The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans.

The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. Here, we investigate how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. Our data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-à-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, we discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages.

Recurrent modification of a conserved cis-regulatory element underlies fruit fly pigmentation diversity.

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages.

Draft Guidance for Industry Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, and Related Dystrophinopathies - Developing Potential Treatments for the Entire Spectrum of Disease.

Background: Duchenne muscular dystrophy (DMD) and related dystrophinopathies are neuromuscular conditions with great unmet medical needs that require the development of effective medical treatments. Objective: To aid sponsors in clinical development of drugs and therapeutic biological products for treating DMD across the disease spectrum by integrating advancements, patient registries, natural history studies, and more into a comprehensive guidance. Methods: This guidance emerged from collaboration between the FDA, the Duchenne community, and industry stakeholders. It entailed a structured approach, involving multiple committees and boards. From its inception in 2014, the guidance underwent revisions incorporating insights from gene therapy studies, cardiac function research, and innovative clinical trial designs. Results: The guidance provides a deeper understanding of DMD and its variants, focusing on patient engagement, diagnostic criteria, natural history, biomarkers, and clinical trials. It underscores patient-focused drug development, the significance of dystrophin as a biomarker, and the pivotal role of magnetic resonance imaging in assessing disease progression. Additionally, the guidance addresses cardiomyopathy's prominence in DMD and the burgeoning field of gene therapy. Conclusions: The updated guidance offers a comprehensive understanding of DMD, emphasizing patient-centric approaches, innovative trial designs, and the importance of biomarkers. The focus on cardiomyopathy and gene therapy signifies the evolving realm of DMD research. It acts as a crucial roadmap for sponsors, potentially leading to improved treatments for DMD.

Barriers to diverse clinical trial participation in Duchenne muscular dystrophy: Engaging Hispanic/Latina caregivers and health professionals.

BACKGROUND: Despite the increasing availability of clinical trials in Duchenne muscular dystrophy, racial/ethnic minorities and other populations facing health disparities remain underrepresented in clinical trials evaluating products for Duchenne. We sought to understand the barriers faced by Hispanic/Latino families specifically and underrepresented groups more generally to clinical trial participation in Duchenne. METHODS: We engaged two participant groups: Hispanic/Latino caregivers of children with Duchenne in the US, including Puerto Rico, and health professionals within the broader US Duchenne community. Caregiver interviews explored attitudes towards and experiences with clinical trials, while professional interviews explored barriers to clinical trial participation among socio-demographically underrepresented families (e.g., low income, rural, racial/ethnic minority, etc.). Interviews were analyzed aggregately and using a thematic analysis approach. An advisory group was engaged throughout the course of the study to inform design, conduct, and interpretation of findings generated from interviews. RESULTS: Thirty interviews were conducted, including with 12 Hispanic/Latina caregivers and 18 professionals. We identified barriers to clinical trial participation at various stages of the enrollment process. In the initial identification of patients, barriers included lack of awareness about trials and clinical trial locations at clinics that were less likely to serve diverse patients. In the prescreening process, barriers included ineligibility, anticipated non-compliance in clinical trial protocols, and language discrimination. In screening, barriers included concerns about characteristics of the trial, as well as mistrust/lack of trust. In consent and recruitment, barriers included lack of timely decision support, logistical factors (distance, time, money), and lack of translated study materials. CONCLUSIONS: Numerous barriers hinder participation in Duchenne clinical trials for Hispanic/Latino families and other populations experiencing health disparities. Addressing these barriers necessitates interventions across multiple stages of the clinical trial enrollment process. Recommendations to enhance participation opportunities include developing clinical trial decision support tools, translating prominent clinical trials educational resources such as ClinicalTrials.gov, fostering trusting family-provider relationships, engaging families in clinical trial design, and establishing ethical guidelines for pre-screening potentially non-compliant patients.

First Regulatory Qualification of a Novel Digital Endpoint in Duchenne Muscular Dystrophy: A Multi-Stakeholder Perspective on the Impact for Patients and for Drug Development in Neuromuscular Diseases.

BACKGROUND: Functional outcome measures used to assess efficacy in clinical trials of investigational treatments for rare neuromuscular diseases like Duchenne muscular dystrophy (DMD) are performance-based tasks completed by the patient during hospital visits. These are prone to bias and may not reflect motor abilities in real-world settings. Digital tools, such as wearable devices and other remote sensors, provide the opportunity for continuous, objective, and sensitive measurements of functional ability during daily life. Maintaining ambulation is of key importance to individuals with DMD. Stride velocity 95th centile (SV95C) is the first wearable acquired digital endpoint to receive qualification from the European Medicines Agency (EMA) to quantify the ambulation ability of ambulant DMD patients aged ≥5 years in drug therapeutic studies; it is also currently under review for the US Food and Drug Administration (FDA) qualification. SUMMARY: Focusing on SV95C as a key example, we describe perspectives of multiple stakeholders on the promise of novel digital endpoints in neuromuscular disease drug development.

Health-Related Quality of Life and Emotional Distress Among Mothers of Sons With Muscular Dystrophy as Compared to Sex- and Age Group-Matched Controls.

The health-related quality of life and emotional distress among mothers of sons with Duchenne or Becker muscular dystrophies (n = 82) were compared to sex- and age group-matched controls (n = 26). Participants self-reported health-related quality of life for themselves and their son(s), emotional distress, and mood/anxiety-related medication. Mothers reported poorer health-related quality of life across all domains of their health-related quality of life, as well as higher levels of emotional distress. Clinically elevated symptoms of anxiety were reported by 39% of mothers. Mothers' report of poorer health-related quality of life for their son(s) was a significant predictor of worse health-related quality of life and emotional distress for themselves across most domains. Additionally, older age of mothers predicted greater energy/less fatigue and lower levels of anxiety. Results highlight the need for screening emotional distress among mothers, as well as consideration for accessible interventions to improve the psychosocial functioning among these families.

Red Light/Green Light, a Dual Fluorescent Protein Reporter System To Study Enhancer-Promoter Specificity in Drosophila.

Enhancers activate gene transcription in spatial and temporal patterns by interactions with gene promoters. These elements typically reside distal to their target promoter, with which they must interact selectively. Additional elements may contribute to enhancer-promoter specificity, including remote control element sequences within enhancers, tethering elements near promoters, and insulator/boundary elements that disrupt off-target interactions. However, few of these elements have been mapped, and as a result, the mechanisms by which these elements interact remain poorly understood. One impediment is their method of study, namely reporter transgenes in which enhancers are placed adjacent to a heterologous promoter, which may circumvent mechanisms controlling enhancer-promoter specificity and long-range interactions. Here, we report an optimized dual reporter transgene system in Drosophila melanogaster that allows the simultaneous comparison of an enhancer's ability to activate proximal and distal fluorescent reporter genes. Testing a panel of fluorescent transgenes in vivo, we found a two-protein combination that allows simultaneous measurement with minimal detection interference. We note differences among four tested enhancers in their ability to regulate a distally placed reporter transgene. These results suggest that enhancers differ in their requirements for promoter interaction and raise important practical considerations when studying enhancer function.

Duchenne and Becker muscular dystrophy carriers: Evidence of cardiomyopathy by exercise and cardiac MRI testing.

BACKGROUND: Varied detection methods have resulted in conflicting reports on the prevalence of cardiac disease in Duchenne and Becker muscular dystrophy carriers (MDC). METHODS: We performed a prospective cohort study of 77 genetically-confirmed MDC mothers, 22 non-carrier mothers, and 25 controls. All participants underwent Cardiopulmonary Exercise Testing (CPET) and Cardiac Magnetic Resonance imaging (CMR). RESULTS: 25% of carriers had ventricular ectopy in recovery of exercise (RecVE) as compared to 1 non-carrier and no controls (p = .003). No difference in age or maximal oxygen consumption was noted. 11 carriers had abnormal (<55%) left ventricular ejection fraction by CMR. Evidence of late gadolinium enhancement (LGE) was noted in 48% of MDC, 1 non-carrier patient and no control subjects (p < .0001). Subset analysis of LGE+ and LGE- subjects revealed differences in age (44.1 v 38.6 yrs.; p = .005), presence of RecVE, (38.9% v 10.5%, p = .004), and high serum creatine kinase (CK) (> 289 U/l; 52.8% v 31.6%, p = .065). CONCLUSION: We describe the prevalence of disease using CPET and CMR in genetically-proven MDC. 49% of carriers had fibrosis, opposed to 5% of non-carriers, highlighting the importance of genetic testing in this population. Despite cardiomyopathy, functional assessment by treadmill was normal, illustrating the discrepancy in cardiac and skeletal muscle impacts. Age, RecVE and serum CK appear to have an important role in predicting cardiomyopathy. Serum CK levels suggest that a systemic higher global disease severity and not tissue heterogeneity may be the etiology for greater cardiac disease and relatively spared skeletal muscle disease in this population. Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT02972580?term=mendell&cond=Duchenne+Muscular+Dystrophy&rank=5; ClinicalTrials.gov Identifier: NCT02972580.

Cis-regulatory evolution integrated the Bric-à-brac transcription factors into a novel fruit fly gene regulatory network.

Gene expression evolution through gene regulatory network (GRN) changes has gained appreciation as a driver of morphological evolution. However, understanding how GRNs evolve is hampered by finding relevant cis-regulatory element (CRE) mutations, and interpreting the protein-DNA interactions they alter. We investigated evolutionary changes in the duplicated Bric-à-brac (Bab) transcription factors and a key Bab target gene in a GRN underlying the novel dimorphic pigmentation of D. melanogaster and its relatives. It has remained uncertain how Bab was integrated within the pigmentation GRN. Here, we show that the ancestral transcription factor activity of Bab gained a role in sculpting sex-specific pigmentation through the evolution of binding sites in a CRE of the pigment-promoting yellow gene. This work demonstrates how a new trait can evolve by incorporating existing transcription factors into a GRN through CRE evolution, an evolutionary path likely to predominate newly evolved functions of transcription factors.