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Clinical-pathological correlations in three patients with fibrodysplasia ossificans progressiva
Accepted Manuscript Clinical-pathological correlations in fibrodysplasia ossificans progressiva
three
patients
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Kelly L. Wentworth, Katherine Bigay, Tea V. Chan, Jennifer P. Ho, Blanca M. Morales, Joseph Connor, Erin Brooks, M. Shahriar Salamat, Henry Charles Sanchez, Geoffrey Wool, Robert J. Pignolo, Frederick S. Kaplan, Edward C. Hsiao PII: DOI: Reference:
S8756-3282(17)30375-7 doi:10.1016/j.bone.2017.10.009 BON 11449
To appear in:
Bone
Received date: Revised date: Accepted date:
30 June 2017 4 October 2017 6 October 2017
Please cite this article as: Kelly L. Wentworth, Katherine Bigay, Tea V. Chan, Jennifer P. Ho, Blanca M. Morales, Joseph Connor, Erin Brooks, M. Shahriar Salamat, Henry Charles Sanchez, Geoffrey Wool, Robert J. Pignolo, Frederick S. Kaplan, Edward C. Hsiao , Clinical-pathological correlations in three patients with fibrodysplasia ossificans progressiva. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bon(2017), doi:10.1016/j.bone.2017.10.009
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ACCEPTED MANUSCRIPT Clinical-Pathological Correlations in Three Patients with Fibrodysplasia Ossificans Progressiva Kelly L. Wentworth1†, Katherine Bigay1*, Tea V. Chan1*, Jennifer P. Ho1*, Blanca M. Morales1, Joseph Connor3, Erin Brooks3, M. Shahriar Salamat3, Henry Charles Sanchez2, Geoffrey Wool6, Robert J. Pignolo4, Frederick S. Kaplan5, and Edward C. Hsiao1†
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Division of Endocrinology, Diabetes, and Metabolism, and the Institute for Human Genetics, Department
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of Medicine, University of California, San Francisco, CA Division of Pathology, University of California, San Francisco, CA
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Department of Medicine, Mayo Clinic, Rochester, MN
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Division of Pathology, University of Wisconsin, Madison, WI
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Departments of Orthopaedic Surgery and Medicine, and the Center for Research in FOP and Related
Disorders at the Perelman School of Medicine of the University of Pennsylvania, Philadelphia, PA 6
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Department of Pathology, University of Chicago, Chicago, IL
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*The 2nd, 3rd and 4th authors all contributed equally to this manuscript
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Abbreviated Title: Autopsy Findings in Patients with FOP Keywords: Fibrodysplasia ossificans progressiva; heterotopic ossification; thoracic insufficiency
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syndrome; restrictive lung disease; cardiomyopathy; autopsy Number of Figures and Tables: 6 figures
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Corresponding authors and person to whom reprint requests should be addressed:
Kelly Wentworth, MD and Edward C. Hsiao, M.D., Ph.D. Department of Endocrinology, Diabetes, and Metabolism Institute for Human Genetics University of California, San Francisco
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ACCEPTED MANUSCRIPT 513 Parnassus Ave, HSE 901 San Francisco, CA 94143-0794 Phone: 415-476-9732 Fax: 415-353-2337
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e-mails:
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[email protected]
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[email protected]
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ACCEPTED MANUSCRIPT Funding and Disclosures: We gratefully acknowledge the funding support for this project. KW received support from a UCSF/Eli and Edythe Broad Fellowship and the Wilsey Family Fellowship to the UCSF Endocrinology, Diabetes, and Metabolism Training Program. EH received research grant support from the Doris Duke Charitable Fund (2014099), the March of Dimes (1-FY14-211), and the Radiant Hope
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Foundation. RJP receives support from the Robert and Arlene Kogod Professorship in Geriatric Medicine
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at the Mayo Clinic, and the Radiant Hope Foundation. KW, KB, JH, TC, FSK, RJP and EH also receive
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research support from Clementia Pharmaceuticals for FOP clinical research studies, which pose no conflicts with this work. The authors have no additional disclosures. We would like to thank the
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International FOP Association for their support of the FOP patient community.
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Authors’ roles: KW, KB, JH, TC, MM, and EH wrote the manuscript. HS, JC, EB, and MSS performed the autopsies, assisted with interpretation of the findings, and provided the post-mortem images. EH, RJP,
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and FSK cared for the patients and provided detailed medical histories. All authors assisted with editing,
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approved the final version of the manuscript, and take responsibility for the integrity of the analysis and
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presentation.
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ACCEPTED MANUSCRIPT ABSTRACT Objective: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder in which heterotopic bone forms in the soft tissues. This often occurs in response to injury or inflammation, leading to joint immobilization and significant disability. There are currently no definitive treatment options for
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this devastating disease. Although the most dramatic phenotype in FOP is the episodic and progressive
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heterotopic ossification, patients report a number of symptoms that affect other organ systems. Post-
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mortem examination of FOP patients may contribute to our understanding of the underlying pathophysiology and complications of this disease. Here, we present the autopsy findings from three
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patients with FOP.
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Findings: Autopsy findings in two of the three patients confirmed that the cause of death was cardiorespiratory failure in the setting of severe thoracic insufficiency from heterotopic ossification. Both
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of these patients also had evidence of right ventricular dilatation likely secondary to thoracic insufficiency.
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The third patient died from complications of a traumatic head injury after a fall but also had post-mortem evidence of thoracic insufficiency syndrome. All three patients had extensive, widespread heterotopic
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ossification and joint deformities consistent with FOP. There was extensive ossification of the spinal
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ligament in these patients, which may contribute to cervical spine rigidity. One patient was diagnosed post-mortem with a brainstem malformation. No additional significant abnormalities were noted in the
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other organ systems. Finally, we also demonstrate that cadaveric skin fibroblasts can be isolated for use as a potential source for future in vitro cell culture studies.
Conclusions: This autopsy case series provides valuable information about the underlying complications of FOP and contributes significantly to our knowledge of this rare yet debilitating disorder. Thoracic insufficiency syndrome, right heart dysfunction, widespread heterotopic ossification, spinal ligament ossification, and CNS malformations were clearly evident; however, most other non-bone tissues appeared to be spared from gross malformations. Finally, the ability to isolate live cells from cadaveric
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ACCEPTED MANUSCRIPT skin is an important technique that will facilitate future studies, particularly as induced pluripotent stem cells and other cell-based technologies evolve. This case series highlights the importance of post-mortem examinations and their contribution to our current knowledge of disease pathophysiology and
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comorbidities.
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ACCEPTED MANUSCRIPT 1. Introduction Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare disorder characterized by massive heterotopic ossification of the connective tissues, leading to progressive joint immobilization and severe disability. Approximately one in two million children worldwide are diagnosed with FOP, typically within the first
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decade of life[1]. FOP is a chronic, progressive disease in which patients develop episodic “flare-ups” of
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painful, localized soft tissue swellings that rapidly progress to heterotopic bone via an endochondral bone
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formation pathway. Flare-ups are sporadic and may occur spontaneously or in response to local injury and inflammation in both the axial and appendicular skeleton. As a consequence of the widespread
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heterotopic ossification and subsequent joint dysfunction, most patients require the use of a wheelchair by the third decade of life[2]. Patients with classical FOP also have congenital malformation of the great toes
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and thumbs, osteochondromas, and variable fusion of the cervical spine facet joints[3, 4]. FOP is a genetic disorder most commonly caused by an autosomal dominant missense mutation
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in the ACVR1/ALK2 gene (c.617 G>A; p.R206H), which encodes activin receptor type Ia/activin-like
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kinase 2 receptor [5]. This point mutation alters the GS activation domain of the kinase receptor, leading to abnormal signaling through the ACVR1 receptor and over-activation of the downstream SMAD 1/5/8
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and p38 signaling pathways. The over-activation of these signaling pathways triggered by BMPs or
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Activin A is responsible for the widespread heterotopic ossification and congenital abnormalities seen in these patients[6-8]. Currently, there are no effective treatments for FOP, and the median age at time of
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death is 42 years with the median estimated lifespan is 56 years[9]. One known complication of FOP is the development of restrictive lung disease as a consequence of the significant heterotopic ossification involving the chest wall, spine, and rib cage[9, 10]. Thoracic insufficiency syndrome, defined as the inability of the thorax to support normal respiration or lung growth, develops as a result of congenital costovertebral malformations and/or extensive heterotopic ossification often accompanied by severe scoliosis[11]. Thoracic insufficiency syndrome is a well-established complication of FOP and remains the leading cause of death in these patients[9, 10].
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ACCEPTED MANUSCRIPT We have little knowledge about whether other organ systems may be affected in FOP. In addition, the advent of new cellular technologies such as induced pluripotent stem cells promises to help reveal many of the physiologic and pathogenic events that contribute to disease progression. Here, we report the autopsy findings of three patients with FOP and the classical R206H mutation who underwent elective
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post-mortem evaluation in order to advance our knowledge of this rare disease, and describe a method for
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harvesting live fibroblasts from cadaveric skin that can be used for future studies.
2. Clinical Case Histories
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2.1 Patient #1
Patient 1 was a 31-year-old woman who was clinically diagnosed with FOP at age 16. Her past medical
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history was significant for hypertension, type 2 diabetes mellitus, obesity, migraine headaches, chronic lower extremity edema, and hypothyroidism. She developed complications of FOP including severe joint
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fusion and immobility requiring the use of a wheelchair by age 28. She also suffered from chronic pain
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(necessitating opioid use), peripheral postural edema, and restrictive lung disease requiring supplemental oxygen. Several months prior to her death, she transitioned to hospice care. Twenty-four hours prior to
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her death, she developed abdominal pain, decreased oral intake, and periods of apnea. She died from
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2.2 Patient #2
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cardiorespiratory failure. A complete autopsy was performed at the request of the family.
Patient 2 was a 53-year-old woman who was clinically diagnosed with FOP at eight years of age. Her disease was relatively mild and she ambulated with the use of a cane for much of her life. Her mobility gradually declined, and she ultimately required the use of a wheelchair due to significant joint immobility. During the months prior to her death, she was mostly confined to her bed. Her medical history was significant for mitral valve prolapse, syncopal episodes of unclear etiology, hypertension, possible atrial fibrillation, chronic headaches, and deep venous thrombosis requiring anticoagulation with warfarin. Based on clinical neurologic evaluation, her headaches were diagnosed as migraines. An MRI at that time
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ACCEPTED MANUSCRIPT revealed diffuse cerebral volume loss with bi-frontal predominance greater than expected for her age and cervical spinal fusion, but no other CNS abnormalities. Her surgical history was significant for a total abdominal hysterectomy and bilateral salpingooophorectomy at age 38 for uterine leimyomata and adenomyosis. Her post-surgical course was
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complicated by symptoms of bowel obstruction secondary to adhesions. At age 53, she was admitted to a
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local hospital for abdominal pain and concern for bowel obstruction. A CT-scan revealed an ileus but no
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anatomical obstruction. She subsequently developed a hospital-acquired left lower lobe pneumonia. An arterial blood gas study at that time showed hypoxia and hypercarbia with a normal pH, suggesting
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chronic hypercarbia. Her condition declined, and she suffered a seizure and cardiac arrest likely secondary to hypoxemic respiratory failure while being transported to a tertiary care hospital.
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Resuscitative attempts were unsuccessful due to the inability to secure an airway given her extensive
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heterotopic ossification. An autopsy was performed at the request of the family and clinicians.
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2.3 Patient #3:
Patient 3 was a 27-year-old woman with malformation of the great toes who was clinically diagnosed
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with FOP during the first decade of life after developing heterotopic ossification in her shoulder. We have
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limited medical history, but she developed kyphoscoliosis and ultimately underwent spinal rod placement during childhood. She sustained several fractures throughout her lifetime. Although the precise history
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surrounding her death is vague, she sustained a traumatic fall and developed intracranial hemorrhage. A limited autopsy was performed since the patient was an organ donor.
3. Autopsy Results 3.1 Patient #1: The official cause of death in this 31-year-old woman was thoracic insufficiency syndrome secondary to FOP. She had the classical congenital bilateral hallux valgus deformity (Fig. 1A). At autopsy, she had prominent heterotopic ossification and ankylosis of the shoulders, elbows, hips, knees, and ankles. Her
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ACCEPTED MANUSCRIPT cervical spine was fixed in a flexed position and she had prominent kyphoscoliosis. Full body postmortem radiographs showed complete fusion of the cervical spine, variable fusion of the thoracolumbar and sacral spine, bilateral fusion of the humeri to the chest wall, and multifocal intramuscular heterotopic ossification most prominent in the bilateral femoral adductor muscles, distal quadriceps and intercostal
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muscles (Fig. 1 B-E). Histologic evaluation revealed some ossified fragments within the fibro-adipose
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soft tissue adjacent to the muscles but there was no dystrophic calcification within the muscle tissue itself.
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Her vertebrae were diffusely osteopenic with narrowed intervertebral disks and extensive ossification of the anterior and posterior longitudinal ligaments of the spine (Fig. 2 B,C).
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The thoracic cavity was severely deformed, and the diaphragm and abdominal organs were displaced superiorly, leading to severe restriction of the chest cavity. The lungs weighed 450g (right) and
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420g (left), which was normal for her age (Fig. 3A)[12]. Lung dimensions are not classically measured during post-mortem examinations, but they appeared small in dimension due to the severe thoracic cavity
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deformity and superior displacement of the organs[13]. Microscopic examination of the lungs showed
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copious intra-alveolar pigmented macrophages with scattered multinucleated giant cells containing refractile polarizable foreign material, which can be seen in chronic aspiration or heart failure (Fig. 3
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B,C). The bronchial cartilage did not show evidence of ossification. The heart was normal in size (360g)
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with patent, non-stenotic coronary arteries. The right ventricle was mildly dilated and extended into the apex of the heart, with a widened tricuspid valve circumference of 12cm (normal female: 10.2-10.9cm).
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These findings were suggestive of mild right-sided heart failure. Microscopic examination of the heart revealed normal myocardium without fibrosis or inflammation. Gross neuropathological examination revealed focal nodularity in the floor of the 4th ventricle at ponto-medullary junction (Fig. 3 D-F). This lesion involved the caudal pons and extended into the rostral medulla. On histology, it consisted mainly of a midline mass of haphazardly arranged islands of gray matter intermixed with bundles of myelinated axons. It displaced the abducens nuclei laterally. The neurons forming the gray matter within the lesion were of the size and morphology similar to neurons of the pontine gray. A more gliotic nodule was found laterally on the right side at the edge of the lateral
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ACCEPTED MANUSCRIPT recess of the 4th ventricle. Some gliosis was also noted in the inferior olivary nuclei while the remaining brainstem nuclei, the ascending and descending tracts, the cerebellum, and the cerebrum lacked any additional lesions. There was also severe neurogenic atrophy of the deltoid muscle which was thought to be secondary to nerve root compression from her kyphosis. There were no other significant neurological
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findings.
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Additional pertinent autopsy findings included marked dependent edema with associated venous
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stasis dermatitis of the lower extremities, and multinodular chronic lymphedematous lesions on her left leg, bilateral gluteal folds, and vulva. She also had an enlarged dorsal cervical fat pad thought to be
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secondary to chronic steroid use. She had diffuse macro and micro-vascular hepatosteatosis likely secondary to obesity (Fig. 4). Her thyroid gland was fibrotic with chronic lymphocytic inflammatory
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changes, consistent with her history of hypothyroidism.
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3.2 Patient #2:
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The official cause of death in this 53-year-old woman was acute respiratory failure secondary to thoracic insufficiency syndrome. Preceding her death, she developed an acute ileus that led to abdominal
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distension and diaphragmatic elevation that further impaired her lung function. At autopsy, the patient had
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marked rigidity of her cervical spine and bilateral extremities. Her arms were held in rigid flexion and her legs in rigid extension. She had pronounced kyphoscoliosis. She had palpable heterotopic bone formation
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noted most prominently in the right biceps muscle, including heterotopic bone that connected the humerus and proximal biceps muscle to the pectoralis muscle. Additionally, she had grossly palpable ossification of the anterior and posterior spinal ligaments and paraspinal muscles which was confirmed by histology (Fig. 2 A). Microscopic evaluation of the vertebrae showed diffuse mild to moderate osteoporosis of the trabecular bone and osseous metaplasia of the intervertebral disc space and spinal longitudinal ligaments. The congenital bilateral hallux valgus malformation was also noted (not shown) The thoracic cavity was notably small. The diaphragm extended to the level of the 4th rib bilaterally and the spleen was displaced superiorly. Her lung weights measured 510g (right) and 325g
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ACCEPTED MANUSCRIPT (left) which was normal for her age; however, the lung volumes were notably reduced. There was evidence of mild-to-moderate pulmonary focal venous intimal fibrosis which was suggestive, but not diagnostic, of pulmonary veno-occlusive disease. There was no evidence of medial or intimal hyperplasia of the pulmonary artery or arterioles to suggest underlying pulmonary hypertension. Her cardiac
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examination revealed a normal-sized heart (301g, normal = 274 +/- 30g). The coronary arteries were
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patent without atherosclerosis. The tricuspid valve leaflets showed moderate age-related thickening. The
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right ventricle was moderately dilated with an average thickness of 0.4cm. The right ventricular outflow tract and pulmonic valves were normal. The left atrium was moderately dilated and the mitral valve
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leaflets showed moderate age-related myxoid degeneration. The left ventricular chamber was normal in size. On microscopic examination, there was bilateral interstitial fibrosis and cardiomyocyte atrophy
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suggesting an underlying cardiomyopathy (Fig. 5).
The gastrointestinal examination showed a markedly dilated cecum and ascending colon without
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any stenosis, intussusception, or other anatomic lesion. Her obstructive symptoms were felt to be
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secondary to a functional ileus or colonic pseudo-obstruction. The liver was normal in size with mild centrilobular steatosis. She had a small 0.4 cm renal stone and microscopic evidence of mild ischemic
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nephropathy due to hyaline arteriolosclerosis from systemic hypertension. The remainder of the
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abdominal organs were normal.
The neuropathological examination revealed scattered hyaline arteriosclerosis of the cerebral and
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cerebellar white matter consistent with her known history of systemic hypertension. No additional neurological abnormalities were identified. She had a micro-papillary carcinoma superimposed upon chronic lymphocytic changes of the thyroid gland. The remainder of the autopsy was unremarkable. Genetic testing on the skin fibroblasts showed the classical ACVR1 (R206H) mutation.
3.3 Patient #3: The official cause of death in this 27-year-old woman was from complications of a traumatic injury. At the time of autopsy, there was significant rigidity of the neck muscles and immobility of the cervical spine.
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ACCEPTED MANUSCRIPT The upper extremities were fixed in flexion at the shoulder, elbow, and wrist joints. There was prominent kyphoscoliosis of the spine and gross deformity of the chest wall. The tissues along the left posterolateral spine and bilateral thighs had palpable ossifications, and there were nodular, hardened areas involving the major joints of the upper extremities and left hip. The major muscle groups in the body had palpable,
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irregular, ossified masses within the muscle and along the tendon sheaths, which was confirmed by post-
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mortem radiographs. There was no reported microscopic evaluation of the muscle tissue.
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There was gross evidence of trauma, including a laceration to the posterior right parietal region of the skull, a fractured right humerus with an overlying wound, and a two centimeter contusion of the
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posterolateral right hip. The right temporal scalp and temporalis muscle had prominent hemorrhage and there was a fracture of the underlying temporal bone that extended into the lower parietal bone with a
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surrounding four centimeter hematoma. There was a small subdural hemorrhage noted bilaterally as well as diffuse, moderate subarachnoid hemorrhage throughout all surfaces of the brain. Microscopic sections
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of the brain demonstrated fresh cerebral contusions and degenerative changes of the cerebellum.
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The pleural and peritoneal spaces contained serosanguinous fluid. The heart weighed 175g with bloody fluid in the pericardial cavity. The coronary arteries were patent and free of atherosclerosis. The
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valves and heart chamber sizes were normal in dimension and the myocardium appeared healthy. The
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chest wall was markedly deformed. The right lung weighed 375 grams and the left lung weighed 455 grams. Both lungs had a small amount of serosanguinous fluid present and mild atelectasis. There were
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patchy areas of mild congestion and slight focal edema without any distinct areas of consolidation. Microscopic sections of the lungs showed focal acute bronchopneumonia. The thyroid gland demonstrated follicular thyroiditis. There were no significant abnormalities in the nervous system, GI, or endocrine systems, although examination was limited due to post-mortem organ donation.
4. Post-Mortem Isolation of Fibroblasts
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ACCEPTED MANUSCRIPT After obtaining consent from the next of kin, human skin was isolated from Patient 2 at the time of autopsy - five days after the patient expired. The cadaver had been refrigerated in the morgue during this time. A 1 cm diameter biopsy was obtained from the skin and dermal fat of the inner thigh. The sample was transported in 100% FBS (Hyclone#SH30070.03) with 2.5mg/ml Penicillin-Streptomycin (Gibco
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#10378016) and 6.25ug/ml Fungizone (Gibco# 15-290-018). The skin samples were washed five times
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with phosphate buffered saline (PBS) containing 2.5mg/ml penicillin-streptomycin and then cut into 1mm
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x 1mm pieces after removing the subcutaneous and dermal fat. Each skin piece was placed in a well of a 6 well plate (BD Falcon# 353046) and covered with a sterile coverslip (TED PELLA, INC# 26022) by
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applying gentle pressure to adhere the skin piece to the plastic surface, as previously described[7]. Primary fibroblast media containing MEM Alpha (Gibco#125710630), 20% FBS, Glutamax, sodium
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pyruvate, 95mM β-mercaptoethanol (Thermo Scientific# 35602), 2.5mg/ml penicillin-streptomycin and 2.5ug/ml Fungizone was passed through a 0.22um filter (Millipore# SCGP-005-25) and added to the
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samples. The pieces of skin were incubated for 5 days at 37°C without changing the media or any
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disruption. After day 5, the media was changed every 2 days with primary fibroblast media containing 0.5mg/ml penicillin-streptomycin until fibroblast outgrowths were obtained. Fibroblasts were successfully
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5. Discussion
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described[7] (Fig. 6).
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isolated approximately 3 weeks after harvesting and genotyping was performed as previously
Early pathological descriptions of FOP have been compiled by Rosenstirn [14], but complete autopsy results in the modern era are lacking. The autopsy results from these three patients clearly showed the highly invasive and aggressive heterotopic ossification associated with FOP. In addition, the results highlight several important comorbid conditions that are associated with progressive FOP. The official cause of death in two of the three patients was cardiorespiratory failure secondary to thoracic insufficiency syndrome. While the cause of death in Patient 3 was from trauma-related injuries, the patient also had evidence of thoracic insufficiency syndrome at autopsy.
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ACCEPTED MANUSCRIPT Thoracic insufficiency is a well-described complication of FOP, and a leading cause of death in this population[10]. In one study evaluating mortality and cardiorespiratory fitness in FOP patients, 54% of patients who died had complications from thoracic insufficiency syndrome. The median age at time of death was 40 years; the median estimated lifespan was 56 years[9]. FOP patients develop thoracic
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insufficiency as a result of extensive heterotopic ossification of the intercostal and paraspinal muscles,
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leading to progressive deformity of the spine and chest wall. The combination of these deformities leads
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to a severely restricted chest cavity and chronic hypoventilation. It is not clear if there are any direct effects of dysregulated ACVR1 signaling in the lung or cardiac parenchyma that may contribute to
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cardiorespiratory dysfunction (5). In severe cases of thoracic insufficiency syndrome, patients may develop pulmonary hypertension and cor pulmonale. One study evaluated 25 patients with FOP aged 5-55
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to determine the extent to which cardiac function was affected in these individuals. In all 25 patients, chest expansion was limited and lung volumes were severely reduced with relatively normal flow rates,
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consistent with restrictive disease [10]. In addition, forty percent of these patients had
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electrocardiographic (EKG) evidence of right-sided cardiac dysfunction despite normal echocardiograms, suggesting that thoracic insufficiency syndrome is associated with a higher incidence of right-sided
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cardiac dysfunction, and that this may be detected on EKG during the early stages of the disease.
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In both patients who expired from thoracic insufficiency syndrome, there was autopsy evidence supporting right-sided cardiac dysfunction. In Patient 1, the cardiac dysfunction was limited to dilatation
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of the right ventricle without any associated cardiomyopathy. Interestingly, she had a normal cardiac evaluation by trans-thoracic echocardiography several months prior to her death. In Patient 2, the cardiac dysfunction was more apparent and included right heart dilatation and scattered, biventricular cardiomyocyte dysfunction, consistent with a formal diagnosis of cardiomyopathy. In both cases, there was no evidence of pulmonary arterial or arteriolar hypertension. There was mild to moderate pulmonary venous intimal fibrosis which could be related to increased left sided heart pressures in Patient 2, and in Patient 1 the microscopic examination of the lungs showed numerous intra-alveolar macrophages which may be suggestive of pulmonary congestion.
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ACCEPTED MANUSCRIPT Our autopsy findings included the discovery of a post-mortem brainstem malformation in Patient 1. The patient did not have any symptoms of brainstem dysfunction prior to autopsy. The literature suggests that patients with FOP can have neurological symptoms including allodynia, neuropathic pain, headaches, and myoclonus, although the pathophysiology behind these symptoms is unclear[15].
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Heterozygous activating somatic mutations of ACVR1, identical to the heterozygous activating germline
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mutations of ACVR1 in FOP, can also be found in tumoral biopsy tissue of pediatric patients with diffuse
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intrinsic pontine glioma (DIPG); however, no patients with FOP have been diagnosed with DIPG [16, 17]. Interestingly, there have been several recent case reports detailing new CNS findings in FOP patients.
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Bertamino et al report a case of a 5-year-old child with FOP who was diagnosed with bilateral dentate lesions and dorsal pontine abnormalities associated with an abnormal soft tissue mass that blocked the 4th
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ventricle and surrounded the ventral pons, causing secondary obstructive hydrocephalus[18]. Kan et al reported on two adults with FOP and neurological symptoms who were found to have T2 hyperintense
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demyelinating white matter lesions resembling multiple sclerosis[19]. Additionally, two infants with FOP
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but without any outward neurological symptoms were noted to have bilateral symmetrical swelling and T2 hyperintensity of the dentate nuclei with dorsal pontine involvement[15]. One patient with an atypical
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FOP mutation (c.982G>C; pG328R) was noted to have cerebral cavernous malformations diagnosed at
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age 32 in the setting of headaches[3]. Another patient with an atypical FOP mutation (c.982G>T, p.G328W) was noted to have anatomic abnormalities of the cerebellum on CT-head in the setting of mild
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cognitive impairment[3]. The ponto-medullary mass noted in our patient was small and did not appear to cause any significant clinical manifestations; however, the identification of CNS involvement on autopsy raises the question of whether there are more widespread CNS effects in patients with FOP that have yet to be identified. In our 3 patients, there was no other evidence of central or peripheral nervous system abnormalities with the exception of nerve compression caused by heterotopic bone. The musculoskeletal findings in all three patients were consistent with the known disease burden in FOP, including widespread heterotopic ossification, joint ankylosis, osteoporosis of the native skeleton, and congenital great toe malformations. These findings also confirm that skeletal muscles of the
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ACCEPTED MANUSCRIPT diaphragm, tongue, and extra-ocular muscles are spared from ossification in FOP. Additionally, there was evidence of vertebral osteopenia and/or osteoporosis in all three cases. Interestingly, autopsy evaluation identified prominent ossification of the anterior and posterior spinal ligaments. This may be an important factor contributing to the neck and spine immobilization seen in these patients. While much of the spinal
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immobility in FOP patients is felt to be from a combination of congenital orthotopic fusion of the facet
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joints of the cervical spine and heterotopic ossification of the ligamentum nuchae and paraspinal muscles,
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it is possible that the ossification of the anterior and posterior spinal ligaments may be an important contributing factor. To our knowledge, this has not been reported previously.
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The remaining autopsy findings in our three patients are thought to be independent from the diagnosis of FOP. One patient had evidence of significant hepatosteatosis, although this was likely a
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consequence of her obesity. All three patients had evidence of peripheral edema with skin changes; however, it is not clear whether this was related to impaired lymphatic drainage in FOP, chronic steroid
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use, right-sided cardiac dysfunction, or complications from their acute illnesses leading to their death.
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Additionally, all three patients had fibrosis and lymphocytic infiltration of the thyroid gland, and one patient had microscopic intra-thyroidal papillary thyroid cancer. While the significance of this endocrine
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abnormality is not clear, there have been studies showing that papillary thyroid cancers may contain
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intralesional heterotopic ossification that is mediated by the BMP/Alk1 signaling pathway[20, 21]. It is unclear whether patients with FOP may be predisposed to papillary thyroid cancer.
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There are some findings that are notably absent. FOP patient surveys suggest an increased incidence of gastrointestinal complaints, and BMP signaling is a critical regulator of gut development[22]. However, no structural gastrointestinal findings were evident on autopsy, including in Patient 2 who had originally presented with symptoms of ileus. Furthermore, atherosclerosis was notably absent in our FOP autopsies. While this may be age related, the absence of atherosclerosis may also suggest that the increased propensity for abnormal bone formation occurs in a tissue-specific manner in FOP. Finally, one of the major challenges in studying FOP and other rare diseases is the accessibility to live cells. While non-invasive techniques have been developed to collect primary cells from urine and
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ACCEPTED MANUSCRIPT blood, these techniques are not useful post-mortem. Our ability to isolate live fibroblasts from an FOP cadaver five days after death extends the window for collecting critical specimens for research and disease modeling, and suggests that consent forms for tissue procurement should include a detailed ethical
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discussion about the implications of this ability.
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6. Conclusions
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This report of autopsy results from three FOP patients shows that the heterotopic ossification is a significant and progressive feature of the disease, and that thoracic insufficiency and right heart
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dysfunction are critical complications with significant medical impact.
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ACCEPTED MANUSCRIPT REFERENCES
1.
Pignolo, R.J., et al., The Natural History of Flare-Ups in Fibrodysplasia Ossificans
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Progressiva (FOP): A Comprehensive Global Assessment. J Bone Miner Res, 2016.
Rocke, D.M., et al., Age- and joint-specific risk of initial heterotopic ossification in
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31(3): p. 650-6.
patients who have fibrodysplasia ossificans progressiva. Clin Orthop Relat Res,
Kaplan, F.S., et al., Classic and atypical fibrodysplasia ossificans progressiva (FOP)
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phenotypes are caused by mutations in the bone morphogenetic protein (BMP) type I
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receptor ACVR1. Hum Mutat, 2009. 30(3): p. 379-90.
clinical and genetic aspects. Orphanet J Rare Dis, 2011. 6: p. 80. Shore, E.M., et al., A recurrent mutation in the BMP type I receptor ACVR1 causes
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inherited and sporadic fibrodysplasia ossificans progressiva. Nat Genet, 2006. 38(5):
progressiva by imparting responsiveness to activin A. Sci Transl Med, 2015. 7(303): p. 303ra137. 7.
Matsumoto, Y., et al., Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show increased mineralization and cartilage formation. Orphanet J Rare Dis, 2013. 8: p. 190.
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Barruet, E., et al., The ACVR1 R206H mutation found in fibrodysplasia ossificans progressiva increases human induced pluripotent stem cell-derived endothelial cell formation and collagen production through BMP-mediated SMAD1/5/8 signaling. Stem Cell Res Ther, 2016. 7(1): p. 115. Kaplan, F.S., et al., Early mortality and cardiorespiratory failure in patients with
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Kussmaul, W.G., et al., Pulmonary and cardiac function in advanced fibrodysplasia
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ossificans progressiva. Clin Orthop Relat Res, 1998(346): p. 104-9. Mayer, O., et al., Thoracic Insufficiency Syndrome. Curr Probl Pediatr Adolesc Health
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Molina, D.K. and V.J. DiMaio, Normal Organ Weights in Women: Part II-The Brain,
Bellemare, J.F., et al., Thoracic dimensions at maximum lung inflation in normal
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Lungs, Liver, Spleen, and Kidneys. Am J Forensic Med Pathol, 2015. 36(3): p. 182-7.
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Rosenstirn, J., A Contribution to the Study of Myositis Ossificans Progressiva. Ann Surg,
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1918. 68(6): p. 591-637. Kitterman, J.A., et al., Neurological symptoms in individuals with fibrodysplasia ossificans progressiva. J Neurol, 2012. 259(12): p. 2636-43. 16.
Taylor, K.R., et al., Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nat Genet, 2014. 46(5): p. 457-61.
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Fontebasso, A.M., et al., Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nat Genet, 2014. 46(5): p. 462-6.
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Bertamino, M., et al., New insights into central nervous system involvement in FOP: Case report and review of the literature. Am J Med Genet A, 2015. 167A(11): p. 281721.
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Kan, L., et al., CNS demyelination in fibrodysplasia ossificans progressiva. J Neurol,
Takeda, M., et al., Papillary thyroid carcinoma with heterotopic ossification is a special
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subtype with extensive progression. Am J Clin Pathol, 2013. 139(5): p. 587-98.
2013. 209(1): p. 14-8.
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Todisco, A., Regulation of Gastric Metaplasia, Dysplasia, and Neoplasia by Bone Morphogenetic Protein Signaling. Cell Mol Gastroenterol Hepatol, 2017. 3(3): p. 339-
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ACCEPTED MANUSCRIPT Figure Captions:
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Fig 1. Skeletal Findings in FOP (Patient 1). Classic hallux valgus malformation of the left foot (A). Views of the cervical spine show complete fusion of the anterior vertebrae and facet joints with marked kyphotic posture of the neck relative to the chest. There are no obvious muscular ossifications in the neck (B, C). The humerus demonstrates extensive muscle ossification with multifocal ossific bridging between the humerus and chest wall. There is deformity and degeneration of both glenohumeral joints. The left humerus demonstrates a healed fracture with one full bone width lateral displacement. There is extensive callus formation around the fracture site and extensive intramuscular ossification proximal and distal to the healed fracture (C,D). The left knee and proximal portion of the left leg demonstrate extensive ossification extending from the patella to the distal femur (E).
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Fig. 2. Anterior and Posterior Ligament Ossification (Patients 1 and 2). Ossification of the anterior and posterior longitudinal ligaments with extension into the intervertebral disc material (fibrous cartilage). ANT= anterior, POST= posterior. Single arrow indicates ossification of posterior ligament, double arrows indicate ossification of posterior ligament- Patient 2(A). Microscopic sections of vertebrae (V), intervertebral disks (IVD), and longitudinal ligaments (arrows) revealed vertebral osteoporosis and extensive ossification of the longitudinal ligaments, evident adjacent to the intervertebral disks. (B) 2x magnification and (C) 4x magnification- Patient 1(B).
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Fig 3. Pulmonary and Brainstem Findings (Patient 1). The left lung weighs 420g which is small for the patient’s size and age. The right lung is normal in size (A). Microscopic sections of the lungs revealed copious intra-alveolar macrophages, many of which were pigmented. Such cells can be seen in cases of heart failure, in which blood backs up into the pulmonary veins causing pulmonary congestion, pulmonary capillary dilation and leakage, and increased hemosiderin-laden macrophages. Scattered multinucleated giant cells containing refractile polarizable foreign material were also noted related to previous aspiration events (B, C). Transverse section of caudal pons. Arrows outline the inferior edge of a somewhat exophytic mass protruding into the 4th ventricle (D). At 40x original magnification the dysplastic gray matter consists of larger neurons that are morphologically similar to neurons in the pontine base (E). LFB/PAS stained transverse section at pontomedullary junction, original magnification 2x. Arrows outline an abnormal aggregate of gray matter with intermixed bundles of myelinated axons. The abducens nuclei are displaced laterally (*).
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Fig. 4. Hepatosteatosis (Patient 1). 20x magnification of the liver parenchyma showing diffuse hepatosteatosis.
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Fig. 5. Cardiomyopathy (Patient 2). Trichrome stain at 200x magnification demonstrates patchy interstitial fibrous (blue) and degenerating cardiac myocytes (atrophy/small myocytes) consistent with cardiomyopathy.
Fig. 6. Fibroblasts derived from FOP cadaver (A, 10x magnification) with confirmatory sequencing of the p.R206H mutation (B, indicated by arrow).
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ACCEPTED MANUSCRIPT Highlights: Thoracic insufficiency syndrome was identified at autopsy in three patients with FOP
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Autopsy findings included spinal ligament ossification and R ventricular dilation
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Brainstem malformation was identified post-mortem in one patient with FOP
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Protocol for post-mortem isolation of fibroblasts in FOP
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three
patients
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Kelly L. Wentworth, Katherine Bigay, Tea V. Chan, Jennifer P. Ho, Blanca M. Morales, Joseph Connor, Erin Brooks, M. Shahriar Salamat, Henry Charles Sanchez, Geoffrey Wool, Robert J. Pignolo, Frederick S. Kaplan, Edward C. Hsiao PII: DOI: Reference:
S8756-3282(17)30375-7 doi:10.1016/j.bone.2017.10.009 BON 11449
To appear in:
Bone
Received date: Revised date: Accepted date:
30 June 2017 4 October 2017 6 October 2017
Please cite this article as: Kelly L. Wentworth, Katherine Bigay, Tea V. Chan, Jennifer P. Ho, Blanca M. Morales, Joseph Connor, Erin Brooks, M. Shahriar Salamat, Henry Charles Sanchez, Geoffrey Wool, Robert J. Pignolo, Frederick S. Kaplan, Edward C. Hsiao , Clinical-pathological correlations in three patients with fibrodysplasia ossificans progressiva. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bon(2017), doi:10.1016/j.bone.2017.10.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Clinical-Pathological Correlations in Three Patients with Fibrodysplasia Ossificans Progressiva Kelly L. Wentworth1†, Katherine Bigay1*, Tea V. Chan1*, Jennifer P. Ho1*, Blanca M. Morales1, Joseph Connor3, Erin Brooks3, M. Shahriar Salamat3, Henry Charles Sanchez2, Geoffrey Wool6, Robert J. Pignolo4, Frederick S. Kaplan5, and Edward C. Hsiao1†
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Division of Endocrinology, Diabetes, and Metabolism, and the Institute for Human Genetics, Department
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of Medicine, University of California, San Francisco, CA Division of Pathology, University of California, San Francisco, CA
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Department of Medicine, Mayo Clinic, Rochester, MN
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Division of Pathology, University of Wisconsin, Madison, WI
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Departments of Orthopaedic Surgery and Medicine, and the Center for Research in FOP and Related
Disorders at the Perelman School of Medicine of the University of Pennsylvania, Philadelphia, PA 6
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Department of Pathology, University of Chicago, Chicago, IL
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*The 2nd, 3rd and 4th authors all contributed equally to this manuscript
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Abbreviated Title: Autopsy Findings in Patients with FOP Keywords: Fibrodysplasia ossificans progressiva; heterotopic ossification; thoracic insufficiency
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syndrome; restrictive lung disease; cardiomyopathy; autopsy Number of Figures and Tables: 6 figures
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Corresponding authors and person to whom reprint requests should be addressed:
Kelly Wentworth, MD and Edward C. Hsiao, M.D., Ph.D. Department of Endocrinology, Diabetes, and Metabolism Institute for Human Genetics University of California, San Francisco
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ACCEPTED MANUSCRIPT 513 Parnassus Ave, HSE 901 San Francisco, CA 94143-0794 Phone: 415-476-9732 Fax: 415-353-2337
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e-mails:
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[email protected]
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[email protected]
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ACCEPTED MANUSCRIPT Funding and Disclosures: We gratefully acknowledge the funding support for this project. KW received support from a UCSF/Eli and Edythe Broad Fellowship and the Wilsey Family Fellowship to the UCSF Endocrinology, Diabetes, and Metabolism Training Program. EH received research grant support from the Doris Duke Charitable Fund (2014099), the March of Dimes (1-FY14-211), and the Radiant Hope
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Foundation. RJP receives support from the Robert and Arlene Kogod Professorship in Geriatric Medicine
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at the Mayo Clinic, and the Radiant Hope Foundation. KW, KB, JH, TC, FSK, RJP and EH also receive
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research support from Clementia Pharmaceuticals for FOP clinical research studies, which pose no conflicts with this work. The authors have no additional disclosures. We would like to thank the
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International FOP Association for their support of the FOP patient community.
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Authors’ roles: KW, KB, JH, TC, MM, and EH wrote the manuscript. HS, JC, EB, and MSS performed the autopsies, assisted with interpretation of the findings, and provided the post-mortem images. EH, RJP,
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and FSK cared for the patients and provided detailed medical histories. All authors assisted with editing,
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approved the final version of the manuscript, and take responsibility for the integrity of the analysis and
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presentation.
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ACCEPTED MANUSCRIPT ABSTRACT Objective: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder in which heterotopic bone forms in the soft tissues. This often occurs in response to injury or inflammation, leading to joint immobilization and significant disability. There are currently no definitive treatment options for
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this devastating disease. Although the most dramatic phenotype in FOP is the episodic and progressive
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heterotopic ossification, patients report a number of symptoms that affect other organ systems. Post-
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mortem examination of FOP patients may contribute to our understanding of the underlying pathophysiology and complications of this disease. Here, we present the autopsy findings from three
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patients with FOP.
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Findings: Autopsy findings in two of the three patients confirmed that the cause of death was cardiorespiratory failure in the setting of severe thoracic insufficiency from heterotopic ossification. Both
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of these patients also had evidence of right ventricular dilatation likely secondary to thoracic insufficiency.
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The third patient died from complications of a traumatic head injury after a fall but also had post-mortem evidence of thoracic insufficiency syndrome. All three patients had extensive, widespread heterotopic
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ossification and joint deformities consistent with FOP. There was extensive ossification of the spinal
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ligament in these patients, which may contribute to cervical spine rigidity. One patient was diagnosed post-mortem with a brainstem malformation. No additional significant abnormalities were noted in the
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other organ systems. Finally, we also demonstrate that cadaveric skin fibroblasts can be isolated for use as a potential source for future in vitro cell culture studies.
Conclusions: This autopsy case series provides valuable information about the underlying complications of FOP and contributes significantly to our knowledge of this rare yet debilitating disorder. Thoracic insufficiency syndrome, right heart dysfunction, widespread heterotopic ossification, spinal ligament ossification, and CNS malformations were clearly evident; however, most other non-bone tissues appeared to be spared from gross malformations. Finally, the ability to isolate live cells from cadaveric
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ACCEPTED MANUSCRIPT skin is an important technique that will facilitate future studies, particularly as induced pluripotent stem cells and other cell-based technologies evolve. This case series highlights the importance of post-mortem examinations and their contribution to our current knowledge of disease pathophysiology and
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comorbidities.
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ACCEPTED MANUSCRIPT 1. Introduction Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare disorder characterized by massive heterotopic ossification of the connective tissues, leading to progressive joint immobilization and severe disability. Approximately one in two million children worldwide are diagnosed with FOP, typically within the first
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decade of life[1]. FOP is a chronic, progressive disease in which patients develop episodic “flare-ups” of
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painful, localized soft tissue swellings that rapidly progress to heterotopic bone via an endochondral bone
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formation pathway. Flare-ups are sporadic and may occur spontaneously or in response to local injury and inflammation in both the axial and appendicular skeleton. As a consequence of the widespread
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heterotopic ossification and subsequent joint dysfunction, most patients require the use of a wheelchair by the third decade of life[2]. Patients with classical FOP also have congenital malformation of the great toes
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and thumbs, osteochondromas, and variable fusion of the cervical spine facet joints[3, 4]. FOP is a genetic disorder most commonly caused by an autosomal dominant missense mutation
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in the ACVR1/ALK2 gene (c.617 G>A; p.R206H), which encodes activin receptor type Ia/activin-like
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kinase 2 receptor [5]. This point mutation alters the GS activation domain of the kinase receptor, leading to abnormal signaling through the ACVR1 receptor and over-activation of the downstream SMAD 1/5/8
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and p38 signaling pathways. The over-activation of these signaling pathways triggered by BMPs or
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Activin A is responsible for the widespread heterotopic ossification and congenital abnormalities seen in these patients[6-8]. Currently, there are no effective treatments for FOP, and the median age at time of
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death is 42 years with the median estimated lifespan is 56 years[9]. One known complication of FOP is the development of restrictive lung disease as a consequence of the significant heterotopic ossification involving the chest wall, spine, and rib cage[9, 10]. Thoracic insufficiency syndrome, defined as the inability of the thorax to support normal respiration or lung growth, develops as a result of congenital costovertebral malformations and/or extensive heterotopic ossification often accompanied by severe scoliosis[11]. Thoracic insufficiency syndrome is a well-established complication of FOP and remains the leading cause of death in these patients[9, 10].
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ACCEPTED MANUSCRIPT We have little knowledge about whether other organ systems may be affected in FOP. In addition, the advent of new cellular technologies such as induced pluripotent stem cells promises to help reveal many of the physiologic and pathogenic events that contribute to disease progression. Here, we report the autopsy findings of three patients with FOP and the classical R206H mutation who underwent elective
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post-mortem evaluation in order to advance our knowledge of this rare disease, and describe a method for
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harvesting live fibroblasts from cadaveric skin that can be used for future studies.
2. Clinical Case Histories
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2.1 Patient #1
Patient 1 was a 31-year-old woman who was clinically diagnosed with FOP at age 16. Her past medical
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history was significant for hypertension, type 2 diabetes mellitus, obesity, migraine headaches, chronic lower extremity edema, and hypothyroidism. She developed complications of FOP including severe joint
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fusion and immobility requiring the use of a wheelchair by age 28. She also suffered from chronic pain
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(necessitating opioid use), peripheral postural edema, and restrictive lung disease requiring supplemental oxygen. Several months prior to her death, she transitioned to hospice care. Twenty-four hours prior to
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her death, she developed abdominal pain, decreased oral intake, and periods of apnea. She died from
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2.2 Patient #2
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cardiorespiratory failure. A complete autopsy was performed at the request of the family.
Patient 2 was a 53-year-old woman who was clinically diagnosed with FOP at eight years of age. Her disease was relatively mild and she ambulated with the use of a cane for much of her life. Her mobility gradually declined, and she ultimately required the use of a wheelchair due to significant joint immobility. During the months prior to her death, she was mostly confined to her bed. Her medical history was significant for mitral valve prolapse, syncopal episodes of unclear etiology, hypertension, possible atrial fibrillation, chronic headaches, and deep venous thrombosis requiring anticoagulation with warfarin. Based on clinical neurologic evaluation, her headaches were diagnosed as migraines. An MRI at that time
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ACCEPTED MANUSCRIPT revealed diffuse cerebral volume loss with bi-frontal predominance greater than expected for her age and cervical spinal fusion, but no other CNS abnormalities. Her surgical history was significant for a total abdominal hysterectomy and bilateral salpingooophorectomy at age 38 for uterine leimyomata and adenomyosis. Her post-surgical course was
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complicated by symptoms of bowel obstruction secondary to adhesions. At age 53, she was admitted to a
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local hospital for abdominal pain and concern for bowel obstruction. A CT-scan revealed an ileus but no
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anatomical obstruction. She subsequently developed a hospital-acquired left lower lobe pneumonia. An arterial blood gas study at that time showed hypoxia and hypercarbia with a normal pH, suggesting
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chronic hypercarbia. Her condition declined, and she suffered a seizure and cardiac arrest likely secondary to hypoxemic respiratory failure while being transported to a tertiary care hospital.
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Resuscitative attempts were unsuccessful due to the inability to secure an airway given her extensive
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heterotopic ossification. An autopsy was performed at the request of the family and clinicians.
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2.3 Patient #3:
Patient 3 was a 27-year-old woman with malformation of the great toes who was clinically diagnosed
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with FOP during the first decade of life after developing heterotopic ossification in her shoulder. We have
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limited medical history, but she developed kyphoscoliosis and ultimately underwent spinal rod placement during childhood. She sustained several fractures throughout her lifetime. Although the precise history
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surrounding her death is vague, she sustained a traumatic fall and developed intracranial hemorrhage. A limited autopsy was performed since the patient was an organ donor.
3. Autopsy Results 3.1 Patient #1: The official cause of death in this 31-year-old woman was thoracic insufficiency syndrome secondary to FOP. She had the classical congenital bilateral hallux valgus deformity (Fig. 1A). At autopsy, she had prominent heterotopic ossification and ankylosis of the shoulders, elbows, hips, knees, and ankles. Her
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ACCEPTED MANUSCRIPT cervical spine was fixed in a flexed position and she had prominent kyphoscoliosis. Full body postmortem radiographs showed complete fusion of the cervical spine, variable fusion of the thoracolumbar and sacral spine, bilateral fusion of the humeri to the chest wall, and multifocal intramuscular heterotopic ossification most prominent in the bilateral femoral adductor muscles, distal quadriceps and intercostal
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muscles (Fig. 1 B-E). Histologic evaluation revealed some ossified fragments within the fibro-adipose
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soft tissue adjacent to the muscles but there was no dystrophic calcification within the muscle tissue itself.
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Her vertebrae were diffusely osteopenic with narrowed intervertebral disks and extensive ossification of the anterior and posterior longitudinal ligaments of the spine (Fig. 2 B,C).
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The thoracic cavity was severely deformed, and the diaphragm and abdominal organs were displaced superiorly, leading to severe restriction of the chest cavity. The lungs weighed 450g (right) and
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420g (left), which was normal for her age (Fig. 3A)[12]. Lung dimensions are not classically measured during post-mortem examinations, but they appeared small in dimension due to the severe thoracic cavity
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deformity and superior displacement of the organs[13]. Microscopic examination of the lungs showed
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copious intra-alveolar pigmented macrophages with scattered multinucleated giant cells containing refractile polarizable foreign material, which can be seen in chronic aspiration or heart failure (Fig. 3
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B,C). The bronchial cartilage did not show evidence of ossification. The heart was normal in size (360g)
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with patent, non-stenotic coronary arteries. The right ventricle was mildly dilated and extended into the apex of the heart, with a widened tricuspid valve circumference of 12cm (normal female: 10.2-10.9cm).
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These findings were suggestive of mild right-sided heart failure. Microscopic examination of the heart revealed normal myocardium without fibrosis or inflammation. Gross neuropathological examination revealed focal nodularity in the floor of the 4th ventricle at ponto-medullary junction (Fig. 3 D-F). This lesion involved the caudal pons and extended into the rostral medulla. On histology, it consisted mainly of a midline mass of haphazardly arranged islands of gray matter intermixed with bundles of myelinated axons. It displaced the abducens nuclei laterally. The neurons forming the gray matter within the lesion were of the size and morphology similar to neurons of the pontine gray. A more gliotic nodule was found laterally on the right side at the edge of the lateral
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ACCEPTED MANUSCRIPT recess of the 4th ventricle. Some gliosis was also noted in the inferior olivary nuclei while the remaining brainstem nuclei, the ascending and descending tracts, the cerebellum, and the cerebrum lacked any additional lesions. There was also severe neurogenic atrophy of the deltoid muscle which was thought to be secondary to nerve root compression from her kyphosis. There were no other significant neurological
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Additional pertinent autopsy findings included marked dependent edema with associated venous
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stasis dermatitis of the lower extremities, and multinodular chronic lymphedematous lesions on her left leg, bilateral gluteal folds, and vulva. She also had an enlarged dorsal cervical fat pad thought to be
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secondary to chronic steroid use. She had diffuse macro and micro-vascular hepatosteatosis likely secondary to obesity (Fig. 4). Her thyroid gland was fibrotic with chronic lymphocytic inflammatory
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changes, consistent with her history of hypothyroidism.
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3.2 Patient #2:
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The official cause of death in this 53-year-old woman was acute respiratory failure secondary to thoracic insufficiency syndrome. Preceding her death, she developed an acute ileus that led to abdominal
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distension and diaphragmatic elevation that further impaired her lung function. At autopsy, the patient had
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marked rigidity of her cervical spine and bilateral extremities. Her arms were held in rigid flexion and her legs in rigid extension. She had pronounced kyphoscoliosis. She had palpable heterotopic bone formation
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noted most prominently in the right biceps muscle, including heterotopic bone that connected the humerus and proximal biceps muscle to the pectoralis muscle. Additionally, she had grossly palpable ossification of the anterior and posterior spinal ligaments and paraspinal muscles which was confirmed by histology (Fig. 2 A). Microscopic evaluation of the vertebrae showed diffuse mild to moderate osteoporosis of the trabecular bone and osseous metaplasia of the intervertebral disc space and spinal longitudinal ligaments. The congenital bilateral hallux valgus malformation was also noted (not shown) The thoracic cavity was notably small. The diaphragm extended to the level of the 4th rib bilaterally and the spleen was displaced superiorly. Her lung weights measured 510g (right) and 325g
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ACCEPTED MANUSCRIPT (left) which was normal for her age; however, the lung volumes were notably reduced. There was evidence of mild-to-moderate pulmonary focal venous intimal fibrosis which was suggestive, but not diagnostic, of pulmonary veno-occlusive disease. There was no evidence of medial or intimal hyperplasia of the pulmonary artery or arterioles to suggest underlying pulmonary hypertension. Her cardiac
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examination revealed a normal-sized heart (301g, normal = 274 +/- 30g). The coronary arteries were
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patent without atherosclerosis. The tricuspid valve leaflets showed moderate age-related thickening. The
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right ventricle was moderately dilated with an average thickness of 0.4cm. The right ventricular outflow tract and pulmonic valves were normal. The left atrium was moderately dilated and the mitral valve
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leaflets showed moderate age-related myxoid degeneration. The left ventricular chamber was normal in size. On microscopic examination, there was bilateral interstitial fibrosis and cardiomyocyte atrophy
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suggesting an underlying cardiomyopathy (Fig. 5).
The gastrointestinal examination showed a markedly dilated cecum and ascending colon without
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any stenosis, intussusception, or other anatomic lesion. Her obstructive symptoms were felt to be
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secondary to a functional ileus or colonic pseudo-obstruction. The liver was normal in size with mild centrilobular steatosis. She had a small 0.4 cm renal stone and microscopic evidence of mild ischemic
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nephropathy due to hyaline arteriolosclerosis from systemic hypertension. The remainder of the
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abdominal organs were normal.
The neuropathological examination revealed scattered hyaline arteriosclerosis of the cerebral and
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cerebellar white matter consistent with her known history of systemic hypertension. No additional neurological abnormalities were identified. She had a micro-papillary carcinoma superimposed upon chronic lymphocytic changes of the thyroid gland. The remainder of the autopsy was unremarkable. Genetic testing on the skin fibroblasts showed the classical ACVR1 (R206H) mutation.
3.3 Patient #3: The official cause of death in this 27-year-old woman was from complications of a traumatic injury. At the time of autopsy, there was significant rigidity of the neck muscles and immobility of the cervical spine.
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ACCEPTED MANUSCRIPT The upper extremities were fixed in flexion at the shoulder, elbow, and wrist joints. There was prominent kyphoscoliosis of the spine and gross deformity of the chest wall. The tissues along the left posterolateral spine and bilateral thighs had palpable ossifications, and there were nodular, hardened areas involving the major joints of the upper extremities and left hip. The major muscle groups in the body had palpable,
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irregular, ossified masses within the muscle and along the tendon sheaths, which was confirmed by post-
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mortem radiographs. There was no reported microscopic evaluation of the muscle tissue.
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There was gross evidence of trauma, including a laceration to the posterior right parietal region of the skull, a fractured right humerus with an overlying wound, and a two centimeter contusion of the
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posterolateral right hip. The right temporal scalp and temporalis muscle had prominent hemorrhage and there was a fracture of the underlying temporal bone that extended into the lower parietal bone with a
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surrounding four centimeter hematoma. There was a small subdural hemorrhage noted bilaterally as well as diffuse, moderate subarachnoid hemorrhage throughout all surfaces of the brain. Microscopic sections
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of the brain demonstrated fresh cerebral contusions and degenerative changes of the cerebellum.
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The pleural and peritoneal spaces contained serosanguinous fluid. The heart weighed 175g with bloody fluid in the pericardial cavity. The coronary arteries were patent and free of atherosclerosis. The
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valves and heart chamber sizes were normal in dimension and the myocardium appeared healthy. The
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chest wall was markedly deformed. The right lung weighed 375 grams and the left lung weighed 455 grams. Both lungs had a small amount of serosanguinous fluid present and mild atelectasis. There were
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patchy areas of mild congestion and slight focal edema without any distinct areas of consolidation. Microscopic sections of the lungs showed focal acute bronchopneumonia. The thyroid gland demonstrated follicular thyroiditis. There were no significant abnormalities in the nervous system, GI, or endocrine systems, although examination was limited due to post-mortem organ donation.
4. Post-Mortem Isolation of Fibroblasts
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ACCEPTED MANUSCRIPT After obtaining consent from the next of kin, human skin was isolated from Patient 2 at the time of autopsy - five days after the patient expired. The cadaver had been refrigerated in the morgue during this time. A 1 cm diameter biopsy was obtained from the skin and dermal fat of the inner thigh. The sample was transported in 100% FBS (Hyclone#SH30070.03) with 2.5mg/ml Penicillin-Streptomycin (Gibco
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#10378016) and 6.25ug/ml Fungizone (Gibco# 15-290-018). The skin samples were washed five times
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with phosphate buffered saline (PBS) containing 2.5mg/ml penicillin-streptomycin and then cut into 1mm
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x 1mm pieces after removing the subcutaneous and dermal fat. Each skin piece was placed in a well of a 6 well plate (BD Falcon# 353046) and covered with a sterile coverslip (TED PELLA, INC# 26022) by
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applying gentle pressure to adhere the skin piece to the plastic surface, as previously described[7]. Primary fibroblast media containing MEM Alpha (Gibco#125710630), 20% FBS, Glutamax, sodium
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pyruvate, 95mM β-mercaptoethanol (Thermo Scientific# 35602), 2.5mg/ml penicillin-streptomycin and 2.5ug/ml Fungizone was passed through a 0.22um filter (Millipore# SCGP-005-25) and added to the
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samples. The pieces of skin were incubated for 5 days at 37°C without changing the media or any
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disruption. After day 5, the media was changed every 2 days with primary fibroblast media containing 0.5mg/ml penicillin-streptomycin until fibroblast outgrowths were obtained. Fibroblasts were successfully
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5. Discussion
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described[7] (Fig. 6).
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isolated approximately 3 weeks after harvesting and genotyping was performed as previously
Early pathological descriptions of FOP have been compiled by Rosenstirn [14], but complete autopsy results in the modern era are lacking. The autopsy results from these three patients clearly showed the highly invasive and aggressive heterotopic ossification associated with FOP. In addition, the results highlight several important comorbid conditions that are associated with progressive FOP. The official cause of death in two of the three patients was cardiorespiratory failure secondary to thoracic insufficiency syndrome. While the cause of death in Patient 3 was from trauma-related injuries, the patient also had evidence of thoracic insufficiency syndrome at autopsy.
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ACCEPTED MANUSCRIPT Thoracic insufficiency is a well-described complication of FOP, and a leading cause of death in this population[10]. In one study evaluating mortality and cardiorespiratory fitness in FOP patients, 54% of patients who died had complications from thoracic insufficiency syndrome. The median age at time of death was 40 years; the median estimated lifespan was 56 years[9]. FOP patients develop thoracic
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insufficiency as a result of extensive heterotopic ossification of the intercostal and paraspinal muscles,
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leading to progressive deformity of the spine and chest wall. The combination of these deformities leads
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to a severely restricted chest cavity and chronic hypoventilation. It is not clear if there are any direct effects of dysregulated ACVR1 signaling in the lung or cardiac parenchyma that may contribute to
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cardiorespiratory dysfunction (5). In severe cases of thoracic insufficiency syndrome, patients may develop pulmonary hypertension and cor pulmonale. One study evaluated 25 patients with FOP aged 5-55
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to determine the extent to which cardiac function was affected in these individuals. In all 25 patients, chest expansion was limited and lung volumes were severely reduced with relatively normal flow rates,
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consistent with restrictive disease [10]. In addition, forty percent of these patients had
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electrocardiographic (EKG) evidence of right-sided cardiac dysfunction despite normal echocardiograms, suggesting that thoracic insufficiency syndrome is associated with a higher incidence of right-sided
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cardiac dysfunction, and that this may be detected on EKG during the early stages of the disease.
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In both patients who expired from thoracic insufficiency syndrome, there was autopsy evidence supporting right-sided cardiac dysfunction. In Patient 1, the cardiac dysfunction was limited to dilatation
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of the right ventricle without any associated cardiomyopathy. Interestingly, she had a normal cardiac evaluation by trans-thoracic echocardiography several months prior to her death. In Patient 2, the cardiac dysfunction was more apparent and included right heart dilatation and scattered, biventricular cardiomyocyte dysfunction, consistent with a formal diagnosis of cardiomyopathy. In both cases, there was no evidence of pulmonary arterial or arteriolar hypertension. There was mild to moderate pulmonary venous intimal fibrosis which could be related to increased left sided heart pressures in Patient 2, and in Patient 1 the microscopic examination of the lungs showed numerous intra-alveolar macrophages which may be suggestive of pulmonary congestion.
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ACCEPTED MANUSCRIPT Our autopsy findings included the discovery of a post-mortem brainstem malformation in Patient 1. The patient did not have any symptoms of brainstem dysfunction prior to autopsy. The literature suggests that patients with FOP can have neurological symptoms including allodynia, neuropathic pain, headaches, and myoclonus, although the pathophysiology behind these symptoms is unclear[15].
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Heterozygous activating somatic mutations of ACVR1, identical to the heterozygous activating germline
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mutations of ACVR1 in FOP, can also be found in tumoral biopsy tissue of pediatric patients with diffuse
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intrinsic pontine glioma (DIPG); however, no patients with FOP have been diagnosed with DIPG [16, 17]. Interestingly, there have been several recent case reports detailing new CNS findings in FOP patients.
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Bertamino et al report a case of a 5-year-old child with FOP who was diagnosed with bilateral dentate lesions and dorsal pontine abnormalities associated with an abnormal soft tissue mass that blocked the 4th
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ventricle and surrounded the ventral pons, causing secondary obstructive hydrocephalus[18]. Kan et al reported on two adults with FOP and neurological symptoms who were found to have T2 hyperintense
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demyelinating white matter lesions resembling multiple sclerosis[19]. Additionally, two infants with FOP
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but without any outward neurological symptoms were noted to have bilateral symmetrical swelling and T2 hyperintensity of the dentate nuclei with dorsal pontine involvement[15]. One patient with an atypical
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FOP mutation (c.982G>C; pG328R) was noted to have cerebral cavernous malformations diagnosed at
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age 32 in the setting of headaches[3]. Another patient with an atypical FOP mutation (c.982G>T, p.G328W) was noted to have anatomic abnormalities of the cerebellum on CT-head in the setting of mild
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cognitive impairment[3]. The ponto-medullary mass noted in our patient was small and did not appear to cause any significant clinical manifestations; however, the identification of CNS involvement on autopsy raises the question of whether there are more widespread CNS effects in patients with FOP that have yet to be identified. In our 3 patients, there was no other evidence of central or peripheral nervous system abnormalities with the exception of nerve compression caused by heterotopic bone. The musculoskeletal findings in all three patients were consistent with the known disease burden in FOP, including widespread heterotopic ossification, joint ankylosis, osteoporosis of the native skeleton, and congenital great toe malformations. These findings also confirm that skeletal muscles of the
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ACCEPTED MANUSCRIPT diaphragm, tongue, and extra-ocular muscles are spared from ossification in FOP. Additionally, there was evidence of vertebral osteopenia and/or osteoporosis in all three cases. Interestingly, autopsy evaluation identified prominent ossification of the anterior and posterior spinal ligaments. This may be an important factor contributing to the neck and spine immobilization seen in these patients. While much of the spinal
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immobility in FOP patients is felt to be from a combination of congenital orthotopic fusion of the facet
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joints of the cervical spine and heterotopic ossification of the ligamentum nuchae and paraspinal muscles,
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it is possible that the ossification of the anterior and posterior spinal ligaments may be an important contributing factor. To our knowledge, this has not been reported previously.
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The remaining autopsy findings in our three patients are thought to be independent from the diagnosis of FOP. One patient had evidence of significant hepatosteatosis, although this was likely a
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consequence of her obesity. All three patients had evidence of peripheral edema with skin changes; however, it is not clear whether this was related to impaired lymphatic drainage in FOP, chronic steroid
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use, right-sided cardiac dysfunction, or complications from their acute illnesses leading to their death.
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Additionally, all three patients had fibrosis and lymphocytic infiltration of the thyroid gland, and one patient had microscopic intra-thyroidal papillary thyroid cancer. While the significance of this endocrine
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abnormality is not clear, there have been studies showing that papillary thyroid cancers may contain
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intralesional heterotopic ossification that is mediated by the BMP/Alk1 signaling pathway[20, 21]. It is unclear whether patients with FOP may be predisposed to papillary thyroid cancer.
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There are some findings that are notably absent. FOP patient surveys suggest an increased incidence of gastrointestinal complaints, and BMP signaling is a critical regulator of gut development[22]. However, no structural gastrointestinal findings were evident on autopsy, including in Patient 2 who had originally presented with symptoms of ileus. Furthermore, atherosclerosis was notably absent in our FOP autopsies. While this may be age related, the absence of atherosclerosis may also suggest that the increased propensity for abnormal bone formation occurs in a tissue-specific manner in FOP. Finally, one of the major challenges in studying FOP and other rare diseases is the accessibility to live cells. While non-invasive techniques have been developed to collect primary cells from urine and
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discussion about the implications of this ability.
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6. Conclusions
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This report of autopsy results from three FOP patients shows that the heterotopic ossification is a significant and progressive feature of the disease, and that thoracic insufficiency and right heart
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dysfunction are critical complications with significant medical impact.
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ACCEPTED MANUSCRIPT REFERENCES
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ACCEPTED MANUSCRIPT Figure Captions:
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Fig 1. Skeletal Findings in FOP (Patient 1). Classic hallux valgus malformation of the left foot (A). Views of the cervical spine show complete fusion of the anterior vertebrae and facet joints with marked kyphotic posture of the neck relative to the chest. There are no obvious muscular ossifications in the neck (B, C). The humerus demonstrates extensive muscle ossification with multifocal ossific bridging between the humerus and chest wall. There is deformity and degeneration of both glenohumeral joints. The left humerus demonstrates a healed fracture with one full bone width lateral displacement. There is extensive callus formation around the fracture site and extensive intramuscular ossification proximal and distal to the healed fracture (C,D). The left knee and proximal portion of the left leg demonstrate extensive ossification extending from the patella to the distal femur (E).
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Fig. 2. Anterior and Posterior Ligament Ossification (Patients 1 and 2). Ossification of the anterior and posterior longitudinal ligaments with extension into the intervertebral disc material (fibrous cartilage). ANT= anterior, POST= posterior. Single arrow indicates ossification of posterior ligament, double arrows indicate ossification of posterior ligament- Patient 2(A). Microscopic sections of vertebrae (V), intervertebral disks (IVD), and longitudinal ligaments (arrows) revealed vertebral osteoporosis and extensive ossification of the longitudinal ligaments, evident adjacent to the intervertebral disks. (B) 2x magnification and (C) 4x magnification- Patient 1(B).
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Fig 3. Pulmonary and Brainstem Findings (Patient 1). The left lung weighs 420g which is small for the patient’s size and age. The right lung is normal in size (A). Microscopic sections of the lungs revealed copious intra-alveolar macrophages, many of which were pigmented. Such cells can be seen in cases of heart failure, in which blood backs up into the pulmonary veins causing pulmonary congestion, pulmonary capillary dilation and leakage, and increased hemosiderin-laden macrophages. Scattered multinucleated giant cells containing refractile polarizable foreign material were also noted related to previous aspiration events (B, C). Transverse section of caudal pons. Arrows outline the inferior edge of a somewhat exophytic mass protruding into the 4th ventricle (D). At 40x original magnification the dysplastic gray matter consists of larger neurons that are morphologically similar to neurons in the pontine base (E). LFB/PAS stained transverse section at pontomedullary junction, original magnification 2x. Arrows outline an abnormal aggregate of gray matter with intermixed bundles of myelinated axons. The abducens nuclei are displaced laterally (*).
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Fig. 4. Hepatosteatosis (Patient 1). 20x magnification of the liver parenchyma showing diffuse hepatosteatosis.
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Fig. 5. Cardiomyopathy (Patient 2). Trichrome stain at 200x magnification demonstrates patchy interstitial fibrous (blue) and degenerating cardiac myocytes (atrophy/small myocytes) consistent with cardiomyopathy.
Fig. 6. Fibroblasts derived from FOP cadaver (A, 10x magnification) with confirmatory sequencing of the p.R206H mutation (B, indicated by arrow).
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ACCEPTED MANUSCRIPT Highlights: Thoracic insufficiency syndrome was identified at autopsy in three patients with FOP
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Autopsy findings included spinal ligament ossification and R ventricular dilation
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Brainstem malformation was identified post-mortem in one patient with FOP
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Protocol for post-mortem isolation of fibroblasts in FOP
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