Genome Wide Set of Human Enhancers Edward Rubin

1 Proteostasis Jeffery W. Kelly, MD Departments of Chemistry and Molecular and Experimental Medicine, and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, CA The chemical information within the polypeptide chain, coand post-translational modifications of the amino acids comprising the protein, such as N-linked glycosylation, and the interactions of the polypeptide with proteostasis network components determine whether a given member of the proteome will fold and function, be degraded, remain natively unfolded, or aggregate and create additional proteostatic challenges for the organism. The outset of the seminar will focus on the intrinsic forces that predispose polypeptides to fold, including conformational propensities, hydrogen bonding, the hydrophobic effect and N-linked glycosylation. The second part of the talk will focus on the extrinsic forces that assist and enable proteome maintenance, and the means by which the proteostasis network enhances protein structure, function and clearance to facilitate life and avoid lossand gain-offunction diseases. The influence of the proteostasis network, comprising transcriptional and translational control of protein synthesis, chaperoneand enzyme-assisted folding, and disaggregation and degradation activities will be covered. Furthermore, the influence of aging-associated signaling pathways on proteome maintenance will be outlined. The lecture will close with a summary of what we have learned about degenerative diseases associated with protein aggregation and loss-of-function diseases associated with excessive mutant protein misfolding and degradation. Specifically, we will focus on how we are ameliorating these diseases with proteostasis regulators, small molecules that readapt the innate biology of proteostasis through signaling pathways that control the proteostasis network. Abstract 2 Genome Wide Set of Human Enhancers Edward Rubin, MD, PhD Director of the DOE Joint Genome Institute (JGI) and Genomics Division at the Lawrence Berkeley National Laboratory, Berkeley CA2 Genome Wide Set of Human Enhancers Edward Rubin, MD, PhD Director of the DOE Joint Genome Institute (JGI) and Genomics Division at the Lawrence Berkeley National Laboratory, Berkeley CA Tissue-specific enhancers are principal regulators of spatiotemporal gene expression and alerations in their activities contribute to many human disorders. Due to their location in non-coding genome regions and limited knowledge about their sequence features, human enhancers have been only minimally annotated. I will describe a large program focused on leveraging extreme evolutionary sequence conservation to identify putative regulatory sequences in the human genome and characterizing their/ in vivo/ enhancer activity in a transgenic mouse assay. To date, we have tested more than 500 such elements including all noncoding human-rodent ultraconserved elements in the human genome. More than 200 of them function as tissue-specific enhancers and reproducibly target gene expression to a broad range of anatomical structures. As a community resource, we have established a database to visualize and query the activity of these enhancer sequences at http://enhancer.lbl.gov/ and will be generating additional data for several thousand enhancers over the next several years. Abstract 3 Modifier Genes: Lessons from Cystic Fibrosis Garry R. Cutting, MD Johns Hopkins University, Baltimore, MD3 Modifier Genes: Lessons from Cystic Fibrosis Garry R. Cutting, MD Johns Hopkins University, Baltimore, MD Most “single gene” or monogenic disorders display phenotype variability that is only partially correlated with allelic variation in the disease-causing gene. Cystic Fibrosis, a disorder due to dysfunction of the CF transmembrane conductance regulator (CFTR), is such an example. Lung disease, the major cause of morbidity and mortality in CF, is highly variable even among individuals with identical CFTR mutations. These observations suggest that modifiers play a substantial role in CF lung disease. However, candidate gene methods to identify genetic modifiers have met with limited success with only two genes demonstrating replicated association with the lung severity. Lack of success may be due to poor selection of candidates or predominance of environmental factors rather than genetic modifiers in CF lung disease variability. To determine the relative contribution of genetic and non-genetic factors to variation, twins and siblings with CF have been recruited by the U.S. CF Twin and Sibling Study. Lung function as measured by the forced expiratory volume in 1 second (FEV1) is predictive of disease progression and survival in CF patients. This measure is more highly correlated in monozygous twins than in dizygous twins and siblings. These correlations infer that heritability (degree to which genes contribute to variation in a trait) of CF lung function is approximately .65. This heritability estimate is unchanged in individuals who carry identical CFTR mutations. These results demonstrate that genetic modifiers play in important role in variation in CF lung disease. The remaining 1/3 of the variation in lung disease can be attributed to other factors including those in the environment such as exposure to second-hand cigarette smoke (SHS). We have demonstrated that SHS exposure can modify lung function by 10%. Furthermore, variation in TGFβ1, a genetic modifier identified by Drumm and colleagues, interacts with SHS to double the deleterious effect upon lung function. To identify new genetic modifiers, the Cystic Fibrosis Modifier Gene Consortium was formed among the CF Twin/Sibling Study, the Genetic Modifier Study from University of North Carolina and Case Western, and the Canadian CF Genetic Modifier Study based in Toronto. The Consortium is currently performing genome-wide association and linkage studies to identify novel modifiers for CF lung disease. Twin and sibling analysis of other CF traits demonstrate that intestinal obstruction (termed meconium ileus) and diabetes display high heritability, thereby justifying genome-wide searches for genetic modifiers of these traits. Thus, studies in CF demonstrate that factors beyond variation in the disease-causing gene underlying disease variability can be quantified and used to inform searches for key modifiers. Abstract 4 Self-Assembling Bioactive Nanostructures for Regenerative Medicine Samuel I. Stupp, PhD Departments of Materials Science and Engineering, Chemistry, and Medicine Institute for BioNanotechnology in Medicine, Northwestern University, Evanston, IL4 Self-Assembling Bioactive Nanostructures for Regenerative Medicine Samuel I. Stupp, PhD Departments of Materials Science and Engineering, Chemistry, and Medicine Institute for BioNanotechnology in Medicine, Northwestern University, Evanston, IL Rapid advances in biology and nanotechnology have led to the possibility of designing bioactive materials for cell signaling in regenerative medicine. The main goal in this new field is to design nanostructures that are molecularly crafted to signal cells both in vitro, or in vivo. The chemistry of such nanostructures should allow them to interact specifically with cell receptors or intracellular organelles, interjecting with signaling pathways. The designs could also offer targeting of nanostructures to specific tissues and organs in order to use systemic delivery of therapies. Ideally, the nanostructures should also disintegrate into nutrients or harmless components within an appropriate time frame after regenerative processes have been triggered by their structure. Our laboratory has developed an extensive family of amphiphilic molecules that self-assemble into nanofiber architectures with capacity to display signals to cells. These systems were designed to be biomimetic of the extracellular matrix and to self-assemble into one-dimensional nanostructures in the presence of electrolytes. They can therefore be delivered clinically as simple injections of aqueous solutions that instantly form networks around cells in vivo. This lecture will illustrate the use of nanoscale molecular features in these systems to regenerate axons in the central nervous system after spinal cord injury and other brain disorders, and will also describe molecular designs targeting bone and cartilage regeneration as well as angiogenesis on demand. In the case of bone, the systems designed have the capacity to both recognize receptors and also mineralize in physiological environments to generate hydroxyapatite crystals that mimic those found in mammalian bone or enamel. The angiogenic materials to be discussed could be useful in cardiovascular therapies such as peripheral vascular disease, wound healing, and cell transplantation. 1. Science 2001, 294 (5527), 1684-1688 2. Proc. Natl. Acad. Sci. USA 2002, 99 (8), 5133-5138 3. Science 2004, 303, 1352-1355 4. J. Amer. Chem. Soc. 2005, 127 (4), 1193-1200 5. Nano Letters 2006, 6 (9), 2086-2090 6. Biomaterials 2007, 28(31), 4608-4618 7. Science 2008, 319 (5871), 1812-1816 8. J. of Neuroscience 2008, 28 (14), 3814-3823 9. J. of Bone and Mineral Research, in press 10. Transplantation, in press Abstract 5 FDA Incentives for Development of Therapies for Rare Diseases Mathew T. Thomas, MD FDA Office of Orphan Products Development5 FDA Incentives for Development of Therapies for Rare Diseases Mathew T. Thomas, MD FDA Office of Orphan Products Development FDA’s Office of Orphan Products Development (OOPD) was created in 1982 to administer the provisions of the 1983 US Orphan Drug Act (ODA) and assist sponsors (individuals, groups or pharmaceutical companies) in the development of products (drugs, biologics, devices, and medical foods) for the diagnosis, prevention or treatment of rare diseases or conditions in the US population. OOPD offers incentives for product development in rare diseases through four major programs: Orphan Designations, Grants, Humanitarian Use Device (HUD) Designations, and Outreach activities. This presentation will discuss these programs, explain the incentives that were developed and how they contribute to OOPD’s mission, review OOPD’s successes, and provide data to demonstrate the utilization of incentives for the development of products for rare bone-related diseases or conditions. Abstract 6 Osteogenesis Imperfecta: What Can We Learn from Mouse Models Antonella Forlino, PhD Department of Biochemistry “A.Castellani”, University of Pavia, Italy6 Osteogenesis Imperfecta: What Can We Learn from Mouse Models Antonella Forlino, PhD Department of Biochemistry “A.Castellani”, University of Pavia, Italy Classical Osteogenesis Imperfecta (OI) is a dominant negative skeletal dysplasia caused by mutations in COL1A1 or COL1A2 genes, coding for the alpha chains of type I collagen. The main OI clinical outcome is bone fragility and deformity. We generated some years ago a knock-in murine model for OI, BrtlIV carrying a Gly349Cys substitution in the α1 chain of type I collagen. BrtlIV shows a moderate or a lethal OI outcome reproducing the phenotypic variability reported for human patients. Taking advantage of that we investigated the molecular basis of this variability by evaluating bone mRNA expression by microarray and bone protein profile by 2-DE and mass spectrometry in the OI murine model BrtlIV. We generated the first reference 2-DE map for calvarial tissue, identifying 164 spots corresponding to 97 distinct proteins. In particular we found an increase in lethal BrtlIV of Gadd153 and a lower expression of the αBcrystallin that indicated an effect of the intracellular machinery on the phenotypic outcome. The higher expression in lethal BrtlIV of the extracellular matrix proteins Prelp, Bmp6 and Bmp7 and the reduced expression of Matrilin 4, Microfibril-associated glycoprotein 2 and Thrombospondin 3, revealed that the extracellular matrix composition also modulates OI phenotype. We then used our murine model to develop a cell therapy treatment which employs in utero transplantation to avoid marrow ablation for this metabolic inborn disorder. The bone marrow cells were isolated from long bones of eGFP-CD1 mice and injected into the liver of E14.5 embryos. Mice were analyzed at 2 m, the age corresponding to the severest BrtlIV bone phenotype, compared to WT. Engraftment with a characteristic patchy distribution was detected in various tissues at sacrifice by inverted microscopy. Confocal microscopy was used to directly quantify the engraftment in long bone diaphysis. The percentage of donor cells was determined by FACS, in both bone marrow and spleen and by Real Time PCR in different tissues. A reduction of the amount of mutated collagen was detected at both trabecular and cortical regions of long bone. The femur length was increased in transplanted mutant mice (p<0.005). PQCT of the distal femoral methaphysis revealed increased total bone and trabecular density in treated versus untreated mutant mice. Micro CT analysis of Brtl mid-shaft femur detected improvement in Total Mineral Content, Cortical Thickness and Cortical Area. The analysis of the treated mice suggested that in utero cell therapy is a promising treatment for classical OI. Abstract 7 Modeling Fibrous Dysplasia Mara Riminucci, MD, PhD Associate Professor of Pathology, University La Sapienza, Rome, Italy Lab Chief, Biomedical Scientific Park, Rome, Italy7 Modeling Fibrous Dysplasia Mara Riminucci, MD, PhD Associate Professor of Pathology, University La Sapienza, Rome, Italy Lab Chief, Biomedical Scientific Park, Rome, Italy Fibrous dysplasia (FD, OMIM#174800) is a genetic disease caused by postzygotic, activating mutations of the GNAS gene, which impair the GTPase activity of Gs-alpha thus resulting in excess intracellular cAMP. FD is a crippling disease occuring either as an isolated mono or polyostotic disorder, or in association with skin and endocrine lesions in the McCune-Albright syndrome. Although remarkable progress has been made over the last few years in our understanding of the disease, several aspects of FD have not been yet fully defined and no specific therapies are available at this time. Suitable experimental in vivo and in vitro models are necessary to dissect the pathogenetic mechanisms of the disease and to test innovative approaches to correct the genetic defect. Towards these ends, we generated a murine model of FD by using a lentivector based approach. A lentiviral vector containing the rat GsalphaR201C cDNA (LV-Gs-alphaR201C) under the control of the constitutive promoter EF1alpha was injected into the perivitelline space of mouse zygotes. Transgenic animals were generated in which multiple bone lesions were reproduced with typical radiographic and histological features of FD. In some animals, skeletal lesions were also associated with extraskeletal diseases thus reproducing the clinical picture of the McCune-Albright syndrome. Furthermore, interesting and unexpected biological features of the disorder have been observed in transgenic mice, which could change our current interpretation of some aspects of the human disease. Abstract 8 Congenital Scoliosis in a Mouse Model of Impaired Matrix-vesicle Calcification Manisha C. Yadav, Ph.D; Sonoko Narisawa, Ph.D; Colin Farquharson, Ph.D and José Luis Millán, Ph.D Sanford Children's Health Research Center, Burnham Institute for Medical Research, La Jolla, CA, USA; Bone Biology Group, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, United Kingdom8 Congenital Scoliosis in a Mouse Model of Impaired Matrix-vesicle Calcification Manisha C. Yadav, Ph.D; Sonoko Narisawa, Ph.D; Colin Farquharson, Ph.D and José Luis Millán, Ph.D Sanford Children's Health Research Center, Burnham Institute for Medical Research, La Jolla, CA, USA; Bone Biology Group, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, United Kingdom Matrix vesicle (MV)-mediated mineralization is believed to be the first step during the regulated process of endochondral ossification. After formation of hydroxyapatite (HA) crystals the MV membranes breakdown and release the preformed HA into the extracellular fluid. Tissue non-specific alkaline phosphatase (TNAP) is the major enzyme that regulates extravesicular growth of HA crystals by restricting the size of the calcification inhibitor inorganic pyrophosphate (PPi). In the absence of TNAP, a build up of PPi causes rickets/osteomalacia characteristic of the most severe forms of hypophosphatasia. However, HA crystal formation inside MVs is not affected, suggesting the involvement of other phosphatase(s) during the initial events leading to MV-induced mineralization. In previous studies, expression of a novel orphan phosphatase, PHOSPHO-1, was reported in mineralizing regions of skeletal tissues. Here we have assessed the phenotypic changes associated with functional ablation of the Phospho1 gene. We examined survival and growth rate of Phospho1 null mutant mice, skeletal abnormalities by radiography, calcification ability and TNAP activity in MVs, circulating levels of PPi and ALP activity in plasma and osteoblast culture media and osteoblastic gene expression by qRT-PCR. Both male and female Phospho1 null mutant mice display stunted growth with short and distorted long bone and prominent thoracic scoliosis. Increased PPi levels were found in the plasma and calvarial osteoblast culture media. MVs showed a decrease in their calcification ability and reduced TNAP activity. We are currently investigating the metabolic changes that lead to these skeletal deformities. To-date the PHOSPHO1 null mice appear to represent a model of congenital scoliosis. Abstract 9 X-Linked Hypophosphatemia (XLH) Farzana Perwad, MD Department of Pediatrics at University of California San Francisco, CA9 X-Linked Hypophosphatemia (XLH) Farzana Perwad, MD Department of Pediatrics at University of California San Francisco, CA XLH is a genetic disorder that is inherited as an X-linked dominant trait, and affected individuals share common clinical features with other hypophosphatemic syndromes such as Autosomal Dominant Hypophosphatemic Rickets (ADHR), Tumor Induced Osteomalacia (TIO) and Autosomal Recessive Hypophosphatemic Rickets (ARHR). This syndrome is characterized by hypophosphatemia due to renal phosphate wasting, inappropriately low or normal serum 1,25(OH)2D concentrations and increased circulating serum FGF-23 concentrations. These biochemical abnormalities contribute to the development of skeletal defects including rickets in children and osteomalacia in adults. Severity of the disease varies considerably even among members of the same family. Hypophosphatemia can occur as early as six to nine months of age. However, XLH patients frequently present with short stature, valgum or varus deformities of the lower extremity, bone pain and dental abscesses. Severely affected individuals can have frontal bossing and spinal cord compression. Enthesopathy is common in middle aged patients. Patients with XLH harbor inactivating mutations in the Phex (Phosphate regulating gene with Homologies to Endopeptidases on the X chromosome) gene. The gene encodes for a 749 amino acid protein that closely resembles members of the endopeptidase family. PHEX protein is a type II integral membrane glycoprotein with a short N terminal cytoplasmic domain, a transmembrane domain and a large extracellular domain. Phex mRNA is predominantly expressed in bone and teeth but the exact function of PHEX is unknown. Studies performed in hyp mice, a mouse model of XLH, show increased production of FGF-23 in bone, suggesting that PHEX negatively regulates FGF-23 production. Several animal studies have confirmed that excess circulating FGF-23 is responsible for the biochemical abnormalities seen in these hypophosphatemic disorders including XLH. This hypothesis was confirmed when the hyp phenotype was completely rescued by crossbreeding hyp mice with the FGF-23 knockout mice. However, the exact mechanism by which PHEX interacts with FGF23 is unknown. Currently there is no curative therapy available to decrease circulating FGF-23 concentrations and thereby improve serum phosphorus and 1,25(OH)2D levels. Therefore, therapy is limited to oral supplementation with phosphate and vitamin D to promote growth in children and prevent skeletal deformation. However, close monitoring is required as this form of supportive therapy can cause hypercalciuria, hypercalcemia, nephrocalcinosis and is a concern for potential long term renal damage. Understanding the relationship between PHEX and FGF-23 and the molecular mechanisms by which FGF-23 regulates phosphate and vitamin D metabolism in the kidney, is crucial to improving clinical outcome and quality of life for patients with XLH. Abstract 10 The Role Of Inflammation In The Pathogenesis Of Bone & Joint Disease In The Mucopolysaccharidoses Calogera M. Simonaro, PhD Genetics and Genomic Sciences at the Mount Sinai School of Medicine in New York, NY10 The Role Of Inflammation In The Pathogenesis Of Bone & Joint Disease In The Mucopolysaccharidoses Calogera M. Simonaro, PhD Genetics and Genomic Sciences at the Mount Sinai School of Medicine in New York, NY The Mucopolysaccharidoses (MPS) are inherited, connective tissue disorders that result from deficiencies of specific lysosomal enzymes required for glycosaminoglycan (GAG) degradation. Among the various organ systems involved, the bones and joints are severely affected. MPS animal models have provided important insights into the causes of bone and joint pathology in these disorders. For example, by 6 months of age an abnormal cellular and molecular profile was seen in the bones and joints of rats with MPS type VI (Maroteaux-Lamy disease), with characteristic increases in cytokines, MMPs, and apoptotic cells. We proposed that GAG storage in the MPS disorders leads to inflammation and apoptosis within cartilage, most likely through activation of the toll-like receptor-4 signaling pathway (Simonaro et al., 2001, 2005, 2008). We have also recently performed gene and protein expression analysis on fibroblast-like synoviocytes (FLS) from MPS VI rats, which similarly revealed that numerous inflammatory molecules were elevated, including several molecules important for TLR4 signaling (e.g., lipoprotein binding protein, CD44, and MyD88). TLR4 reporter cells lines have been generated, and will be used to investigate whether GAGs directly activate TLR4 or the lipopolysaccharide signaling pathway in vitro. In addition, MPS VII/TLR4 double mutants are being generated to evaluate the in vivo effects of GAGs on this important signaling pathway. We have also found that treatment of normal FLS and chondrocytes with GAGs leads to proliferation and apoptosis, respectively. This correlated with the production of the “pro-survival” lipid, sphingosine-1-phosphate, in FLS, and the “proapoptotic” lipid, ceramide, in chondrocytes. Both lipids have been implicated in signaling. These findings have important implications for the pathogenesis and treatment of MPS, and have defined a novel mechanism of GAG-stimulated disease that may be occurring in other common bone disorders. Abstract 11 A Novel Genetic Form of Hypoparathyroidism is Caused by DominantNegative Mutations in the Transcription Factor GCMB Michael Mannstadt, MD Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston MA11 A Novel Genetic Form of Hypoparathyroidism is Caused by DominantNegative Mutations in the Transcription Factor GCMB Michael Mannstadt, MD Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston MA Identification of genetic causes of hypoparathyroidism (HP) is important for enhancing our understanding of parathyroid biology and could help define new drug targets. HP is characterized by hypocalcemia and low/inadequately normal PTH levels. Mutations in several different genes were previously discovered as the cause of autosomal-dominant (AD) or autosomal-recessive (AR) forms of HP, but most sporadic forms remain unresolved at the molecular level. AD-HP can be caused by heterozygous activating mutations in the calcium-sensing receptor or by heterozygous mutations in the PTH gene that impair intracellular processing of the nascent protein. AR-HP can be caused by homozygous mutations in the genes encoding PTH or glial cells missing B (GCMB), a transcription factor specific for the parathyroid gland. Consistent with these findings, GCMB-null mice lack parathyroid glands, whereas heterozygous carriers of inactivating GCMB mutations appear to be healthy, both in mice and man. We identified a novel mechanism for AD-HP in two unrelated families. Two different heterozygous, single nucleotide deletions in the last GCMB exon (mutA and mutB) were identified in genomic DNA of the affected members of both families. Both mutations lead to a shift in the open reading frame resulting in the replacement of the second transactivation domain located within the carboxyl-terminal region of GCMB with unrelated amino acid sequence. Consistent with the mode of inheritance, the mutant GCMB proteins exhibited in vitro dominant-negative properties in luciferase-based reporter assays, while two previously reported homozygous GCMB mutations (R47L and G63S) that are the cause of an autosomal recessive form of HP did not affect function of wild-type (WT) GCMB. We generated three antibodies in rabbits against peptides corresponding to GCMB residues 111-130 (N1), 225-245 (C1) and 481-500 (C2). WT-GCMB was well expressed in fibroblast DF-1 cells as assessed by Western blot analysis of cell lysates using antibodies N1, C1, or C2. As expected, when using antibody N1, Western analysis of lysates from cells transfected with mutA and mutB revealed a slightly larger protein band than lysates from cells expressing WT-GCMB; no mutant protein was detected when using antibody C2 that is directed against the portion of the protein that is replaced in GCMBmutA and GCMBmutB. GFP-tagged mutant GCMB demonstrated normal nuclear localization. In summary, two novel, heterozygous mutations in the parathyroid-specific transcription factor GCMB were identified in the affected members of two families with autosomal-dominant hypoparathyroidism. In vitro analyses revealed a dominant-negative effect of the mutant GCMB proteins, thus providing evidence for a novel disease mechanism. Abstract 12 Melorheostosis Deborah Wenkert, MD Shriners Hospital for Children, St Louis, USA12 Melorheostosis Deborah Wenkert, MD Shriners Hospital for Children, St Louis, USA Melorheostosis (OMIM #155950) is a rare (0.9 per million) sclerosing bone disease reported ~300 times in the literature. It features monostotic or polyostotic, asymmetric irregular dense linear bony thickening of the cortex of one or more long bones. This radiographic finding, named for its flowing candle wax appearance, begins with an endosteal hyperostosis. The disorder, typically unilateral, most often involves the extremities, scapula, and pelvis in an apparently sclerotomal distribution. Although it can be a radiographic finding without clinical consequences, melorheostosis can be associated with pain, asymmetrical growth, soft tissue fibrosis, vascular anomalies, and contractures. On occasion, melorheostosis can be found in association with other sclerosing bone disorders when it is called “mixed sclerosing bone dystrophy” or with the LEMD3 associated disorders, osteopoikilosis or Buschke-Ollendorff Syndrome. The cause and pathogenic basis of isolated sporadic melorheostosis, however, is not known. Current theories include postzygotic mutation with mosaicism, reaction to trauma, infection or sensory neuropathy, and autoimmune disease. There have been 10 new papers regarding melorheostosis so far in medline in 2008 including reviews and case reports. Two papers revealed possible clues to pathogenesis (elevated FGF-23 levels in one patient, coexistence of tricho-dentoosseous syndrome and melorheostosis in another). The Melorheostosis Association website, on which >130 people with melorheostosis have posted medical histories, may give additional clues. Although review of these histories contains questions of accuracy and ascertainment bias, a review of histories posted before 8/27/2008 yielded 121 nonfamilial, nonaxial cases: Only 6 patients clearly indicated bilaterality of x-ray findings; 98 had unilateral involvement. Twice as many had lower as had upper extremity involvement (77 vs 37) and only 6 indicated both upper and lower extremity involvement. Only 16 patients indicated they experienced no pain; 93 complained of pain. Other complications included limb shortening (17), decreased range of motion (37), swelling (14), and only 1 patient mentioned Raynaud’s phenomena. Thus, the patients who posted their histories of sporadic melorheostosis on the melorheostosis website had more complaints of pain but otherwise mimic the clinical characteristics of the disease as reported in the literature. Interestingly, the distribution of patients across the United States is similar to census data which may argue against an infectious or local toxic etiology for the disease.