The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

An ectopic enhancer restores CFTR expression through de novo chromatin looping.

Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by both ubiquitous and cell-type selective cis-regulatory elements (CREs). These CREs include extragenic and intronic enhancers that bind lineage-specific transcription factors, and architectural protein-marked structural elements. Deletion of the airway-selective -35 kb enhancer in 16HBE14o- lung epithelial cells was shown earlier to disrupt normal enhancer-promoter looping at the CFTR locus and nearly abolish its expression. Using a 16HBE14o- clone that lacks the endogenous -35 kb CRE, we explore the impact of relocating the functional core of this element to an ectopic site in intron 1. The -35 kb sequence establishes a de novo enhancer signature in chromatin at the insertion site, and augments CFTR expression, albeit not fully restoring WT levels. The relocated -35 kb enhancer also initiates de novo chromatin contacts with the CFTR promoter and other known CFTR CREs. These results are broadly relevant to therapeutic gene editing.

Human Molecular Genetics and the long road to treating cystic fibrosis.

The causative gene in cystic fibrosis (CF) was identified in 1989, 3 years before the publication of the first issue of Human Molecular Genetics. The cystic fibrosis transmembrane conductance regulator (CFTR) gene was among the first underlying a common inherited disorder to be cloned, and hence, its subsequent utilization toward a cure for CF provides a roadmap for other monogenic diseases. Over the past 30 years, the advances that built upon knowledge of the gene and the CFTR protein to develop effective therapeutics have been remarkable, and yet, the setbacks have also been challenging. Technological progress in other fields has often circumvented the barriers. This review focuses on key aspects of CF diagnostics and current approaches to develop new therapies for all CFTR mutations. It also highlights the major research advances that underpinned progress toward treatments and considers the remaining obstacles.

Transcriptomic analysis of lung development in wildtype and CFTR-/- sheep suggests an early inflammatory signature in the CF distal lung.

The precise molecular events initiating human lung disease are often poorly characterized. Investigating prenatal events that may underlie lung disease in later life is challenging in man, but insights from the well-characterized sheep model of lung development are valuable. Here, we determine the transcriptomic signature of lung development in wild-type sheep (WT) and use a sheep model of cystic fibrosis (CF) to characterize disease associated changes in gene expression through the pseudoglandular, canalicular, saccular, and alveolar stages of lung growth and differentiation. Using gene ontology process enrichment analysis of differentially expressed genes at each developmental time point, we define changes in biological processes (BP) in proximal and distal lung from WT or CF animals. We also compare divergent BP in WT and CF animals at each time point. Next, we establish the developmental profile of key genes encoding components of ion transport and innate immunity that are pivotal in CF lung disease and validate transcriptomic data by RT-qPCR. Consistent with the known pro-inflammatory phenotype of the CF lung after birth, we observe upregulation of inflammatory response processes in the CF sheep distal lung during the saccular stage of prenatal development. These data suggest early commencement of therapeutic regimens may be beneficial.

Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-ß signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies.

Cross-talk between enhancers, structural elements and activating transcription factors maintains the 3D architecture and expression of the CFTR gene.

Robust protocols to examine 3D chromatin structure have greatly advanced knowledge of gene regulatory mechanisms. Here we focus on the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which provides a paradigm for validating models of gene regulation built upon genome-wide analysis. We examine the mechanisms by which multiple cis-regulatory elements (CREs) at the CFTR gene coordinate its expression in intestinal epithelial cells. Using CRISPR/Cas9 to remove CREs, individually and in tandem, followed by assays of gene expression and higher-order chromatin structure (4C-seq), we reveal the cross-talk and dependency of two cell-specific intronic enhancers. The results suggest a mechanism whereby the locus responds when CREs are lost, which may involve activating transcription factors such as FOXA2. Also, by removing the 5' topologically-associating domain (TAD) boundary, we illustrate its impact on CFTR gene expression and architecture. These data suggest a multi-layered regulatory hierarchy that is highly sensitive to perturbations.

A Distracted Scientist: The Life and Contributions of John Senders.

OBJECTIVE: To provide an evaluative and personal overview of the life and contributions of Professor John Senders and to introduce this Special Issue dedicated to his memory. BACKGROUND: John Senders made many profound contributions to HF/E. These various topics are exemplified by the range of papers which compose the Special Issue. Collectively, these works document and demonstrate the impact of his many valuable research works. METHOD: The Special Issue serves to summarize Senders' collective body of work as can be extracted from archival sources. This introductory paper recounts a series of remembrances derived from personal relationships, as well as the products of cooperative investigative research. RESULTS: This collective evaluative process documents Senders' evident and deserved status in the highest pantheon of HF/E pioneers. It records his extraordinary life, replete with accounts of his insights and joie de vivre in exploring and explaining the world which surrounded him. APPLICATIONS: Senders' record of critical contributions provides the example, par excellence, of the successful and fulfilling life in science. It encourages all, both researchers and practitioners alike, in their own individual search for excellence.

Krüppel-like factor 5 regulates wound repair and the innate immune response in human airway epithelial cells.

A complex network of transcription factors regulates genes involved in establishing and maintaining key biological properties of the human airway epithelium. However, detailed knowledge of the contributing factors is incomplete. Here we characterize the role of Krüppel-like factor 5 (KLF5), in controlling essential pathways of epithelial cell identity and function in the human lung. RNA-seq following siRNA-mediated depletion of KLF5 in the Calu-3 lung epithelial cell line identified significant enrichment of genes encoding chemokines and cytokines involved in the proinflammatory response and also components of the junctional complexes mediating cell adhesion. To determine direct gene targets of KLF5, we defined the cistrome of KLF5 using ChIP-seq in both Calu-3 and 16HBE14o- lung epithelial cell lines. Occupancy site concordance analysis revealed that KLF5 colocalized with the active histone modification H3K27ac and also with binding sites for the transcription factor CCAAT enhancer-binding protein beta (C/EBPß). Depletion of KLF5 increased both the expression and secretion of cytokines including IL-1ß, a response that was enhanced following exposure to Pseudomonas aeruginosa lipopolysaccharide. Calu-3 cells exhibited faster rates of repair after KLF5 depletion compared with negative controls in wound scratch assays. Similarly, CRISPR-mediated KLF5-null 16HBE14o- cells also showed enhanced wound closure. These data reveal a pivotal role for KLF5 in coordinating epithelial functions relevant to human lung disease.

Sexual Well-Being After Nipple-Sparing Mastectomy: Does Preservation of the Nipple Matter?

INTRODUCTION: The primary aim of this study was to evaluate patient-reported outcome measures in patients undergoing mastectomy with and without breast reconstruction (immediate or delayed) with and without nipple preservation. METHODS: All female patients undergoing mastectomy between 2011 and 2015 at Mayo Clinic Rochester were identified and were mailed the BREAST-Q survey. Breast satisfaction, psychosocial well-being, and sexual well-being were evaluated and compared by surgery type using Wilcoxon rank-sum tests for univariate analysis and linear regression for multivariable analysis adjusting for potential confounders. RESULTS: Of 1547 patients, 771 completed the BREAST-Q survey (response rate 50%). Of these 771 respondents, 237 (31%) did not have reconstruction, 198 (26%) had nipple-sparing mastectomy with reconstruction (NSM), and 336 (44%) had skin-sparing mastectomy with reconstruction (SSM) ± nipple-areolar complex (NAC) reconstruction (via surgery ± tattoo). Patients with breast reconstruction had consistently higher BREAST-Q scores versus those without. Comparing NSM with all SSMs, there was no difference in satisfaction with breasts (mean 71.8 vs. 70.2, p = 0.21) or psychosocial well-being (mean 81.9 vs. 81.3, p = 0.47); however, sexual well-being was significantly higher in the NSM group on univariate (mean 64.5 vs. 58.0, p = 0.002) and multivariable (ß = -4.69, p = 0.03) analysis. Sexual well-being scores were similar for NSM and the SSM subgroups with any type of NAC reconstruction. CONCLUSIONS: This study demonstrates that NSM positively impacts patient sexual well-being after breast reconstruction compared with SSM, particularly SSM without nipple reconstruction or tattoo. SSM with any type of NAC reconstruction achieved similar satisfaction and sexual well-being to those undergoing NSM.

BACH1, the master regulator of oxidative stress, has a dual effect on CFTR expression.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a topologically associated domain (TAD) in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB domain and CNC homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here, we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (∼8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.

Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells.

The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanisms controlling its transcription are likely divergent between them. Here, we determine how two extragenic CREs that are prominent in epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at -44 and -35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here, we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter, suggesting non-redundant enhancers. The deletions also greatly reduced promoter interactions with the 5' TAD boundary. We show substantial recruitment of RNAPII to the -35 kb element and identify CEBPß as a key activator of airway expression of CFTR, likely through occupancy at this CRE and the gene promoter.

OTX2 regulates CFTR expression during endoderm differentiation and occupies 3' cis-regulatory elements.

BACKGROUND: Cell-specific and developmental mechanisms contribute to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene; however, its developmental regulation is poorly understood. Here we use human induced pluripotent stem cells differentiated into pseudostratified airway epithelial cells to study these mechanisms. RESULTS: Changes in gene expression and open chromatin profiles were investigated by RNA-seq and ATAC-seq, and revealed that alterations in CFTR expression are associated with differences in stage-specific open chromatin. Additionally, two novel open chromatin regions, at +19.6 kb and +22.6 kb 3' to the CFTR translational stop signal, were observed in definitive endoderm (DE) cells, prior to an increase in CFTR expression in anterior foregut endoderm (AFE) cells. Chromatin studies in DE and AFE cells revealed enrichment of active enhancer marks and occupancy of OTX2 at these sites in DE cells. Loss of OTX2 in DE cells alters histone modifications across the CFTR locus and results in a 2.5-fold to 5-fold increase in CFTR expression. However, deletion of the +22.6 kb site alone does not affect CFTR expression in DE or AFE cells. CONCLUSIONS: These results suggest that a network of interacting cis-regulatory elements recruit OTX2 to the locus to impact CFTR expression during early endoderm differentiation.

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.

A functional genomics approach to investigate the differentiation of iPSCs into lung epithelium at air-liquid interface.

The availability of robust protocols to differentiate induced pluripotent stem cells (iPSCs) into many human cell lineages has transformed research into the origins of human disease. The efficacy of differentiating iPSCs into specific cellular models is influenced by many factors including both intrinsic and extrinsic features. Among the most challenging models is the generation of human bronchial epithelium at air-liquid interface (HBE-ALI), which is the gold standard for many studies of respiratory diseases including cystic fibrosis. Here, we perform open chromatin mapping by ATAC-seq and transcriptomics by RNA-seq in parallel, to define the functional genomics of key stages of the iPSC to HBE-ALI differentiation. Within open chromatin peaks, the overrepresented motifs include the architectural protein CTCF at all stages, while motifs for the FOXA pioneer and GATA factor families are seen more often at early stages, and those regulating key airway epithelial functions, such as EHF, are limited to later stages. The RNA-seq data illustrate dynamic pathways during the iPSC to HBE-ALI differentiation, and also the marked functional divergence of different iPSC lines at the ALI stages of differentiation. Moreover, a comparison of iPSC-derived and lung donor-derived HBE-ALI cultures reveals substantial differences between these models.

Functional genomics analysis of human colon organoids identifies key transcription factors.

Organoids are a valuable three-dimensional (3D) model to study the differentiated functions of the human intestinal epithelium. They are a particularly powerful tool to measure epithelial transport processes in health and disease. Though biological assays such as organoid swelling and intraluminal pH measurements are well established, their underlying functional genomics are not well characterized. Here we combine genome-wide analysis of open chromatin by ATAC-Seq with transcriptome mapping by RNA-Seq to define the genomic signature of human intestinal organoids (HIOs). These data provide an important tool for investigating key physiological and biochemical processes in the intestinal epithelium. We next compared the transcriptome and open chromatin profiles of HIOs with equivalent data sets from the Caco2 colorectal carcinoma line, which is an important two-dimensional (2D) model of the intestinal epithelium. Our results define common features of the intestinal epithelium in HIO and Caco2 and further illustrate the cancer-associated program of the cell line. Generation of Caco2 cysts enabled interrogation of the molecular divergence of the 2D and 3D cultures. Overrepresented motif analysis of open chromatin peaks identified caudal type homeobox 2 (CDX2) as a key activating transcription factor in HIO, but not in monolayer cultures of Caco2. However, the CDX2 motif becomes overrepresented in open chromatin from Caco2 cysts, reinforcing the importance of this factor in intestinal epithelial differentiation and function. Intersection of the HIO and Caco2 transcriptomes further showed functional overlap in pathways of ion transport and tight junction integrity, among others. These data contribute to understanding human intestinal organoid biology.

The FOXA1 transcriptional network coordinates key functions of primary human airway epithelial cells.

The differentiated functions of the human airway epithelium are coordinated by a complex network of transcription factors. These include the pioneer factors Forkhead box A1 and A2 (FOXA1 and FOXA2), which are well studied in several tissues, but their role in airway epithelial cells is poorly characterized. Here, we define the cistrome of FOXA1 and FOXA2 in primary human bronchial epithelial (HBE) cells by chromatin immunoprecipitation with deep-sequencing (ChIP-seq). Next, siRNA-mediated depletion of each factor is used to investigate their transcriptome by RNA-seq. We found that, as predicted from their DNA-binding motifs, genome-wide occupancy of the two factors showed substantial overlap; however, their global impact on gene expression differed. FOXA1 is an abundant transcript in HBE cells, while FOXA2 is expressed at low levels, and both these factors likely exhibit autoregulation and cross-regulation. FOXA1 regulated loci are involved in cell adhesion and the maintenance of epithelial cell identity, particularly through repression of genes associated with epithelial to mesenchymal transition (EMT). FOXA1 also directly targets other transcription factors with a known role in the airway epithelium such as SAM-pointed domain-containing Ets-like factor (SPDEF). The intersection of the cistrome and transcriptome for FOXA1 revealed enrichment of genes involved in epithelial development and tissue morphogenesis. Moreover, depletion of FOXA1 was shown to reduce the transepithelial resistance of HBE cells, confirming the role of this factor in maintaining epithelial barrier integrity.

Recent advances in developing therapeutics for cystic fibrosis.

Despite hope that a cure was imminent when the causative gene was cloned nearly 30 years ago, cystic fibrosis (CF [MIM: 219700]) remains a life-shortening disease affecting more than 70 000 individuals worldwide. However, within the last 6 years the Food and Drug Administration's approval of Ivacaftor, the first drug that corrects the defective cystic fibrosis transmembrane conductance regulator protein [CFTR (MIM: 602421)] in patients with the G551D mutation, marks a watershed in the development of novel therapeutics for this devastating disease. Here we review recent progress in diverse research areas, which all focus on curing CF at the genetic, biochemical or physiological level. In the near future it seems probable that development of mutation-specific therapies will be the focus, since it is unlikely that any one approach will be efficient in correcting the more than 2000 disease-associated variants. We discuss the new drugs and combinations of drugs that either enhance delivery of misfolded CFTR protein to the cell membrane, where it functions as an ion channel, or that activate channel opening. Next we consider approaches to correct the causative genetic lesion at the DNA or RNA level, through repressing stop mutations and nonsense-mediated decay, modulating splice mutations, fixing errors by gene editing or using novel routes to gene replacement. Finally, we explore how modifier genes, loci elsewhere in the genome that modify CF disease severity, may be used to restore a normal phenotype. Progress in all of these areas has been dramatic, generating enthusiasm that CF may soon become a broadly treatable disease.

An organoid model to assay the role of CFTR in the human epididymis epithelium.

Organoid cultures derived from primary human tissues facilitate the study of disease processes and the development of new therapeutics. Most men with cystic fibrosis (CF) are infertile due to defects in the epididymis and vas deferens; however, the causative mechanisms are still unclear. We used human epididymis epithelial cell (HEE) organoids and polarized HEE cell cultures to assay the CF transmembrane conductance regulator (CFTR) in the human epididymis. 3D HEE organoids and polarized 2D HEE cell cultures on membrane inserts were established from human caput epididymis. Single-cell RNA sequencing (scRNA-seq) was performed to map cell type-specific gene expression in the organoids. Using forskolin (FSK) to activate CFTR and inhibitor CFTRinh172 to block its activity, we assessed how CFTR contributes to organoid swelling and epithelial barrier function. The scRNA-seq data showed key caput epididymis cell types present in HEE organoid cultures. FSK at 10 µM induced HEE organoid swelling by 20% at 16 h, while 5 and 10 µM CFTRinh172 treatment significantly reduced HEE organoid size. In transepithelial resistance (TER) measurements, FSK reduced TER, while inhibition of CFTR increased TER; also, depletion of CFTR with specific siRNAs significantly increased TER. FSK treatment significantly increased the flux of 4-kDa but not 70-kDa dextran, suggesting activation of CFTR mainly enhances transcellular diffusion. We have demonstrated that CFTR contributes to the maintenance of HEE cell TER and that cultured HEE organoids are a useful model to investigate human epididymis function. These results facilitate progress in elucidating how CFTR-dependent cellular processes impair fertility in CF.

Coordinate regulation of ELF5 and EHF at the chr11p13 CF modifier region.

E74-like factor 5 (ELF5) and ETS-homologous factor (EHF) are epithelial selective ETS family transcription factors (TFs) encoded by genes at chr11p13, a region associated with cystic fibrosis (CF) lung disease severity. EHF controls many key processes in lung epithelial function so its regulatory mechanisms are important. Using CRISPR/Cas9 technology, we removed three key cis-regulatory elements (CREs) from the chr11p13 region and also activated multiple open chromatin sites with CRISPRa in airway epithelial cells. Deletion of the CREs caused subtle changes in chromatin architecture and site-specific increases in EHF and ELF5. CRISPRa had most effect on ELF5 transcription. ELF5 levels are low in airway cells but higher in LNCaP (prostate) and T47D (breast) cancer cells. ATAC-seq in these lines revealed novel peaks of open chromatin at the 5' end of chr11p13 associated with an expressed ELF5 gene. Furthermore, 4C-seq assays identified direct interactions between the active ELF5 promoter and sites within the EHF locus, suggesting coordinate regulation between these TFs. ChIP-seq for ELF5 in T47D cells revealed ELF5 occupancy within EHF introns 1 and 6, and siRNA-mediated depletion of ELF5 enhanced EHF expression. These results define a new role for ELF5 in lung epithelial biology.

Episodic and Chronic Migraine in Primary Care.

OBJECTIVE: To inform migraine care model development by assessing differences between patients with chronic migraine (CM) and episodic migraine (EM) in the current state of treatment, disability, patient satisfaction, and quality improvement opportunities. BACKGROUND: Efficient and focused use of scarce resources will be needed to address challenges within large populations of migraine patients. METHODS: We deployed a cross-sectional survey study of randomly selected migraine patients within a community primary care practice. RESULTS: There were 516 survey respondents (516/1804 [30%] response rate). CM patients were more likely than EM patients to report care from a neurologist (76/110 [69%] vs 229/406 [56%]; P = .0026), and higher disability according to the Migraine Disability Assessment and Headache Impact Test - 6 questionnaires (P < .0001). CM patients were less likely than EM patients to report overall satisfaction with care (16/110 [38%] vs 156/406 [66%], P = .0002), satisfaction with access to care (17/110 [33%] vs 176/406 [68%], P < .0001), and advice they needed (16/110 [31%] vs 160/406 [62%], P < .0001). Most patients with migraine had been offered triptan medications 377/516 (78%). Overall, 156/516 (31%) of individuals were currently taking any medication for migraine prevention, and 208/516 (40%) including botulinum toxin injections. CM patients were more likely to be taking preventive medication (39/110 [36%] vs 117/406 [29%], P = .0191) and report familiarity with the diagnosis of medication-overuse headache than patients with EM (80/110 [81%] vs 256/406 [69%], P = .0178). CONCLUSIONS: We observed differences between patients with chronic and EM and expected care delivery improvement opportunities for migraine patients in primary care. CM patients report higher levels of disability and less satisfaction with access to perceived needed medical advice and care. These findings support the need to further develop and study novel care models to efficiently and effectively deliver high-quality care and expertise in limited supply to a diverse migraine population.