Metabolomic Biomarkers Are Associated With Area of the Pons in Fragile X Premutation Carriers at Risk for Developing FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder that affects movement and cognition in male and female carriers of a premutation allele (55–200 CGG repeats; PM) in the fragile X mental retardation (FMR1) gene. It is currently unknown how the observed brain changes are associated with metabolic signatures in individuals who develop the disorder over time. The primary objective of this study was to investigate the correlation between longitudinal changes in the brain (area of the pons, midbrain, and MCP width) and the changes in the expression level of metabolic biomarkers of early diagnosis and progression of FXTAS in PM who, as part of an ongoing longitudinal study, emerged into two distinct categories. These included those who developed symptoms of FXTAS (converters, CON) at subsequent visits and those who did not meet the criteria of diagnosis (non-converters, NCON) and were compared to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern Blot and PCR analysis. Magnetic Resonance Imaging (MRIs) acquisition was obtained on a 3T Siemens Trio scanner and metabolomic profile was obtained by ultra-performance liquid chromatography, accurate mass spectrometer, and an Orbitrap mass analyzer. Our findings indicate that differential metabolite levels are linked with the area of the pons between healthy control and premutation groups. More specifically, we observed a significant association of ceramides and mannonate metabolites with a decreased area of the pons, both at visit 1 (V1) and visit 2 (V2) only in the CON as compared to the NCON group suggesting their potential role in the development of the disorder. In addition, we found a significant correlation of these metabolic signatures with the FXTAS stage at V2 indicating their contribution to the progression and pathogenesis of FXTAS. Interestingly, these metabolites, as part of lipid and sphingolipid lipids pathways, provide evidence of the role that their dysregulation plays in the development of FXTAS and inform us as potential targets for personalized therapeutic development.

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Evidence of mitochondrial dysfunction in fragile X-associated tremor/ataxia syndrome

Biochemical Journal ◽

10.1042/bj20091960 ◽

2010 ◽

Vol 429 (3) ◽

pp. 545-552 ◽

Cited By ~ 104

Author(s):

Catherine Ross-Inta ◽

Alicja Omanska-Klusek ◽

Sarah Wong ◽

Cedrick Barrow ◽

Dolores Garcia-Arocena ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Neurodegenerative Disorder ◽

Late Onset ◽

Fragile X ◽

Mitochondrial Protein ◽

Additional Treatment ◽

Cgg Repeat ◽

Cgg Repeats ◽

Nitrative Stress ◽

Ataxia Syndrome

FXTAS (fragile X-associated tremor/ataxia syndrome) is a late-onset neurodegenerative disorder that affects individuals who are carriers of premutation expansions (55–200 CGG repeats) in the 5′ untranslated region of the FMR1 (fragile X mental retardation 1) gene. The role of MD (mitochondrial dysfunction) in FXTAS was evaluated in fibroblasts and brain samples from premutation carriers with and without FXTAS symptoms, with a range of CGG repeats. This study resulted in several important conclusions: (i) decreased NAD- and FAD-linked oxygen uptake rates and uncoupling between electron transport and synthesis of ATP were observed in fibroblasts from premutation carriers; (ii) a lower expression of mitochondrial proteins preceded both in age and in CGG repeats the appearance of overt clinical involvement; (iii) the CGG repeat size required for altered mitochondrial protein expression was also smaller than that required to produce brain intranuclear inclusions from individuals with the premutation who died, suggesting that MD is an incipient pathological process occurring in individuals who do not display overt features of FXTAS; and (iv) on the basis of the CGG repeats, MD preceded the increase in oxidative/nitrative stress damage, indicating that the latter is a late event. MD in carriers of small CGG repeats, even when the allele size is not sufficient to produce FXTAS, may predispose them to other disorders (e.g. Parkinson's disease) that are likely to involve MD, and to environmental stressors, which may trigger the development of FXTAS symptoms. Detection of MD is of critical importance to the management of FXTAS, since it opens up additional treatment options for this disorder.

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Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats

Nature Communications ◽

10.1038/s41467-021-21021-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Magdalena Derbis ◽

Emre Kul ◽

Daria Niewiadomska ◽

Michał Sekrecki ◽

Agnieszka Piasecka ◽

...

Keyword(s):

Motor Behavior ◽

Neurodegenerative Disorder ◽

Therapeutic Potential ◽

Cell Damage ◽

Fragile X ◽

Nuclear Proteins ◽

Mirna Biogenesis ◽

Loop Formation ◽

Cgg Repeats ◽

Initiation Of Translation

AbstractFragile X-associated tremor/ataxia syndrome (FXTAS) is an incurable neurodegenerative disorder caused by expansion of CGG repeats in the FMR1 5’UTR. The RNA containing expanded CGG repeats (rCGGexp) causes cell damage by interaction with complementary DNA, forming R-loop structures, sequestration of nuclear proteins involved in RNA metabolism and initiation of translation of polyglycine-containing protein (FMRpolyG), which forms nuclear insoluble inclusions. Here we show the therapeutic potential of short antisense oligonucleotide steric blockers (ASOs) targeting directly the rCGGexp. In nuclei of FXTAS cells ASOs affect R-loop formation and correct miRNA biogenesis and alternative splicing, indicating that nuclear proteins are released from toxic sequestration. In cytoplasm, ASOs significantly decrease the biosynthesis and accumulation of FMRpolyG. Delivery of ASO into a brain of FXTAS mouse model reduces formation of inclusions, improves motor behavior and corrects gene expression profile with marginal signs of toxicity after a few weeks from a treatment.

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Molecular Pathogenesis and Peripheral Monitoring of Adult Fragile X-Associated Syndromes

International Journal of Molecular Sciences ◽

10.3390/ijms22168368 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8368

Author(s):

Luis M. Valor ◽

Jorge C. Morales ◽

Irati Hervás-Corpión ◽

Rosario Marín

Keyword(s):

Neurodegenerative Disorder ◽

Late Onset ◽

Fragile X ◽

Endocrine System ◽

Molecular Pathogenesis ◽

Adult Women ◽

Adult Men ◽

Cgg Repeats ◽

Reproductive Impairment ◽

Ataxia Syndrome

Abnormal trinucleotide expansions cause rare disorders that compromise quality of life and, in some cases, lifespan. In particular, the expansions of the CGG-repeats stretch at the 5’-UTR of the Fragile X Mental Retardation 1 (FMR1) gene have pleiotropic effects that lead to a variety of Fragile X-associated syndromes: the neurodevelopmental Fragile X syndrome (FXS) in children, the late-onset neurodegenerative disorder Fragile X-associated tremor-ataxia syndrome (FXTAS) that mainly affects adult men, the Fragile X-associated primary ovarian insufficiency (FXPOI) in adult women, and a variety of psychiatric and affective disorders that are under the term of Fragile X-associated neuropsychiatric disorders (FXAND). In this review, we will describe the pathological mechanisms of the adult “gain-of-function” syndromes that are mainly caused by the toxic actions of CGG RNA and FMRpolyG peptide. There have been intensive attempts to identify reliable peripheral biomarkers to assess disease progression and onset of specific pathological traits. Mitochondrial dysfunction, altered miRNA expression, endocrine system failure, and impairment of the GABAergic transmission are some of the affectations that are susceptible to be tracked using peripheral blood for monitoring of the motor, cognitive, psychiatric and reproductive impairment of the CGG-expansion carriers. We provided some illustrative examples from our own cohort. Understanding the association between molecular pathogenesis and biomarkers dynamics will improve effective prognosis and clinical management of CGG-expansion carriers.

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FMRP Levels in Human Peripheral Blood Leukocytes Correlates with Intellectual Disability

Diagnostics ◽

10.3390/diagnostics11101780 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1780

Author(s):

Mark Roth ◽

Lucienne Ronco ◽

Diego Cadavid ◽

Blythe Durbin-Johnson ◽

Randi J. Hagerman ◽

...

Keyword(s):

Mental Retardation ◽

Intellectual Disability ◽

Peripheral Blood ◽

Fragile X ◽

Repeat Expansion ◽

Repeat Number ◽

Cgg Repeat ◽

Fragile X Mental Retardation ◽

Cgg Repeats ◽

Fmr1 Mrna

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. FXS is an X-linked, neurodevelopmental disorder caused by a CGG trinucleotide repeat expansion in the 5′ untranslated region (UTR) of the Fragile X Mental Retardation gene, FMR1. Greater than 200 CGG repeats results in epigenetic silencing of the gene leading to the deficiency or absence of Fragile X mental retardation protein (FMRP). The loss of FMRP is considered the root cause of FXS. The relationship between neurological function and FMRP expression in peripheral blood mononuclear cells (PBMCs) has not been well established. Assays to detect and measure FMR1 and FMRP have been described; however, none are sufficiently sensitive, precise, or quantitative to properly characterize the relationships between cognitive ability and CGG repeat number, FMR1 mRNA expression, or FMRP expression measured in PBMCs. To address these limitations, two novel immunoassays were developed and optimized, an electro-chemiluminescence immunoassay and a multiparameter flow cytometry assay. Both assays were performed on PMBCs isolated from 27 study participants with FMR1 CGG repeats ranging from normal to full mutation. After correcting for methylation, a significant positive correlation between CGG repeat number and FMR1 mRNA expression levels and a significant negative correlation between FMRP levels and CGG repeat expansion was observed. Importantly, a high positive correlation was observed between intellectual quotient (IQ) and FMRP expression measured in PBMCs.

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Cascade Testing for Fragile X Syndrome in a Rural Setting in Cameroon (Sub-Saharan Africa)

Genes ◽

10.3390/genes11020136 ◽

2020 ◽

Vol 11 (2) ◽

pp. 136

Author(s):

Karen Kengne Kamga ◽

Séraphin Nguefack ◽

Khuthala Minka ◽

Edmond Wonkam Tingang ◽

Alina Esterhuizen ◽

...

Keyword(s):

Fragile X Syndrome ◽

Fragile X ◽

Autism Spectrum ◽

Sub Saharan Africa ◽

Rural Setting ◽

Premature Menopause ◽

Molecular Testing ◽

Full Mutation ◽

Cgg Repeat ◽

Cgg Repeats

Fragile X Syndrome (FXS), an X-linked dominant monogenic condition, is the main genetic cause of intellectual disability (ID) and autism spectrum disorder (ASD). FXS is associated with an expansion of CGG repeat sequence in the Fragile X Mental Retardation gene 1 (FMR1) on chromosome X. Following a neuropediatric assessment of two male siblings who presented with signs of FXS that was confirmed with molecular testing, we provided cascade counselling and testing to the extended family. A total of 46 individuals were tested for FXS; among them, 58.70% (n = 27) were females. The mean age was 9.4 (±5) years for children and 45.9 (±15.9) years for adults. Pedigree analysis suggested that the founder of these families was likely a normal transmitting male. Four out of 19 males with clinical ID were confirmed to have a full mutation for FXS, while 14/27 females had a pathologic CGG expansion (>56 CGG repeats) on one of their X chromosomes. Two women with premature menopause were confirmed of being carriers of premutation (91 and 101 CGG repeats). We also identified maternal alleles (91 and 126 CGG repeats) which expanded to a full mutation in their offspring (>200 CGG repeats). This study is a rare report on FXS from Africa and illustrates the case scenario of implementing genetic medicine for a neurogenetic condition in a rural setting.

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Lack of a Clear Behavioral Phenotype in an Inducible FXTAS Mouse Model Despite the Presence of Neuronal FMRpolyG-Positive Aggregates

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.599101 ◽

2020 ◽

Vol 7 ◽

Author(s):

Saif N. Haify ◽

Ruchira S. D. Mankoe ◽

Valerie Boumeester ◽

Esmay C. van der Toorn ◽

Rob F. M. Verhagen ◽

...

Keyword(s):

Mouse Model ◽

Rna Binding ◽

Neurodegenerative Disorder ◽

Rna Binding Proteins ◽

Fragile X ◽

Brain Regions ◽

Intranuclear Inclusions ◽

Behavioral Deficits ◽

Cgg Repeat ◽

Progressive Cerebellar Ataxia

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a rare neurodegenerative disorder caused by a 55–200 CGG repeat expansion in the 5′ untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. FXTAS is characterized by progressive cerebellar ataxia, Parkinsonism, intention tremors and cognitive decline. The main neuropathological hallmark of FXTAS is the presence of ubiquitin-positive intranuclear inclusions in neurons and astrocytes throughout the brain. The molecular pathology of FXTAS involves the presence of 2 to 8-fold elevated levels of FMR1 mRNA, and of a repeat-associated non-AUG (RAN) translated polyglycine peptide (FMRpolyG). Increased levels of FMR1 mRNA containing an expanded CGG repeat can result in cellular toxicity by an RNA gain-of-function mechanism. The increased levels of CGG repeat-expanded FMR1 transcripts may create RNA foci that sequester important cellular proteins, including RNA-binding proteins and FMRpolyG, in intranuclear inclusions. To date, it is unclear whether the FMRpolyG-positive intranuclear inclusions are a cause or a consequence of FXTAS disease pathology. In this report we studied the relation between the presence of neuronal intranuclear inclusions and behavioral deficits using an inducible mouse model for FXTAS. Neuronal intranuclear inclusions were observed 4 weeks after dox-induction. After 12 weeks, high numbers of FMRpolyG-positive intranuclear inclusions could be detected in the hippocampus and striatum, but no clear signs of behavioral deficits related to these specific brain regions were found. In conclusion, the observations in our inducible mouse model for FXTAS suggest a lack of correlation between the presence of intranuclear FMRpolyG-positive aggregates in brain regions and specific behavioral phenotypes.

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Screening for FMR1 CGG Repeat Expansion in Thai Patients with Autism Spectrum Disorder

BioMed Research International ◽

10.1155/2021/4359308 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Areerat Hnoonual ◽

Charunee Jankittunpaiboon ◽

Pornprot Limprasert

Keyword(s):

Autism Spectrum Disorder ◽

Fragile X ◽

Single Gene ◽

Autism Spectrum ◽

Spectrum Disorder ◽

Fmr1 Gene ◽

Normal Male ◽

Cgg Repeat ◽

Cgg Repeats ◽

Normal Controls

Autism spectrum disorder (ASD) is a complex disorder with a heterogeneous etiology. Fragile X syndrome (FXS) is recognized as the most common single gene mutation associated with ASD. FXS patients show some autistic behaviors and may be difficult to distinguish at a young age from autistic children. However, there have been no published reports on the prevalence of FXS in ASD patients in Thailand. In this study, we present a pilot study to analyze the CGG repeat sizes of the FMR1 gene in Thai autistic patients. We screened 202 unrelated Thai patients (168 males and 34 females) with nonsyndromic ASD and 212 normal controls using standard FXS molecular diagnosis techniques. The distributions of FMR1 CGG repeat sizes in the ASD and normal control groups were similar, with the two most common alleles having 29 and 30 CGG repeats, followed by an allele with 36 CGG repeats. No FMR1 full mutations or premutations were found in either ASD individuals or the normal controls. Interestingly, three ASD male patients with high normal CGG and intermediate CGG repeats (44, 46, and 53 CGG repeats) were identified, indicating that the prevalence of FMR1 intermediate alleles in Thai ASD patients was approximately 1% while these alleles were absent in the normal male controls. Our study indicates that CGG repeat expansions of the FMR1 gene may not be a common genetic cause of nonsyndromic ASD in Thai patients. However, further studies for mutations other than the CGG expansion in the FMR1 gene are required to get a better information on FXS prevalence in Thai ASD patients.

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Beyond Trinucleotide Repeat Expansion in Fragile X Syndrome: Rare Coding and Noncoding Variants in FMR1 and Associated Phenotypes

Genes ◽

10.3390/genes12111669 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1669

Author(s):

Cedrik Tekendo-Ngongang ◽

Angela Grochowsky ◽

Benjamin D. Solomon ◽

Sho T. Yano

Keyword(s):

Copy Number ◽

Trinucleotide Repeat ◽

De Novo ◽

Fragile X ◽

Copy Number Variants ◽

Repeat Expansion ◽

Full Mutation ◽

Cgg Repeat ◽

Testing Strategies ◽

Cgg Repeats

FMR1 (FMRP translational regulator 1) variants other than repeat expansion are known to cause disease phenotypes but can be overlooked if they are not accounted for in genetic testing strategies. We collected and reanalyzed the evidence for pathogenicity of FMR1 coding, noncoding, and copy number variants published to date. There is a spectrum of disease-causing FMR1 variation, with clinical and functional evidence supporting pathogenicity of five splicing, five missense, one in-frame deletion, one nonsense, and four frameshift variants. In addition, FMR1 deletions occur in both mosaic full mutation patients and as constitutional pathogenic alleles. De novo deletions arise not only from full mutation alleles but also alleles with normal-sized CGG repeats in several patients, suggesting that the CGG repeat region may be prone to genomic instability even in the absence of repeat expansion. We conclude that clinical tests for potentially FMR1-related indications such as intellectual disability should include methods capable of detecting small coding, noncoding, and copy number variants.

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Unique AGG Interruption in the CGG Repeats of the FMR1 Gene Exclusively Found in Asians Linked to a Specific SNP Haplotype

Genetics Research International ◽

10.1155/2016/8319287 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Pornprot Limprasert ◽

Janpen Thanakitgosate ◽

Kanoot Jaruthamsophon ◽

Thanya Sripo

Keyword(s):

Genetic Background ◽

Fragile X ◽

Cgg Repeat ◽

Direct Dna Sequencing ◽

Asian Populations ◽

Cgg Repeats ◽

Normal Individuals ◽

The Common ◽

Almost All ◽

Agg Interruptions

Fragile X syndrome (FXS) is the most common inherited intellectual disability. It is caused by the occurrence of more than 200 pure CGG repeats in the FMR1 gene. Normal individuals have 6–54 CGG repeats with two or more stabilizing AGG interruptions occurring once every 9- or 10-CGG-repeat blocks in various populations. However, the unique (CGG)6AGG pattern, designated as 6A, has been exclusively reported in Asians. To examine the genetic background of AGG interruptions in the CGG repeats of the FMR1 gene, we studied 8 SNPs near the CGG repeats in 176 unrelated Thai males with 19–56 CGG repeats. Of these 176 samples, we identified AGG interruption patterns from 95 samples using direct DNA sequencing. We found that the common CGG repeat groups (29, 30, and 36) were associated with 3 common haplotypes, GCGGATAA (Hap A), TTCATCGC (Hap C), and GCCGTTAA (Hap B), respectively. The configurations of 9A9A9, 10A9A9, and 9A9A6A9 were commonly found in chromosomes with 29, 30, and 36 CGG repeats, respectively. Almost all chromosomes with Hap B (22/23) carried at least one 6A pattern, suggesting that the 6A pattern is linked to Hap B and may have originally occurred in the ancestors of Asian populations.

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Human Cerebral Cortex Proteome of Fragile X-Associated Tremor/Ataxia Syndrome

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.600840 ◽

2021 ◽

Vol 7 ◽

Author(s):

Katharine Nichole Holm ◽

Anthony W. Herren ◽

Sandra L. Taylor ◽

Jamie L. Randol ◽

Kyoungmi Kim ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Structural Integrity ◽

Rna Binding ◽

Neurodegenerative Disorder ◽

Fragile X ◽

Protein Abundance ◽

Cgg Repeat ◽

Phosphoserine Aminotransferase ◽

Brain Volume Loss ◽

Ataxia Syndrome

Background: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder associated with premutation CGG-repeat expansions (55–200 repeats) in the 5′ non-coding portion of the fragile X mental retardation 1 (FMR1) gene. Core features of FXTAS include progressive tremor/ataxia, cognitive decline, variable brain volume loss, and white matter disease. The principal histopathological feature of FXTAS is the presence of central nervous system (CNS) and non-CNS intranuclear inclusions.Objective: To further elucidate the molecular underpinnings of FXTAS through the proteomic characterization of human FXTAS cortexes.Results: Proteomic analysis of FXTAS brain cortical tissue (n = 8) identified minor differences in protein abundance compared to control brains (n = 6). Significant differences in FXTAS relative to control brain predominantly involved decreased abundance of proteins, with the greatest decreases observed for tenascin-C (TNC), cluster of differentiation 38 (CD38), and phosphoserine aminotransferase 1 (PSAT1); proteins typically increased in other neurodegenerative diseases. Proteins with the greatest increased abundance include potentially novel neurodegeneration-related proteins and small ubiquitin-like modifier 1/2 (SUMO1/2). The FMRpolyG peptide, proposed in models of FXTAS pathogenesis but only identified in trace amounts in the earlier study of FXTAS inclusions, was not identified in any of the FXTAS or control brains in the current study.Discussion: The observed proteomic shifts, while generally relatively modest, do show a bias toward decreased protein abundance with FXTAS. Such shifts in protein abundance also suggest altered RNA binding as well as loss of cell–cell adhesion/structural integrity. Unlike other neurodegenerative diseases, the proteome of end-stage FXTAS does not suggest a strong inflammation-mediated degenerative response.

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