangxi Previncial Children's Hospital, Jiangxi, China.
| | - Taoyun Ji
- Department of Pediatrics, Peking University First Hospital, Beijing, China.
| | - Yu Zhang
- Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China.
| | - Zhiming Xiong
- State Key Lab of Medical Genetics, Central South University, Changsha, China.
| | - Liwen Wang
- Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China.
| | - Xiru Wu
- Department of Pediatrics, Peking University First Hospital, Beijing, China.
| | - Gary J Latham
- Research & Technology Development, Asuragen, Inc., Austin, TX, USA.
| | - Yuwu Jiang
- Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China. .,Department of Pediatrics, Peking University First Hospital, Beijing, China.
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McIntosh N, Gane LW, McConkie-Rosell A, Bennett RL. Genetic Counseling for Fragile X Syndrome: Recommendations of the National Society of Genetic Counselors. J Genet Couns 2015; 9:303-25. [PMID: 26141473 DOI: 10.1023/a:1009454112907] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The National Society of Genetic Counselors' (NSGC) recommendations for fragile X syndrome (FXS) genetic counseling are intended to assist health care professionals who provide genetic counseling for individuals and families in whom the diagnosis of FXS is strongly suspected or has been made. The recommendations are the opinions of genetic counselors with expertise in FXS counseling and are based on clinical experience, a review of pertinent English language medical articles, and reports of expert committees. These recommendations should not be construed as dictating an exclusive course of management, nor does use of such recommendations guarantee a particular outcome. These recommendations do not displace a health care provider's professional judgment based on the clinical circumstances of a particular client.
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50
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Lyons JI, Kerr GR, Mueller PW. Fragile X Syndrome: Scientific Background and Screening Technologies. J Mol Diagn 2015; 17:463-71. [PMID: 26162330 DOI: 10.1016/j.jmoldx.2015.04.006] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2014] [Revised: 03/05/2015] [Accepted: 04/02/2015] [Indexed: 11/26/2022] Open
Abstract
Fragile X is the most common inherited cause of mental retardation with a prevalence of 1 in 4000 for males and 1 in 5000 to 8000 for females. The American College of Medical Genetics and Genomics has recommended diagnostic testing for fragile X in symptomatic persons, women with ovarian dysfunction, and persons with tremor/ataxia syndrome. 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. These technologic approaches include destabilizing the CGG-repeat region with betaine and using chimeric CGG-targeted PCR primers, using heat pulses to destabilize C-G bonds in the PCR extension step, and using melting curve analysis to differentiate expanded CGG repeats from normals. 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.
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Affiliation(s)
- Justine I Lyons
- Molecular Risk Assessment Laboratory, Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, Atlanta, Georgia
| | - Gregory R Kerr
- Molecular Risk Assessment Laboratory, Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, Atlanta, Georgia
| | - Patricia W Mueller
- Molecular Risk Assessment Laboratory, Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, Atlanta, Georgia.
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