Biology subjects

Jones, T. I.

Publications and source records attributed to Jones, T. I..

6 recordsLinked to original sources

bioRxiv2026

The D4Z4caster DNA methylation signature identifies individuals at epigenetic risk for developing facioscapulohumeral muscular dystrophy (FSHD)

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic dysregulation at the chromosome 4q35 D4Z4 repeat array under specific permissive genetic conditions. Due to the complexity, expense, and general inaccessibility of FSHD genetic testing, many individuals displaying characteristic muscle weakness are never genetically confirmed and at-risk relatives cannot get screened. We previously developed a targeted bisulfite sequencing (BSS) protocol using the Sanger method to determine DNA methylation levels at specific D4Z4 loci relevant to distinguishing forms of FSHD from non-FSHD that can be used with DNA isolated from saliva, thereby reducing cost and increasing accessibility compared to traditional D4Z4 deletion testing that uses DNA isolated from blood. MethodsHere, we adapt the D4Z4 BSS protocol to next-generation sequencing (NGS) to increase sequencing depth and further reduce cost, validate both sequencing technologies against several cohorts of genetically defined samples, and introduce the D4Z4caster software for computing DNA methylation signatures with diagnostic utility from raw sequencing data. ResultsBoth Sanger and NGS BSS methods using D4Z4caster were validated as providing high sensitivity and specificity, with geometric mean of sensitivity and specificity (G-mean) >95% and area-under-the ROC curve (AUC) of 0.99. The NGS method allows for higher throughput and increased read depth, while the Sanger method allows faster processing of individual samples. Importantly, the NGS method could identify FSHD1 cases that are likely mosaic and would otherwise be missed. ConclusionsD4Z4caster methylation signatures can accurately detect contracted FSHD1-permissive chromosome 4q35 alleles, hypomethylation of D4Z4 arrays indicative of FSHD2, and SNPs that are important for diagnostic use. This workflow is amenable to transitioning to clinical settings for an accurate, low-cost FSHD molecular diagnostic test that could be accessible worldwide. What is already known on this topicCurrently accepted genetic diagnostics for FSHD1 are complex and expensive and can mischaracterize certain complex genetic cases. These diagnostics all require high molecular weight genomic DNA typically freshly isolated from blood, highly specialized equipment, and additional testing for FSHD2, making FSHD diagnostics the most expensive among neuromuscular diseases and inaccessible to much of the world. However, the epigenetic status of the 4q35 and 10q26 D4Z4 repeat arrays, as determined by DNA methylation status using our bisulfite sequencing-based protocol, distinguishes genetically FSHD1, FSHD2, and non-FSHD samples. Additionally, since our protocol is PCR-based, it can utilize DNA isolated from multiple sources, including saliva and buccal swabs. What this study addsThis study validates the relevant DNA methylation signatures against several large cohorts of genetically-confirmed FSHD and non-FSHD samples and optimizes the DNA methylation data analysis for the greater accuracy required for diagnostic utility, including the exclusion of nonpathogenic chromosome 10q or 4A166 contractions. In addition, we introduce the D4Z4caster analysis software, which runs in a portable and scalable Docker container, and provides increased quantitative accuracy important for: 1) confirming likely clinical cases of FSHD that do not meet the currently accepted genetic definition of FSHD1 or FSHD2, 2) identifying FSHD1 somatic mosaicism, and 3) potential prognostic applications. How this study might affect research, practice or policyFSHD1 is genetically defined by a D4Z4 array at the 4q35 locus that is contracted to 1-10 repeat units. However, disease penetrance is influenced by repeat number, epigenetic modifications, and genetic background, causing a misalignment of current genetic diagnosis with clinical diagnosis. This study will improve the accuracy of epigenetic analysis for determining cases of genetic FSHD, help broaden the definition of genetic FSHD to more accurately correspond to clinical FSHD, and allow identification of those at risk for developing clinical FSHD in affected families and in large population studies now being performed and proposed. In addition, it will better inform how an individuals epigenetic status is interpreted for potential prognostic value. Overall, this methodology is: 1) significantly less expensive than current clinically-approved FSHD diagnostic technologies, 2) more accessible due to compatibility with DNA isolated from multiple sources including saliva, and 3) compatible with the current sequencing equipment and workflow for DNA isolation used in commercial clinical laboratories. Together, these advantages will help move the technology toward becoming an approved molecular diagnostic test for FSHD in the USA, Europe, and countries currently lacking clear access to testing.

genetics↗

bioRxiv2026

Identification of compounds that repress DUX4 expression in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic dysregulation of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Thus, most FSHD therapeutic approaches target DUX4. Our previous study identified the chromatin remodeling factor BAZ1A (bromodomain adjacent to zinc finger domain protein 1A) as a promising target for therapeutic development. Here we used an artificial intelligence-based screening pipeline to identify molecules predicted to bind the BAZ1A bromodomain, and validated hit compounds using FSHD-specific assays in FSHD myocytes. One compound, termed C06, emerged as a potent and specific repressor of DUX4 and DUX4 target gene expression. Interestingly, while C06 exhibited binding to BAZ1A in vitro, it can also inhibit multiple kinases, including p38, an upstream activator of DUX4. Despite this, at low doses C06 was an equally effective and more specific repressor of DUX4 than losmapimod, which is a robust and specific p38 inhibitor. Thus, C06 is a useful tool for potent and specific DUX4 suppression, and a viable candidate for further development. Our results highlight both the utility and limitations of AI for targeted drug discovery, and the importance of using an FSHD-specific functional screening strategy for selecting relevant candidates.

pharmacology and toxicology↗

bioRxiv2025

Transgenic mouse models for investigating human DUX4 expression during development and its roles in FSHD pathophysiology

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy caused by aberrant expression of the DUX4 retrogene, and it affects skeletal muscles primarily in the face, shoulder, and limbs. In healthy individuals, DUX4 is expressed in early development and is subsequently silenced in most somatic tissues. The spatiotemporal pattern of DUX4 mis-expression beyond the cleavage stage in FSHD is poorly understood because DUX4 is not well conserved beyond primates. Here, we generated Cre reporter mouse lines with human DUX4 regulatory elements to investigate the cell lineages derived from DUX4-expressing cells in embryos and adults. Intriguingly, we found that DUX4-expressing cell lineages were present in embryonic forelimb, hindlimb, and face. In adults, the reporter was expressed strongly in testis and to a lesser extent in other tissues, including weak, sporadic expression in skeletal muscles, reminiscent of mosaic DUX4 expression in FSHD. Within skeletal muscles, DUX4 lineage cells include pericytes, an interstitial cell that contributes to muscle regeneration and repair. Overall, this study introduces a new research tool for the field, and provides new insight into potential developmental mechanisms underlying FSHD pathophysiology. Summary statementCre reporter mouse lines with human DUX4 regulatory elements are discovery tools for developmental processes and mechanisms underlying FSHD pathophysiology.

developmental biology↗

bioRxiv2025

Custom-Built Electrodes Perform Comparably to a Discontinued Commercial Electrode for Neuromuscular Electrical Stimulation in Mice

ObjectiveA commercial electrode used for transcutaneous neuromuscular electrical stimulation (NMES) in mice is no longer available. In response, we developed two low- cost, customizable alternatives--a 3D-Printed Electrode and a Pen Electrode assembled from off-the-shelf jumper wire components--and evaluated their performance relative to the discontinued commercial standard. MethodsWe conducted in vivo NMES of the left hindlimb ankle dorsiflexors in C57BL/6J mice using three electrode types: a Simple Electrode (previously available as BS4 50-6824, Harvard Apparatus), our novel 3D-Printed Electrode, and our novel Pen Electrode. Contractile torque was recorded during twitch and tetanic contractions, and electrode performance was evaluated based on peak torque values. A repeated measures ANOVA was used to compare torque generated by each animal for the three different types of electrodes. ResultsBoth custom electrodes generated twitch and tetanic responses comparable to those of the Commercial electrode. No statistically significant differences were observed in peak torque values in either twitch (p = 0.181) or tetany (p = 0.438). ConclusionCustom-built 3D-printed and Pen-style electrodes offer low-cost, accessible alternatives to discontinued commercial devices for murine NMES studies. These tools can facilitate continued use of electrical stimulation protocols in preclinical neuromuscular research.

physiology↗

bioRxiv2023

Validation of the association between MRI and gene signatures in facioscapulohumeral dystrophy muscle: implications for clinical trial design

Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression, but reproducibility across studies needs further validation. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA. Together with moderate-to-strong correlations of gene signatures and MRI characteristics between the TA muscles bilaterally, these results suggest a whole muscle model of disease progression and provide a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design.

molecular biology↗

bioRxiv2018

Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity

BackgroundAll types of facioscapulohumeral muscular dystrophy (FSHD) are caused by the aberrant myogenic activation of the somatically silent DUX4 gene, which initiates a cascade of cellular events ultimately leading to FSHD pathophysiology. Therefore, FSHD is a dominant gain-of-function disease that is amenable to modeling by DUX4 overexpression. However, there is large variability in the patient population. Typically, progressive skeletal muscle weakness becomes noticeable in the second or third decade of life, yet there are many genetically FSHD individuals who develop symptoms much later in life or remain relatively asymptomatic throughout their lives. Conversely, in rare cases, FSHD may present clinically prior to 5-10 yrs of age, ultimately manifesting as a very severe early onset form of the disease. Thus, there is a need to control the timing and severity of pathology in FSHD-like models.\n\nMethodsWe have recently described a line of conditional DUX4 transgenic mice, FLExDUX4, that develop a myopathy upon induction of human DUX4-fl expression in skeletal muscle. Here, we use the FLExDUX4 mouse crossed with the skeletal muscle-specific and tamoxifen inducible line ACTAl-MerCreMer to generate a highly versatile bi-transgenic mouse model with chronic, low-level DUX4-fl expression and mild pathology, that can be induced to develop more severe FSHD-like pathology in a dose-dependent response to tamoxifen. We identified conditions to reproducibly generate models exhibiting mild, moderate, or severe DUX4-dependent pathophysiology, and characterized their progression.\n\nResultsWe assayed DUX4-fl mRNA and protein levels, fitness, strength, global gene expression, histopathology, and immune response, all of which are consistent with an FSHD-like myopathic phenotype. Importantly, we identified sex-specific and muscle-specific differences that should be considered when using these models for preclinical studies.\n\nConclusionsThe ACTA1-MCM;FLExDUX4 bi-transgenic mouse model expresses a chronic low level of DUX4-fl and has mild pathology and detectable muscle weakness. The onset and progression of moderate to severe pathology can be controlled via tamoxifen injection to provide consistent and readily screenable phenotypes for assessing therapies targeting DUX4-fl mRNA and protein. Thus, these FSHD-like mouse models can be used to study a range of DUX4-fl expression and pathology dependent upon investigator need, through controlled mosaic expression of DUX4.

cell biology↗