The Comparative Pathology Workbench: Interactive visual analytics for biomedical data.

Pathologists need to compare histopathological images of normal and diseased tissues between different samples, cases, and species. We have designed an interactive system, termed Comparative Pathology Workbench (CPW), which allows direct and dynamic comparison of images at a variety of magnifications, selected regions of interest, as well as the results of image analysis or other data analyses such as scRNA-seq. This allows pathologists to indicate key diagnostic features, with a mechanism to allow discussion threads amongst expert groups of pathologists and other disciplines. The data and associated discussions can be accessed online from anywhere in the world. The Comparative Pathology Workbench (CPW) is a web-browser-based visual analytics platform providing shared access to an interactive "spreadsheet" style presentation of image and associated analysis data. The CPW provides a grid layout of rows and columns so that images that correspond to matching data can be organised in the form of an image-enabled "spreadsheet". An individual workbench can be shared with other users with read-only or full edit access as required. In addition, each workbench element or the whole bench itself has an associated discussion thread to allow collaborative analysis and consensual interpretation of the data. The CPW is a Django-based web-application that hosts the workbench data, manages users, and user-preferences. All image data are hosted by other resource applications such as OMERO or the Digital Slide Archive. Further resources can be added as required. The discussion threads are managed using WordPress and include additional graphical and image data. The CPW has been developed to allow integration of image analysis outputs from systems such as QuPath or ImageJ. All software is open-source and available from a GitHub repository.

Disparities in Air Pollutants Across Racial, Ethnic, and Poverty Groups at US Public Schools.

We investigate socioeconomic disparities in air quality at public schools in the contiguous US using high resolution estimates of fine particulate matter (PM2.5) and nitrogen dioxide (NO2) concentrations. We find that schools with higher proportions of people of color (POC) and students eligible for the federal free or reduced lunch program, a proxy for poverty level, are associated with higher pollutant concentrations. For example, we find that the median annual NO2 concentration for White students, nationally, was 7.7 ppbv, compared to 9.2 ppbv for Black and African American students. Statewide and regional disparities in pollutant concentrations across racial, ethnic, and poverty groups are consistent with nationwide results, where elevated NO2 concentrations were associated with schools with higher proportions of POC and higher levels of poverty. Similar, though smaller, differences were found in PM2.5 across racial and ethnic groups in most states. Racial, ethnic, and economic segregation across the rural-urban divide is likely an important factor in pollution disparities at US public schools. We identify distinct regional patterns of disparities, highlighting differences between California, New York, and Florida. Finally, we highlight that disparities exist not only across urban and non-urban lines but also within urban environments.

Trans-cortical vessels in the mouse temporal bulla bone are a means to recruit myeloid cells in chronic otitis media and limit peripheral leukogram changes.

Chronic otitis media, inflammation of the middle ear, is a sequel to acute otitis media in ∼8% of children. Chronic otitis media with effusion is the most common cause of childhood deafness and is characterised by effusion of white blood cells into the auditory bulla cavity. Skull flat bones have trans-cortical vessels which are responsible for the majority of blood flow in and out of the bone. In experimental models of stroke and aseptic meningitis there is preferential recruitment of myeloid cells (neutrophils and monocytes) from the marrow in skull flat bones. We report trans-cortical vessels in the mouse temporal bone connect to the bulla mucosal vasculature and potentially represent a means to recruit myeloid cells directly into the inflamed bulla. The mutant mouse strains Junbo (Mecom Jbo/+ ) and Jeff (Fbxo11 Jf/+ ) develop chronic otitis spontaneously; Mecom Jbo/+ mice have highly cellular neutrophil (90%) rich bulla exudates whereas Fbxo11 Jf/+ mice have low cellularity serous effusions (5% neutrophils) indicating differing demand for neutrophil recruitment. However we found peripheral leukograms of Mecom Jbo/+ and Fbxo11 Jf/+ mice are similar to their respective wild-type littermate controls with healthy bullae and infer preferential mobilization of myeloid cells from temporal bulla bone marrow may mitigate the need for a systemic inflammatory reaction. The cytokines, chemokines and haematopoietic factors found in the inflamed bulla represent candidate signalling molecules for myeloid cell mobilization from temporal bone marrow. The density of white blood cells in the bulla cavity is positively correlated with extent of mucosal thickening in Mecom Jbo/+ , Fbxo11 Jf/+ , and Eda Ta mice and is accompanied by changes in epithelial populations and bone remodelling. In Mecom Jbo/+ mice there was a positive correlation between bulla cavity WBC numbers and total bacterial load. The degree of inflammation varies between contralateral bullae and between mutant mice of different ages suggesting inflammation may wax and wane and may be re-initiated by a new wave of bacterial infection. Clearance of white blood cells and inflammatory stimuli from the bulla cavity is impaired and this may create a pro-inflammatory feedback loop which further exacerbates otitis media and delays its resolution.

The EDA-deficient mouse has Zymbal's gland hypoplasia and acute otitis externa.

In mice, rats, dogs and humans, the growth and function of sebaceous glands and eyelid Meibomian glands depend on the ectodysplasin signalling pathway. Mutation of genes encoding the ligand EDA, its transmembrane receptor EDAR and the intracellular signal transducer EDARADD leads to hypohidrotic ectodermal dysplasia, characterised by impaired development of teeth and hair, as well as cutaneous glands. The rodent ear canal has a large auditory sebaceous gland, the Zymbal's gland, the function of which in the health of the ear canal has not been determined. We report that EDA-deficient mice, EDAR-deficient mice and EDARADD-deficient rats have Zymbal's gland hypoplasia. EdaTa mice have 25% prevalence of otitis externa at postnatal day 21 and treatment with agonist anti-EDAR antibodies rescues Zymbal's glands. The aetiopathogenesis of otitis externa involves infection with Gram-positive cocci, and dosing pregnant and lactating EdaTa females and pups with enrofloxacin reduces the prevalence of otitis externa. We infer that the deficit of sebum is the principal factor in predisposition to bacterial infection, and the EdaTa mouse is a potentially useful microbial challenge model for human acute otitis externa.

Limb development genes underlie variation in human fingerprint patterns.

Fingerprints are of long-standing practical and cultural interest, but little is known about the mechanisms that underlie their variation. Using genome-wide scans in Han Chinese cohorts, we identified 18 loci associated with fingerprint type across the digits, including a genetic basis for the long-recognized "pattern-block" correlations among the middle three digits. In particular, we identified a variant near EVI1 that alters regulatory activity and established a role for EVI1 in dermatoglyph patterning in mice. Dynamic EVI1 expression during human development supports its role in shaping the limbs and digits, rather than influencing skin patterning directly. Trans-ethnic meta-analysis identified 43 fingerprint-associated loci, with nearby genes being strongly enriched for general limb development pathways. We also found that fingerprint patterns were genetically correlated with hand proportions. Taken together, these findings support the key role of limb development genes in influencing the outcome of fingerprint patterning.

Comparison of effusion cell block and biopsy immunohistochemistry in mesothelial hyperplasia, mesothelioma, and carcinoma in dogs.

BACKGROUND: Determining the cause of effusions is challenging and might require a biopsy. Whether cell blocks from effusions are representative of biopsies requires investigation. A previously developed immunohistochemical panel aids in the differentiation of hyperplastic and neoplastic mesothelium in canine biopsies but has not been investigated in effusions. OBJECTIVES: The study aimed to assess cell blocks as an alternative to biopsies and determine whether immunohistochemistry helps distinguish hyperplastic mesothelium, mesothelioma, and carcinoma. METHODS: Effusions and biopsies were collected from five dogs with mesothelial hyperplasia (group MH), six with mesothelioma (group M), and five with carcinoma (group C). Immunohistochemistry (IHC) for cytokeratin, vimentin, Wilm's tumor protein 1 (WT1), desmin, glucose transporter 1 (GLUT1), and insulin-like growth factor II mRNA-binding protein 3 (IMP3) was performed. Sections were scored for staining intensity and the percentage of positively stained cells. RESULTS: In paired cell blocks and biopsies, vimentin and WT1 staining were positively correlated for intensity and the percentage of positive cells, although not all paired results were identical. The intensity of IMP3 staining in cell blocks was higher in group M than in group C (P = 0.012), and WT1 staining was higher in group MH than in group C (P = 0.020). For biopsies, the intensity of WT1 staining was higher in group MH than in group C (P = 0.031). In group C, WT1 was negative in all cell blocks and biopsies, and desmin was negative in four of five cases. CONCLUSIONS: IHC results for the cell blocks and biopsies were comparable for potentially useful markers, such as WT1, which helped discriminate between groups. IHC provided additional information, although results were not always definitive. Further studies on a larger population are required.

Methods for the Administration of EDAR Pathway Modulators in Mice.

Genetic deficiency of ectodysplasin A (EDA) causes X-linked hypohidrotic ectodermal dysplasia, a congenital condition characterized by the absence or abnormal formation of sweat glands, teeth, and several skin appendages. Stimulation of the EDA receptor (EDAR) with agonists in the form of recombinant EDA or anti-EDAR antibodies can compensate for the absence of Eda in a mouse model of Eda deficiency, provided that agonists are administered in a timely manner during fetal development. Here we provide detailed protocols for the administration of EDAR agonists or antagonists, or other proteins, by the intravenous, intraperitoneal, and intra-amniotic routes as well as protocols to collect blood, to visualize sweat gland function, and to prepare skulls in mice.

Cytological and microbiological characteristics of middle ear effusions in brachycephalic dogs.

BACKGROUND: Middle ear effusion is common in brachycephalic dogs with similarities to otitis media with effusion in children. Association with the cranial and eustachian tube morphology and bacterial infection is suspected in both species. HYPOTHESIS/OBJECTIVES: To determine cytological and bacteriological features of middle ear effusions in dogs, provide information on histological features, and further assess the dog as a model of the human disease. ANIMALS: Sixteen live dogs, 3 postmortem cases of middle ear effusion, and 2 postmortem controls. METHODS: Prospective; clinical investigation using computed tomography, magnetic resonance imaging, video-otoscopy, myringotomy; cytological assessment of 30 and bacteriology of 28 effusions; histology and immunohistochemistry (CD3 for T-lymphocytes, Pax5 for B lymphocytes and MAC387 for macrophages) of 10 middle ear sections. RESULTS: Effusions were associated with neurological deficits in 6/16 (38%) and concurrent atopic dermatitis and otitis externa in 9/16 (56%) of live cases. Neutrophils and macrophages predominated on cytology (median 60 [range 2%-95.5%] and 27 [2%-96.5%]) whether culture of effusions was positive or not. In histology sections, the mucosa was thickened in affected dogs but submucosal gland dilatation occurred in affected and unaffected dogs. There was no bacterial growth from 22/28 (79%) of effusions. Bacteria isolated from the other 6 (21%) were predominantly Staphylococcus pseudintermedius (4/6, 67%). CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical, morphological, and cytological findings in middle ear effusions of dogs and people suggest similar pathogeneses. Middle ear effusion of dogs could be a useful model of human otitis media with effusion. Such comparisons can improve understanding and management across species.

Role of ectodysplasin signalling in middle ear and nasal pathology in rat and mouse models of hypohidrotic ectodermal dysplasia.

Patients with mutations in the ectodysplasin receptor signalling pathway genes - the X-linked ligand ectodysplasin-A (EDA), the receptor EDAR or the receptor adapter EDARADD - have hypohidrotic ectodermal dysplasia (HED). In addition to having impaired development of teeth, hair, eccrine sweat glands, and salivary and mammary glands, HED patients have ear, nose and throat disease. The mouse strains Tabby (EdaTa ) and downless (Edardl-J/dl-J ) have rhinitis and otitis media due to loss of submucosal glands in the upper airway. We report that prenatal correction of EDAR signalling in EdaTa mice with the agonist anti-EDAR antibody rescues the auditory-tube submucosal glands and prevents otitis media, rhinitis and nasopharyngitis. The sparse- and wavy-haired (swh) rat strain carries a mutation in the Edaradd gene and has similar cutaneous HED phenotypes to mouse models. We report that auditory-tube submucosal glands are smaller in the homozygous mutant Edaraddswh/swh than those in unaffected heterozygous Edaraddswh/+ rats, and that this predisposes them to otitis media. Furthermore, the pathogenesis of otitis media in the rat HED model differs from that in mice, as otitis media is the primary pathology, and rhinitis is a later-onset phenotype. These findings in rodent HED models imply that hypomorphic as well as null mutations in EDAR signalling pathway genes may predispose to otitis media in humans. In addition, this work suggests that the recent successful prenatal treatment of X-linked HED (XLHED) in humans may also prevent ear, nose and throat disease, and provides diagnostic criteria that distinguish HED-associated otitis media from chronic otitis media with effusion, which is common in children.

Chronic otitis media is initiated by a bulla cavitation defect in the FBXO11 mouse model.

Auditory bulla cavitation defects are a cause of otitis media, but the normal cellular pattern of bulla mesenchyme regression and its failure are not well understood. In mice, neural-crest-derived mesenchyme occupies the bulla from embryonic day 17.5 (E17.5) to postnatal day 11 (P11) and then regresses to form the adult air-filled bulla cavity. We report that bulla mesenchyme is bordered by a single layer of non-ciliated epithelium characterized by interdigitating cells with desmosome cell junctions and a basal lamina, and by Bpifa1 gene expression and laminin staining of the basal lamina. At P11-P12, the mesenchyme shrinks: mesenchyme-associated epithelium shortens, and mesenchymal cells and extracellular matrix collagen fibrils condense, culminating in the formation of cochlea promontory mucosa bordered by compact non-ciliated epithelial cells. FBXO11 is a candidate disease gene in human chronic otitis media with effusion and we report that a bulla cavitation defect initiates the pathogenesis of otitis media in the established mouse model Jeff (Fbxo11Jf/+ ). Persistent mesenchyme in Fbxo11Jf/+ bullae has limited mesenchymal cell condensation, fibrosis and hyperplasia of the mesenchyme-associated epithelium. Subsequent modification forms fibrous adhesions that link the mucosa and the tympanic membrane, and this is accompanied by dystrophic mineralization and accumulation of serous effusion in the bulla cavity. Mouse models of bulla cavitation defects are important because their study in humans is limited to post-mortem samples. This work indicates new diagnostic criteria for this otitis media aetiology in humans, and the prospects of studying the molecular mechanisms of murine bulla cavitation in organ culture.

Transcript Analysis Reveals a Hypoxic Inflammatory Environment in Human Chronic Otitis Media With Effusion.

Chronic otitis media with effusion (COME) is the most common cause of childhood hearing loss in the developed world. Underlying pathophysiology is not well understood, and in particular the factors that lead to the transition from acute to chronic inflammation. Here we present the first genome-wide transcript analysis of white blood cells in the effusion of children with COME. Analysis of microarray data for enriched pathways reveals upregulation of hypoxia pathways, which is confirmed using real-time PCR and determining VEGF protein titres. Other pathways upregulated in both mucoid and serous effusions include Toll-like receptor signaling, complement, and RANK-RANKL. Cytology reveals neutrophils and macrophages predominated in both serous and mucoid effusions, however, serous samples had higher lymphocyte and eosinophil differential counts, while mucoid samples had higher neutrophil differential counts. Transcript analysis indicates serous fluids have CD4+ and CD8+ T-lymphocyte, and NK cell signatures. Overall, our findings suggest that inflammation and hypoxia pathways are important in the pathology of COME, and targets for potential therapeutic intervention, and that mucoid and serous COME may represent different immunological responses.

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

Loss of the homeostatic protein BPIFA1, leads to exacerbation of otitis media severity in the Junbo mouse model.

Otitis Media (OM) is characterized by epithelial abnormalities and defects in innate immunity in the middle ear (ME). Although, BPIFA1, a member of the BPI fold containing family of putative innate defence proteins is abundantly expressed by the ME epithelium and SNPs in Bpifa1 have been associated with OM susceptibility, its role in the ME is not well characterized. We investigated the role of BPIFA1 in protection of the ME and the development of OM using murine models. Loss of Bpifa1 did not lead to OM development. However, deletion of Bpifa1 in Evi1Jbo/+ mice, a model of chronic OM, caused significant exacerbation of OM severity, thickening of the ME mucosa and increased collagen deposition, without a significant increase in pro-inflammatory gene expression. Our data suggests that BPIFA1 is involved in maintaining homeostasis within the ME under steady state conditions and its loss in the presence of inflammation, exacerbates epithelial remodelling leading to more severe OM.

Macrophage colony-stimulating factor increases hepatic macrophage content, liver growth, and lipid accumulation in neonatal rats.

Signaling via the colony-stimulating factor 1 receptor (CSF1R) controls the survival, differentiation, and proliferation of macrophages. Mutations in CSF1 or CSF1R in mice and rats have pleiotropic effects on postnatal somatic growth. We tested the possible application of pig CSF1-Fc fusion protein as a therapy for low birth weight (LBW) at term, using a model based on maternal dexamethasone treatment in rats. Neonatal CSF1-Fc treatment did not alter somatic growth and did not increase the blood monocyte count. Instead, there was a substantial increase in the size of liver in both control and LBW rats, and the treatment greatly exacerbated lipid droplet accumulation seen in the dexamethasone LBW model. These effects were reversed upon cessation of treatment. Transcriptional profiling of the livers supported histochemical evidence of a large increase in macrophages with a resident Kupffer cell phenotype and revealed increased expression of many genes implicated in lipid droplet formation. There was no further increase in hepatocyte proliferation over the already high rates in neonatal liver. In conclusion, treatment of neonatal rats with CSF1-Fc caused an increase in liver size and hepatic lipid accumulation, due to Kupffer cell expansion and/or activation rather than hepatocyte proliferation. Increased liver macrophage numbers and expression of endocytic receptors could mitigate defective clearance functions in neonates. NEW & NOTEWORTHY This study is based on extensive studies in mice and pigs of the role of CSF1/CSF1R in macrophage development and postnatal growth. We extended the study to neonatal rats as a possible therapy for low birth weight. Unlike our previous studies in mice and pigs, there was no increase in hepatocyte proliferation and no increase in monocyte numbers. Instead, neonatal rats treated with CSF1 displayed reversible hepatic steatosis and Kupffer cell expansion.

Understanding the aetiology and resolution of chronic otitis media from animal and human studies.

Inflammation of the middle ear, known clinically as chronic otitis media, presents in different forms, such as chronic otitis media with effusion (COME; glue ear) and chronic suppurative otitis media (CSOM). These are highly prevalent diseases, especially in childhood, and lead to significant morbidity worldwide. However, much remains unclear about this disease, including its aetiology, initiation and perpetuation, and the relative roles of mucosal and leukocyte biology, pathogens, and Eustachian tube function. Chronic otitis media is commonly modelled in mice but most existing models only partially mimic human disease and many are syndromic. Nevertheless, these models have provided insights into potential disease mechanisms, and have implicated altered immune signalling, mucociliary function and Eustachian tube function as potential predisposing mechanisms. Clinical studies of chronic otitis media have yet to implicate a particular molecular pathway or mechanism, and current human genetic studies are underpowered. We also do not fully understand how existing interventions, such as tympanic membrane repair, work, nor how chronic otitis media spontaneously resolves. This Clinical Puzzle article describes our current knowledge of chronic otitis media and the existing research models for this condition. It also identifies unanswered questions about its pathogenesis and treatment, with the goal of advancing our understanding of this disease to aid the development of novel therapeutic interventions.

Non-Typeable Haemophilus influenzae Infection of the Junbo Mouse.

Acute otitis media, inflammation of the middle ear bulla, is the most common bacterial infection in children. For one of the principal otopathogens, non-typeable Haemophilus influenzae (NTHi), animal models allow us to investigate host-microbial interactions relevant to the onset and progression of infection and to study treatment of middle ear disease. We have established a robust model of NTHi middle ear infection in the Junbo mouse. Intranasal inoculation with NTHi produces high rates of bulla infection and high bacterial titers in bulla fluids; bacteria can also spread down the respiratory tract to the mouse lung. An innate immune response is detected in the bulla of Junbo mice following NTHi infection, and bacteria are maintained in some ears at least up to day 56 post-inoculation. The Junbo/NTHi infection model facilitates studies on bacterial pathogenesis and antimicrobial intervention regimens and vaccines for better treatment and prevention of NTHi middle ear infection. © 2017 by John Wiley & Sons, Inc.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

Ectodysplasin signalling deficiency in mouse models of hypohidrotic ectodermal dysplasia leads to middle ear and nasal pathology.

Hypohidrotic ectodermal dysplasia (HED) results from mutation of the EDA, EDAR or EDARADD genes and is characterized by reduced or absent eccrine sweat glands, hair follicles and teeth, and defective formation of salivary, mammary and craniofacial glands. Mouse models with HED also carry Eda, Edar or Edaradd mutations and have defects that map to the same structures. Patients with HED have ear, nose and throat disease, but this has not been investigated in mice bearing comparable genetic mutations. We report that otitis media, rhinitis and nasopharyngitis occur at high frequency in Eda and Edar mutant mice and explore the pathogenic mechanisms related to glandular function, microbial and immune parameters in these lines. Nasopharynx auditory tube glands fail to develop in HED mutant mice and the functional implications include loss of lysozyme secretion, reduced mucociliary clearance and overgrowth of nasal commensal bacteria accompanied by neutrophil exudation. Heavy nasopharynx foreign body load and loss of gland protection alters the auditory tube gating function and the auditory tubes can become pathologically dilated. Accumulation of large foreign body particles in the bulla stimulates granuloma formation. Analysis of immune cell populations and myeloid cell function shows no evidence of overt immune deficiency in HED mutant mice. Our findings using HED mutant mice as a model for the human condition support the idea that ear and nose pathology in HED patients arises as a result of nasal and nasopharyngeal gland deficits, reduced mucociliary clearance and impaired auditory tube gating function underlies the pathological sequelae in the bulla.

A genome-wide association scan implicates DCHS2, RUNX2, GLI3, PAX1 and EDAR in human facial variation.

We report a genome-wide association scan for facial features in ∼6,000 Latin Americans. We evaluated 14 traits on an ordinal scale and found significant association (P values<5 × 10(-8)) at single-nucleotide polymorphisms (SNPs) in four genomic regions for three nose-related traits: columella inclination (4q31), nose bridge breadth (6p21) and nose wing breadth (7p13 and 20p11). In a subsample of ∼3,000 individuals we obtained quantitative traits related to 9 of the ordinal phenotypes and, also, a measure of nasion position. Quantitative analyses confirmed the ordinal-based associations, identified SNPs in 2q12 associated to chin protrusion, and replicated the reported association of nasion position with SNPs in PAX3. Strongest association in 2q12, 4q31, 6p21 and 7p13 was observed for SNPs in the EDAR, DCHS2, RUNX2 and GLI3 genes, respectively. Associated SNPs in 20p11 extend to PAX1. Consistent with the effect of EDAR on chin protrusion, we documented alterations of mandible length in mice with modified Edar funtion.