Abstract

Fragile X Syndrome is an X-linked genetic disorder resulting from the impairment of transcription of the FMR1 gene, producing less to no FMRP protein. FMRP is a translational inhibitor that plays a key role in neurological development. Recent research has contributed to a more accurate understanding of the heredity, diagnosis, treatment, and molecular impact of both the pre- and full mutation of Fragile X Syndrome. Fragile X Syndrome is unique compared to other genetic disorders typically covered during undergraduate biology courses due to the complex heredity, the molecular changes in the non-coding region of the gene, and the varying phenotypes presented. This review provides insight into the current understanding of Fragile X Syndrome to assist instructors and students using the Fragile States case study or faculty looking to introduce this complex genetic disorder as a way to engage students with the different aspects of biology. Students will be able to explore the complexities of diagnosis due to the disorder’s range of symptoms. Students can also investigate epigenetic gene regulation, specifically how methylation affects the transcription of FMR1. The resulting lack of FMRP has been shown to promote neurotoxicity, and students can investigate how this arises due to the disruption of sphingosine metabolism.

Primary Image: The enzyme DNMT1 interacts with DNA, as produced by enzymlogic.com. Used under license CC BY-SA 2.0 DEED.

Citation

Introduction

FMR1 codes for Fragile X messenger ribonucleoprotein 1, previously known as Fragile X Mental Retardation Protein (FMRP), which has been characterized as a translational inhibitor (1). Fragile X Syndrome (FXS) arises from the methylation of CGG repeats in the 5’ untranslated region (UTR), resulting in the silencing of the FMR1 gene. The CGG repeats in the 5’ UTR add a regulatory region to the FMR1 gene in addition to the promoter and enhancer regions (2). The CGG repeats prevent access to the transcription start site from the RNA polymerases needed to transcribe FMR1. Alleles with expanded CGG repeats (>200) are characterized as loss-of-function mutations (2). Without FMRP, neurological development is impaired, resulting in Autism Spectrum Disorder and other developmental delays (3). It is estimated that 1 in 7,000 males and 1 in 11,000 females have the full FXS mutation (4).

More limited CGG expansion (50–199 repeats) in the 5’ UTR of FMR1 is categorized as a premutation. Individuals with the premutation show some gene methylation but can still produce limited FMRP for the cell, resulting in individuals experiencing Fragile X-associated tremor/ataxia syndrome (FXTAS) and Primary Ovarian Insufficiency. The partial silencing as a result of the CGG repeats in the 5’ untranslated region of FMR1 can also cause anxiety, depression, and hypothyroidism (4). While individuals with the full mutation can also show these phenotypes, intellectual disabilities and developmental delays are more prevalent. An estimated 1 in 130 to 250 females and 1 in 250 to 800 males have the premutation for FXS (4).

The lesson “Fragile States: A Case Study Exploring Genetics and Molecular Biology through the Lens of Fragile X Syndrome” walks students through the genetics, regulation, metabolism, and neurobiology implicated in those with FXS (5). In this way, discussing FXS as a complex genetic disorder provides ways to extend and apply material introduced in many undergraduate biology curricula. This paper summarizes current research and clinical understanding to support instructors and students in exploring FXS.

Diagnosis

Full Fragile X Syndrome (FXS), arising when an individual has greater than 200 CGG repeats within a regulatory region of the FMR1 gene, appears early in a child's life and is characterized as a neurodevelopmental disorder. These children are often diagnosed with Autism Spectrum Disorder, an intellectual disability, language deficit, attention deficit, hyperactivity disorder, or a combination (6). A diagnosis of autism or any overlapping phenotype should prompt the physician to test for FXS, especially since it is the leading genetic cause of autism, accounting for 2–12% of the diagnoses (7). Besides neurodevelopmental disorders, those with complete silencing of FMR1 also present with protruding ears, flat feet, hyperextensible joints, and macroorchidism (4). Those with FXS also have an increased risk of hernias and mitral valve prolapse (4). Individuals with the premutation (50–199 repeats) present with a later onset and can develop ovarian insufficiency or Fragile X-associated tremor/ataxia syndrome. Premutation “carriers” (discussed further below) also have a higher risk of depression, anxiety, and hypothyroidism (4).

Due to the large variability in the presentation of symptoms in Fragile X and related syndromes, physicians had difficulty identifying patients as having the pre- or full mutation at the FMR1 locus before the availability of DNA testing. The disorder initially got its name “Fragile X” due to the karyotype of the patient's X chromosome. A section of the chromosome was visualized not to be completely disconnected but appeared fragile or broken (8). Initial testing for the disorder consisted of cytogenetic testing during the 1970s. With the genomic revolution, testing in the 1990s improved to utilizing PCR and Southern blots for detection. Currently, to quantify the CGG repeats an individual has, triple primer PCR and capillary electrophoresis are used (7). Current prenatal diagnostic measures include amniocentesis and chorionic villus sampling (9). Research into how FXS affects metabolic pathways has also identified metabolites to help diagnose the condition and provide treatment possibilities (10).

In part 1 of the Fragile States case study, students are asked to determine the causes and conditions associated with FXS (5). A deeper understanding of the phenotypes associated with the disorder helps expose students to the interconnectedness of biological systems by exploring how the disruption of function in one gene causes a range of phenotypes. Effects on sphingolipid metabolism are described in part 5 of the case.

Carriers and Heredity

Fragile X Syndrome (FXS) is characterized as a single-gene disorder, but changes at the FMR1 locus produce a range of phenotypes. Initially, FXS was described as an X-linked dominant disorder (11). However, with epidemiological reports showing an increase in males diagnosed with FXS, the classification of the disorder as X-linked dominant was no longer supported (4). Since FMR1 is on the X chromosome, biological males have only one gene copy. In contrast, biological females have two copies. If the disorder were X-linked dominant, biological males and biological females would have an equal likelihood of diagnosis. With further advancements in testing and diagnosis, more individuals displayed an extensive range of phenotypes based on varying levels of methylation of the 5’ UTR of FMR1 and the multiple downstream players that FMRP regulates (12). Based on this, the disorder appears to follow a non-Mendelian inheritance pattern (4). The current description of FXS may shift to reflect variable expressivity.

Another complicating factor in the genetic literature of FXS is the use of the term “carrier.” Most commonly, when the term “carrier” is used in genetics, it refers to an individual with one allele containing a given deleterious mutation, but the other allele is functional; thus, the individual would not show an altered phenotype. Within the Fragile X field, individuals with the premutation may be called “carriers,” but they may still show symptoms due to partial methylation of the FMR1 gene (13). This understanding of how FXS is passed down through generations results from the determination that the CGG repeats of FMR1 retain their capacity to be methylated and also tend to expand in number over time (13). In the past, diagnosis was not usually made until an individual presented with phenotypes typical of the full silencing of FMR1 despite generations of family members with the phenotypic traits of partial FMR1 silencing (14). This increased understanding of heredity and increased education of FXS symptoms can help provide families with earlier diagnoses and interventions.

Insight on the complex heredity of FXS can support broadening one's understanding of the genetic basis of disease. In part 2 of the Fragile States case study, students are asked to construct a pedigree of FXS inheritance through multiple generations (5). Increased knowledge of heredity is crucial here since FXS is shown not to follow the typical Mendelian inheritance of most disorders taught in a classroom.

Molecular Biology and Epigenetic Regulation

The Fragile X Syndrome (FXS) disorder results from an array of CGG repeats inserted into the 5’ untranslated region of FMR1. The triplet repeats are targets of DNA methylation, which typically prevents transcription. The gene is completely silenced with the full mutation characterized by more than 200 CGG repeats. The coding region of FMR1 remains unaffected by the Fragile X mutation (3). In individuals without these large numbers of repeats in the 5’ UTR, FMR1 follows the central dogma when the gene is transcribed and then translated into FMRP protein in the cytoplasm of neuronal cells (Figure 1).

Currently, it is understood that there is an extensive range of phenotypes present even in individuals with partial methylation of a premutation allele (50–199 CGG repeats). Surprisingly, FMR1 messenger RNA (mRNA) transcript levels were found actually to increase in individuals with the premutation (13). A hypothesis for how this occurs reflects multiple transcription start sites for FMR1 and the cellular machinery bypassing the start sites that were blocked due to the methylation (13). FMR1 encodes multiple isoforms that use these different transcription start sites. Therefore, those with the premutation may be expressing different isoforms of FMR1 (13). Despite the increase in transcription, those with the premutation still experience decreased translation of FMRP, possibly due to a lack of polyadenylation of these FMR1 transcripts. The use of different transcription start sites is hypothesized to, in turn, change the polyadenylation site of the transcript (13). Polyadenylation is essential for the stability of the mRNA transcript (13). Without the polyadenylated tail, the mRNA would be degraded upon leaving the nucleus before binding to a ribosome to initiate translation. Therefore, the change in the adenylation site could result in a less stable transcript and decreased translation of FMR1 (13).

Insight into these molecular mechanisms can provide possible treatment avenues to explore for those with FXS. Since the gene's coding region remains unaffected, treatments should target regulation to overcome the silencing caused by methylated CGG repeats and allow FMR1 to be transcribed and its mRNA polyadenylated. Current research is targeting the epigenetic factor DNA methyltransferase I (DNMT1) to explore its potential to interfere with the methylation and re-activate FMR1. By perturbing DNMT1, methylation levels in the FMR1 5’UTR decreased, suggesting selective inhibition of DNMT1 as a possible FXS treatment. This work also led to the creation of a screening process that utilizes the CRISPR-Cas9 system to identify loci that work to maintain the methylation of FMR1. This method yielded a list of 155 genes that regulate FMR1 (2). Additional genes involved in FMR1 inactivation include both SMARCD1 and DPF3, which function in the SWI/SNF chromatin remodeling complex. ZNF217 and CTBP2 are transcriptional co-repressors, complexes that work to silence transcription, that were also found to contribute to the inactivation of FMR1. These genes can serve as potential treatment targets for reactivating FMR1 in those with FXS (2).

In the Fragile States case study part 3, students are asked about the regulation of FMR1 (5). Not only does this provide an opportunity to learn more about the central dogma of molecular biology, but FXS also provides an opportunity to investigate epigenetic regulation outside of the coding region of a gene.

FMRP In Translation

FMRP plays a regulatory role in neurological cells. The primary function of FMRP is to act as a translational inhibitor. Current work suggests that there are multiple mechanisms by which FMRP can accomplish inhibition. The first mechanism involves FMRP binding to mature mRNA and preventing the ribosome from translocating. FMRP contains KH and RGG box domains for RNA binding, allowing it to bind to multiple segments on the transcript and act as a roadblock for the ribosome (3). As a result, the polypeptide is not elongated and therefore nonfunctional. The second mechanism in which FMRP blocks translation is by preventing the tRNA from binding to the ribosome. FMRP binds to the 60S subunit. Therefore, the tRNA cannot assemble with the ribosome, and the ribosome itself will stall, preventing polypeptide synthesis (Figure 2).

In individuals with Fragile X Syndrome, translational products of some genes will accumulate and, as a result, prevent the proper formation of neuronal circuits. Current research supports the idea that FMRP can target and bind to recognition sites within specific mRNAs, blocking translation products from accumulating (15). These sequences are present in multiple genes known to underlie Autism Spectrum Disorder. In addition, FMRP-regulated genes linked to autism tend to have transcripts that are longer in length. Since these transcripts are longer, the untangling mechanism of topoisomerase is essential for their transcription. FMRP has been shown to bind to the transcript's coding region and directly interact with topoisomerase to prevent the expression of these autism-linked genes (3, 16).

An increased understanding of translation regulation allows students to grasp the implications if the production of the translational inhibitor FMRP is blocked. Insight into the healthy-state function of a gene or pathway is essential for determining the implications of the function being impaired in the diseased state.

Sphingosine Metabolism

The Purkinje cells located in the cerebellum help regulate and control movement. Sphingosine, a type of lipid, is essential for the proper membrane structure of these cells (17). In individuals with Fragile X Syndrome (FXS), the silencing of FMR1 affects the metabolism of sphingosine, which results in impaired cerebellum function and individuals struggling with movement and coordination. Tremors, decreased motor function, and trouble balancing are all symptoms of individuals with Fragile X-associated tremor/ataxia syndrome (FXTAS) (10).

Along with membrane function, the regulation of sphingosine metabolism also helps prevent cell neurotoxicity levels from rising. Typically, the synthesis and degradation of ceramides to sphingolipids occur to maintain normal levels of these metabolites. Ceramide synthase catalyzes the reaction of sphingosine to ceramide. In individuals with FXS, the function of ceramide synthase is affected. This is hypothesized to be a genetic interaction since both mice and Drosophila with FXTAS were shown to have significant elevation of the substrate for ceramide synthases (10). Specifically, sphingosine does not bind to ceramide synthase, resulting in elevated levels of sphingosine. In contrast, ceramide levels decrease (10). This ratio of substrate to product results in the increased neurotoxicity that plays a role in FXTAS development (Figure 3).

The Fragile States case study, specifically part 5, highlights sphingosine metabolism, the pathway's key role in neurological development, and how it is affected by silencing FMRP (5). Sphingosine metabolism may not be covered in many undergraduate courses, but the pathway plays an important role in many functions throughout the body (18). The process and regulation of metabolic pathways, which are often first learned through the study of glucose metabolism, can be applied by students to understand the pathways of sphingosine metabolism and to extend that understanding to FXS.

Conclusion

Fragile X Syndrome (FXS) results from methylation of CGG repeats in the 5’ untranslated region of the FMR1 gene. This example of a genetic disorder in which the coding region remains unaltered is likely of interest to instructors and students. There is a range of phenotypes in those with FXS due to the number of biochemical processes FMRP (the FMR1 gene product) regulates. Moreover, carriers (those with a premutation) can still be affected due to the partial silencing of FMR1. Due to an increased understanding of heredity and mechanism, FXS should be seen as an example of variable expressivity. Silencing of FMR1 has downstream effects on metabolism, including the sphingosine pathway, mediated through the loss of FMRP’s effects as a translational inhibitor. This lesson is able to provide the background for the Fragile States case study that provides an in-depth look at the molecular effects of a complex genetic disorder.

Acknowledgments

This case study was developed as an extension of the authors’ work with the Genomics Education Partnership. We thank Anam Ali for her review of the manuscript. Finally, we wish to offer special thanks to Dr. Debra Walther for her review and suggestions for this manuscript.

References

1. National Center for Biotechnology Information. 2024. FMR1 fragile X messenger ribonucleoprotein 1 [Homo sapiens (human)]. Retrieved from https://www.ncbi.nlm.nih.gov/gene/2332 (accessed 13 January 2024).
2. Vershkov D, Yilmaz A, Yanuka O, Nielsen AL, Benvenisty N. 2022. Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome. Stem Cell Rep 17:1048–1058. doi:10.1016/j.stemcr.2022.03.011.
3. Richter JD, Zhao X. 2021. The molecular biology of FMRP: New insights into fragile X syndrome. Nat Rev Neurosci 22:209–222. doi:10.1038/s41583-021-00432-0.
4. Stone WL, Basit H, Shah M, Los E. 2023. Fragile X syndrome. StatPearls Publishing, Treasure Island, FL.
5. Hark AT, Washco LE. 2023. Fragile states: A case study exploring genetics, molecular biology, and biochemistry through the lens of fragile X syndrome. CourseSource 10. doi:10.24918/cs.2023.33.
6. Kaufmann WE, Kidd SA, Andrews HF, Budimirovic DB, Esler A, Haas-Givler B, Stackhouse T, Riley C, Peacock G, Sherman SL, Brown WT, Berry-Kravis E. 2017. Autism spectrum disorder in fragile X syndrome: Cooccurring conditions and current treatment. Pediatrics 139:S194–S206. doi:10.1542/peds.2016-1159F.
7. Acero-Garcés DO, Saldarriaga W, Cabal-Herrera AM, Rojas CA, Hagerman RJ. 2023. Fragile X syndrome in children. Colomb Medica 54:e3005089. doi:10.25100/cm.v54i2.5089.
8. Saldarriaga W, Tassone F, González-Teshima LY, Forero-Forero JV, Ayala-Zapata S, Hagerman R. 2014. Fragile X syndrome. Colomb Medica 45:190–198.
9. Cleveland Clinic. n.d. Fragile X syndrome. Retrieved from https://my.clevelandclinic.org/health/diseases/5476-fragile-x-syndrome (accessed 8 January 2023).
10. Kong HE, Lim J, Zhang F, Huang L, Gu Y, Nelson DL, Allen EG, Jin P. 2019. Metabolic pathways modulate the neuronal toxicity associated with fragile X-associated tremor/ataxia syndrome. Hum Mol Genet 28:980–991. doi:10.1093/hmg/ddy410.
11. Garber KB, Visootsak J, Warren ST. 2008. Fragile X syndrome. Eur J Hum Genet 16:666–672. doi:10.1038/ejhg.2008.61.
12. Flanagan K, Baradaran-Heravi A, Yin Q, Dao Duc K, Spradling AC, Greenblatt EJ. 2022. FMRP-dependent production of large dosage-sensitive proteins is highly conserved. Genetics 221:iyac094. doi:10.1093/genetics/iyac094.
13. Willemsen R, Levenga J, Oostra BA. 2011. CGG repeat in the FMR1 gene: Size matters. Clin Genet 80:214–225. doi:10.1111/j.1399-0004.2011.01723.x.
14. Ranjan R, Jha S, Prajjwal P, Chaudhary A, Dudeja P, Vora N, Mateen MA, Yousuf MA, Chaudhary B. 2023. Neurological, psychiatric, and multisystemic involvement of fragile X syndrome along with its pathophysiology, methods of screening, and current treatment modalities. Cureus 15:e35505. doi:10.7759/cureus.35505.
15. Athar YM, Joseph S. 2020. RNA-binding specificity of the human fragile X mental retardation protein. J Mol Biol 432:3851–3868. doi:10.1016/j.jmb.2020.04.021.
16. King MK, Jope RS. 2013. Lithium treatment alleviates impaired cognition in a mouse model of fragile X syndrome. Genes, Brain Behav 12:723–731. doi:10.1111/gbb.12071.
17. Paul MS, Limaiem F. 2022. Histology, Purkinje cells. StatPearls Publishing, Treasure Island, FL.
18. Mei M, Liu M, Mei Y, Zhao J, Li Y. 2023. Sphingolipid metabolism in brain insulin resistance and neurological diseases. Front Endocrinol 14:1243132. doi:10.3389/fendo.2023.1243132.