Authors: Dejan B. Budimirovic, Annette Schlageter, Stela Filipovic-Sadic, Dragana D. Protic, Eran Bram, E. Mark Mahone, Kimberly Nicholson, Kristen Culp, Kamyab Javanmardi, Jon Kemppainen, Andrew Hadd, Kevin Sharp, Tatyana Adayev, Giuseppe LaFauci, Carl Dobkin, Lili Zhou, William Ted Brown, Elizabeth Berry-Kravis, Walter E Kaufmann, and Gary J. Latham


A genotype refers to the genetic characteristics of a person and a phenotype refers to the physical or observable traits of a person. Genotypes (genetics) can influence phenotypes (physical traits). For example, the genetics behind Fragile X syndrome (FMR1 gene, lack of FMRP) influences the physical expression of the gene as a phenotype. The physical expression (phenotype) of FXS could be physical traits like eye color or other observable traits like cognition that can be measured through IQ testing.

We know that FMRP is expressed throughout our body, including our blood, tissues, and brain. Levels of FMRP in the blood of patients with FXS have been positively correlated with cognitive performance, specifically intelligence quotient (IQ) and adaptive behavior. When two things are positively correlated, they move together; if one goes up, so does the other and if one goes down, so does the other. Higher levels of FMRP in the blood have been positively correlated with higher cognition, IQ, and adaptive behavior (life skills).


Dr. Dejan Budimirovic and study team partnered with Asuragen through an NIH-funded study to research the genotype-protein-phenotype in Fragile X syndrome. Research is needed to understand the different levels of FMRP throughout the body and how those levels may impact things like cognition, IQ, and co-occurring conditions like autism, or ASD. There is a newer series of FMR1-based molecular methods that can measure and report features of the gene and protein expression across different types of samples (blood and check cells) with greater sensitivity and specificity.

This study used nine complementary assays for assessing FMR1 DNA, RNA, and protein, using a reference set of well-characterized FMR1 cell lines.

The team used the molecular data from the assays paired with the neurobehavioral profiles of each sample to better understand the relationship between genotype and phenotype(s) of FXS. There were two cohorts in this study; a reference cohort and a clinical cohort.

The Reference Cohort included 11 individuals, with and without FXS, and their samples were used to create control materials and data for each of the assays used.

The Clinical Cohort included 42 patients with a range of FMR1 expansions. Each member of the Clinical Cohort had detailed genetic workup including testing, assays, and FRMP measurements, along with a neurobehavioral profile that included various assessments. Each member of the Clinical Cohort had both whole blood and buccal cell specimens collected. Buccal cells were collected using a cheek swab similar to a Q-Tip. DNA and RNA were isolated from the samples, and additional analyses including CGG Repeat Genotyping, AGG Interruption Analysis, PCR, Southern Blot, FMR1 mRNA, and quantitative FMRP occurred. The neurobehavioral data for the Clinical Cohort included capturing the following; presence of ASD, anxiety, and intellectual disability, level of adaptive skills, presence and severity of challenging behaviors, overall clinical severity of FXS as determined by a caregiver-informed, clinician-rated scale, and use of antipsychotics.

The nine assays were compared in the Reference Cohort to understand if the molecular profile can indicate clinical presentation of FXS. This was successful; the molecular analyses matched the clinical notes characterizing each of the individuals. Having shown a link between FMR1 molecular work and detailed clinical annotations, the study team then selected a random group of patients from the Clinical Cohort to run the same experiment, going one step further in diving the group between those with FXS + ASD and those with FXS only.

This study confirms the relationship between the number of repeats and an individual with FX’s traits with greater precision than before; more repeats lead to more methylation, causing lower protein levels and more severe traits. For example, individuals with FXS with low levels of FMRP are more likely to also have a diagnosis of ASD and severe intellectual disability.

This study also suggests that FMRP levels seem to be a strong candidate for a biomarker of overall clinical severity in FXS.

The research also dove into the relationship between FRMP and anxiety, including a strong correlation of unspecified anxiety only in low functioning males with low FMRP and ASD, and social avoidance in females and higher functioning males without ASD and higher FMRP levels.

The study results also support the existence of a more severe neurobehavioral phenotype mainly in males with FXS. This is characterized by severe ID, ASD diagnosis, and severe irritability, aggression, agitation, and self-injury types of often anxiety-driven behavior.

Understanding the different phenotypes in FXS and their relationship to things like FMRP level can help researchers, clinicians, and families select treatment options and supports. Much more research is needed to better understand the genotype-phenotype relationship in FXS, but this is a great start.

Why This Matters

This is the first study to integrate FMR1 DNA, RNA and protein analyses and to correlate these molecular measures with multiple neurobehavioral parameters, including ASD diagnosis, in order to refine genotype-phenotype correlations in FXS. This is “bench to bedside” research in a true sense; understanding the link between FMRP, methylation, and characteristics of FXS may help researchers, clinicians, and families identify who would benefit most from certain treatments. This work also confirmed prior with in FXS showing FMRP is a biomarker of overall clinical severity in FXS. Knowing this, FMRP could be used to help stratify patients with FXS in future clinical trials.

Next Steps

More research needs to be done in a larger group of individuals to confirm these findings and further explore the link between genotype-phenotype. Further exploring both size and methylation mosaicism is necessary as well. We also need better outcome measures to measure anxiety and other behaviors in FXS so we can accurately measure changes in behaviors across treatments/interventions.


Read the full publication in Brain Sciences and the helpful article summarizing this work in Spectrum.

Acknowledgements: Since 2013, these results were presented at the following conferences: 1st through 4th International Conference on FMR1 premutation in 2013, 2015, 2017, and 2019, respectively; ACMG Annual Clinical Genetics Meeting 2014; 15th and 16th International Fragile X Conferences in 2016 and 2018, respectively; Gordon Research Conference “Fragile X and Autism-Related Disorders” in 2016; International Conference on Autism and Fragile-X Associated Disorders in 2019, and American Academy of Child & Adolescent Psychiatry Annual Meeting in 2019.

Funding: The authors acknowledged their funding source, NICHD grant 1R43HD082900-01A1 (Latham PI) subcontract to Kennedy Krieger Institute (PI: Budimirovic).

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