Myodural bridges have been described in various species as connective tissue structures “bridging” small cranio-cervical muscles to the dura. Myodural bridges are thought to stabilize the dural sac during head and neck movements and promote cerebrospinal fluid motion; however, their role in neurological diseases has not yet been established. We report ultrasonographic visualization, necropsy, histopathologic and ultrastructural findings of myodural bridges in horses with hereditary equine regional dermal asthenia (HERDA), an equine model of Ehlers-Danlos syndromes. Five HERDA and 5 control horses were studied. Post-mortem examination and ultrasonographic studies (3 HERDA and 4 controls) demonstrated that the atlanto-occipital and atlanto-axial myodural bridges are dynamic structures “moving” the dura. En block resection of the myodural bridges (4 HERDA and 5 controls) was accomplished and histopathology showed myofiber degeneration in 3 HERDA horses and 1 control. Ultrastructural examination revealed loosely packed collagen fibrils with abnormal orientation in all HERDA horses compared to mild abnormalities in 2 controls. Our study provides necropsy and ultrasonographic evidence of the dynamic aspect of the myodural bridges as dural sac stabilizers. Myodural bridges may be pathologically altered in connective tissue disease as evidenced by the ultrastructural morphology in the HERDA myodural bridge. They have been extensively studied in human cadavers and were first described by Hack et al. (1995)1. Scali et al. (2015) further pioneered detailed insight into these complicated structures using E12 sheet plastination in human cadavers with in situ visualization of those bridging structures5. The insight into the composition and arrangement of the posterior atlanto-occipital membrane and cerebrospinal dura was a break-through in the anatomy of the craniocervical junction and aided in the understanding of certain neurogenic disorders5. Myodural bridges have since been described in several animal species, including rhesus macaques, European rabbits, dogs, cats, Norway rats, guinea pigs, and the Indoasian finless porpoise6. According to both a recent thesis and a recent conference report, the existence of MDBs has been established in horses by cadaver studies8. The MDBs are theorized to stabilize the dura mater during extension of the head and neck, thus serving as dural tension monitors, preventing infolding of the dura mater and disruption of the flow of cerebrospinal fluid (CSF)12. The MDBs are also thought to function as a pump in conjunction with the suboccipital musculature to power CSF flow13. Myodural bridge dysfunction has been implicated as a cause of chronic headache and is theorized to be a contributor to the chronic headache phenotype observed in people with Ehlers-Danlos syndromes (EDS)20.Myodural bridges (MDBs) are bilateral dense bands of connective tissue that connect the suboccipital musculature with the dura mater at the atlanto-occipital (AO) and atlanto-axial (AA) levels22. Hereditary equine regional dermal asthenia is an autosomal recessive disorder that occurs in Quarter Horses and is caused by a mutation in peptidyl-prolyl cis-trans isomerase B (PPIB) encoding for cyclophilin B (Cyp B)23. Similar to people with EDS, horses with HERDA have fragile, hyperextensible skin as well as joint hypermobility24.Our main objective was to further describe MDBs in horses, and to demonstrate that they are abnormal in horses with connective tissue disease. Horses with hereditary equine regional dermal asthenia (HERDA) were used as a naturally occurring animal model of EDS due to their similarities to people with kyphoscoliotic EDS (kEDS)A total of 10 horses (5 HERDA and 5 control) were evaluated. The HERDA group consisted of 4 Quarter Horses and 1 Paint Horse. The HERDA group contained 2 juveniles , 2 adults , and 1 geriatric horse (21-years-old), of which 3 were mares and 2 were geldings. The 10-year-old HERDA horse was not euthanized because it was client-owned; however, ultrasonographic examination was performed. The control horses consisted of 3 Quarter Horses, 1 Arabian, and 1 Morgan. The control group contained 3 juveniles , 1 adult (6-years-old), and 1 geriatric horse (18-years-old), of which 4 were mares and 1 was a gelding. Control horses were donated due to non-neurologic health concerns which consisted of orthopedic and ophthalmic disease in this group. Genetic testing and physical examination were required for study enrollment for Quarter Horses and Paint Horses. Genetic testing was performed on 3 HERDA horses prior to study enrollment. All were confirmed to be homozygous positive, H/H, for the PPIB mutation via DNA testing conducted on hair root samples at UC Davis Veterinary Genetics Laboratory. The remaining 2 HERDA horses were tested using hair root samples at Michigan State University and were also homozygous positive, H/H, for the PPIB mutation. All HERDA horses displayed a phenotype consistent with the disorder, including skin hyperextensibility and fragility, abnormal scarring, and abnormal joint hypermobility. All 3 control Quarter Horses were confirmed to be homozygous negative, N/N, for the PPIB mutation via genetic testing on hair root samples at Michigan State University and were phenotypically normal. Carriers of the PPIB mutation, H/N, were excluded from the study. All horses used in this study were evaluated with the permission of the Michigan State University Institutional Animal Care and Use Committee. Experimental protocols were approved by, and performed in accordance with, the guidelines and regulations set forth by the Michigan State University Institutional Animal Care and Use Committee.Static and dynamic neurologic examinations were performed on 3 HERDA horses and 4 control horses. Examination included observation of mentation, cranial nerve examination, assessment of spinal reflexes and muscle symmetry, and gait and postural assessment. Two HERDA horses were unable to be examined due to the early point at which they were evaluated. One control horse was unable to be examined due to a comminuted second phalangeal fracture.26. Ultrasonographic imaging allows for visualization of motion of both the MDBs and the dura mater. Three HERDA horses and 4 control horses were imaged. Two HERDA horses and 1 control were not imaged due to the early point at which they were enrolled in the study, during which horses were solely assessed for presence of the MDB. For this study, 6 horses were imaged with a Mindray TE7 ultrasound and a 2–4 MHz cardiac probe. One horse was imaged with an Esaote ultrasound and a convex 1–8 MHz probe. To image the MDBs, frequency ranged from 3.4 to 4.2 MHz and depth ranged from 10 to 13 cm. An approximately 40 × 15 cm area was clipped and cleaned with alcohol. Horses were positioned with their heads in neutral position, which was defined as 105° ± 5°. Head position was measured with a HALO digital goniometer  at the angle between the ventral mandible and the jugular groove. Probe position and anatomic landmarks were selected based on past techniques developed for ultrasound-guided AO and AA centesis for CSF collection in standing horses;28 however, our imaging focused on the rectus capitis dorsalis minor (RCDmi) and the myodural attachments. Ultrasonographic imaging was conducted during head and neck movement between neutral, flexion, and extension to demonstrate the synchronized movements of the MDB with the dura mater. Flexion was defined as 90° ± 5°and extension was defined as 115 ± 5°.A novel method for ultrasonographic examination of the MDBs was developed. Past studies in people have attempted to use MRI imaging to visualize the MDBs; however, ultrasound provides a more dynamic means of imaging. Prior MRI and ultrasonographic studies in horses have identified the AO membrane, but did not identify the MDBsIn order to confirm that the structures viewed on ultrasound were in fact the MDBs, ultrasound guided injection of latex dye was performed at the AO and AA levels immediately following euthanasia in 2 control horses. An 18-gauge spinal needle was used to inject 2–3 mL of blue latex dye under ultrasound guidance.Four HERDA and 5 control horses were humanely euthanized to allow for anatomic examination of the MDBs. One HERDA horse, a 10-year-old Paint mare, was client-owned and was not euthanized for this study. Necropsy was performed within 1 hour of euthanasia in all but 1 control horse. Necropsy examination of one horse, a 6-year-old control, was performed at the Cummings School of Veterinary Medicine at Tufts University. All other necropsy examinations were performed at the Michigan State University Veterinary Diagnostic Laboratory. Bilateral AO and AA MDBs were successfully identified in all horses.um. Resulting sections were stained with H&E, as well as Masson’s trichrome. Slides from 2 initial HERDA horses (aged 2-years and 3-years-old) and 1 control horse (6-years-old) were processed at Rhode Island Hospital Department of Neuropathology and photographed with a SPOT Insight 2Mp Monochrome FireWire Digital Camera and SPOT imaging software. All other specimens were processed at the Michigan State University Veterinary Diagnostic Laboratory where they were photographed with an Olympus BX45 microscope with an Olympus DP71 camera and Olympus cellSens software. Efforts were made to section all specimens in the same orientation, and to include the RCDmi, MDB, and dura mater in each section.The AO MDBs were collected at necropsy from 4 HERDA horses and 5 control horses and were submitted for histopathologic examination. Specimens were fixed for a minimum of 24 hours in 10% neutral buffered formalin and were routinely processed for histopathologic examination, embedded in paraffin, and sectioned at 5 31. These antibodies are validated for use in horses, and equine lymph node was used as a positive control32. Antigen retrieval was accomplished via immersion in a citrate buffer for 30 minutes, while 3,3′-Diaminobenzidine was used as a chromogen and hematoxylin as a counterstain. Bond Polymer Refine Detection  was the IHC kit used for both CD3 and CD20. All IHC was performed at the Michigan State University Veterinary Diagnostic Laboratory.CD3 (T cells) and CD20 (B cells) immunohistochemistry (IHC) was performed on 4 HERDA and 5 control MDBs to better evaluate the types of cells present. Tissues were formalin fixed and paraffin embedded. Dewaxing was performed with Bond Max DeWax . Sections were stained using a Bond-Max autostainer . Slides were incubated with rabbit anti-human polyclonal anti-CD20 antibody  for B cells at a dilution of 1:200 for 30 minutes and with rabbit anti-human polyclonal anti-CD3 antibody  for T cells at a dilution of 1:200 for 30 minutes33. A Power Tome Ultramicrotome  was used to cut 70 nm thin sections and post stained with uranyl acetate and lead citrate. One HERDA horse sample was processed at Rhode Island Hospital where upgraded alcohols were used for dehydration followed by four changes of propylene oxide. This sample was then embedded in Epon-Araldite rather than Spurr, prior to sectioning, staining, and imaging at Michigan State University Center for Advanced Microscopy34. No other deviations from protocol were introduced for this sample. All other samples were imaged at Michigan State University Center for Advanced Microscopy. The MDB was imaged at 14,000×, 27,000×, and 40,000× in longitudinal and cross-section using a JEOL 100CX Transmission Electron Microscope  at an accelerating voltage of 100 kV. Imaging at higher or lower magnification was performed when indicated.The AO MDBs from 4 HERDA and 5 control horses were submitted for transmission electron microscopy. Connective tissue from the cranioventral aspect of the bridge, adjacent to the junction of the dense connective tissue and dura mater, was submitted for processing. Samples were formalin fixed for 24 hours prior to transfer to glutaraldehyde in preparation for TEM processing at Michigan State University Center for Advanced Microscopy. Two samples, 1 HERDA horse and 1 control, were removed from paraffin blocks, due to a lack of tissue left in formalin after histopathology. Due to the durability of connective tissue, this method was deemed acceptable. After primary fixation in glutaraldehyde, samples were washed with 0.1 M cacodylate buffer. Samples were then postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in a gradient series of acetone, and infiltrated and embedded in SpurrSarcocystis neurona at the Michigan State University Veterinary Diagnostic Laboratory due to the presence of sarcocysts in the RCDmi and abnormalities in the neurologic examination.Cerebrospinal fluid was obtained via centesis of the cerebellomedullary cistern immediately following euthanasia in 2 HERDA horses and 4 controls and was stored at −80 °C for future molecular analysis. Cerebrospinal fluid from 1 HERDA horse, a 21-year-old mare, was submitted for a western blot for antibody to S. neurona was excluded due to a negative western blot performed on CSF; however, disease due to Neospora hughesi is still possible.The results of all control horse examinations were within normal limits. Abnormalities were noted in 1 HERDA horse, a 21-year-old Quarter Horse mare, that displayed mild truncal sway and a base-narrow, single-track gait in the hind limbs when blindfolded with the head elevated. This horse had cervical spine osteoarthritis, and this gait may be attributed to cervical spine pathology; however, an additional lumbosacral lesion cannot be ruled out. Equine protozoal myeloencephalitis due to The MDBs, dura mater, dentate ligaments, subarachnoid space, and spinal cord were easily visible at both the AO and AA interspaces Fig. . Dural mMyodural bridges were discovered in the horse at both the AO and AA levels. At the AO level, the MDBs originated from the RCDmi. En block resection of the AO and AA MDBs was accomplished in all horses Fig.  and maniThe MDBs were composed of areas of dense connective tissue interspersed with loose connective tissue, nervous tissue, adipose tissue, and vasculature Fig. . SynoviaInflammatory changes were present in 1 HERDA horse MDB. This horse, a 3-year-old Quarter Horse, displayed large aggregates of lymphocytes, as well as lymphocytic and plasmacytic perivascular cuffing. The presence of both T and B lymphocytes was confirmed via CD3 and CD20 IHC Fig. . The remS. neurona antibodies; results were negative. The sarcocysts were suspected to be Sarcocystis fayeri, a commonly occurring sarcocyst incidentally found in the muscle of horses. No evidence of sarcocyst rupture was observed. Lack of periodic acid-Schiff staining ruled out polysaccharide storage myopathy as a cause of myofiber degeneration. A 6-year-old HERDA mare displayed rare degenerate myofibers with phagocytosis of necrotic material by macrophages and mild infiltrates of eosinophils in the RCDmi. A single sarcocyst was observed within the RCDmi of this horse. A third HERDA horse, a 3-year-old gelding, had a focal area of myofiber degeneration with macrophage infiltration. The musculature was normal in all but one control, a 3-year-old Quarter Horse gelding that had extensive necrosis of myofibers and replacement by macrophages within the RCDmi. Additionally, it was observed at necropsy that this horse had a grossly visible area of necrosis in the superficial cervical musculature. On histology, the semispinalis capitis was found to have a focal area of myofiber necrosis and extensive phagocytosis of necrotic debris by macrophages. This horse had a history of trauma sustained during trailering.Myofiber degeneration was observed in the RCDmi in 3 HERDA horses and 1 control. A 21-year-old HERDA mare had myofiber degeneration with secondary phagocytosis of necrotic material by macrophages, as well as multiple sarcocysts within the muscle. Due to the presence of sarcocysts, CSF was submitted for a western blot to test for Differences in collagen fibril size, fibril packing density, and fibril orientation were subjectively compared between HERDA and control horses. The results are summarized in Table 6. Our study provides necropsy (post-mortem) and ultrasonographic findings that are directed to the dynamic aspect of the MDBs as dural sac stabilizers in horses. Further, this is the first study to report that MDBs may be pathologically altered in disease and can specifically be pathologically altered in connective tissue disease as evidenced by the ultrastructural histology in the HERDA MDB. Severe ultrastructural abnormalities in the collagen fibril orientation were present only within the HERDA MDB but not in the controls. It is conceivable that the function of the MDBs is impaired in HERDA horses, and we speculate in EDS patients as well.The results of this study support that MDBs are a conserved anatomic structure across mammalian species35. At the AO level, the equine MDBs originate from the RCDmi, a muscle analogous to the RCPmi in people35. As in people, the MDBs attach via the posterior, or dorsal, atlanto-occipital membrane-spinal dura complex36. At the AA level, the MDBs originated from the RCDma, a muscle analogous to the RCPma in people. The obliquus capitis caudalis, analogous to the obliquus capitis inferior in people, also contributes to the MDB in the horse35. The anatomy of the equine MDB attachments at the AO and AA levels is virtually identical to that of other land-dwelling mammals, including the attachment through the dorsal atlanto-occipital membrane-spinal dura complex at the AO level. This anatomy differs from the description by Zheng, et al. (2017) of the Indoasian finless porpoise, a marine mammal with an AO MDB which does not attach via the posterior atlanto-occipital membrane-spinal dura complex, and instead inserts directly onto the dura mater via tendon like projections from the RCDmi. The Indoasian finless porpoise also has a muscular attachment at the AA level which includes the lateral rectus capitis dorsal muscle, a muscle not found in the land-dwelling mammals described by Zheng et al.6.While horses with HERDA are known to have many similarities to people with EDS, the neurologic status of HERDA horses has not previously been documented. The anatomy of the equine craniocervical region shares many similarities with people, making them an excellent model for examination of the MDBs9 and is supported by our own in-vivo ultrasound imaging showing all anatomical elements involved in the human study, the nerve roots, the dentate ligaments, the dural sac, and the MDBs. Ultrasonographic imaging in horses should encourage respective studies of the cervical spine in people to evaluate the configuration of the dural sac during head and spine movement.Ultrasonography gives dynamic insights into how the anchoring elements of the spine function, the intradural filaments and emerging spinal nerve roots stabilize and center the spinal cord inside the dural sac. The extradural MDBs connect the dura to the surrounding head and neck muscles. This study corroborates the likelihood that MDBs allow the dural sac to remain patent even with neck and head movement. This physiological concept has been previously suggested based on histologic and necropsy findings in people11. The ultrastructure of the MDB has not yet been examined in people; however, there is a large body of work examining the ultrastructure of other tissues, particularly skin, in the EDS population. People with EDS often display collagen fibrils described as whirling within bundles, as well as hook-like or twisted fibrils. Collagen fibrils with irregular edges in cross-section, as well as loosely packed collagen fibrils, are also common findings in EDS37. Similar findings have been described in the skin and corneas of HERDA horses38. The HERDA MDBs examined in this study displayed characteristics, such as decreased fibril packing density and abnormal fibril orientation, that are commonly observed in the skin of people with EDS. Additionally, granulo-filamentous deposits were observed in the interfibrillar space in the HERDA population, which are commonly associated with kEDS in people37. The abnormal ultrastructural collagen fibril orientation seen in HERDA horses but not controls may suggest “defective” MDB dynamics in HERDA. A recent article by Zheng et al. (2018) of anatomical and histologic analyses of AO and AA MDBs in human cadavers did show that MDBs in fact display parallel collagen fibers. This group used a method sensitive to fibril orientation, the “fiber quad staining” method, to detect reticular fibers, elastic fibers and collagen fibers. They found that the MDB was mainly made of collagen type 1 fibers, fibers which are most common, and observed in tendons as well. These authors suggest that MDBs are high-tensile tendon-like structures39.Histologically, the human and equine MDB share many similarities. The MDBs of both species contain connective tissue, adipose tissue, vasculature, and peripheral nervous tissue11. This finding further supports an anatomical and functional significance, as well as the clinical relevance of the myodural bridges’ function as a passive and active stabilizer of the cervical spinal cord across species. The active contribution of the MDBs in the neuromuscular stabilization of the spinal cord has been previously discussed by Scali, Pontell and Enix et al.  as evidenced by the presence of neural tissues within the MDB structures using IHC staining with anti-neurofilament protein antibodies12.Peripheral nerves in the MDBs have also been described in human cadaver histology, and this study was able to demonstrate the presence of peripheral nerve associated with the connective tissue core of the equine AO MDB via histopathology21.Further ultrasonographic studies are being conducted to evaluate the presence of dural infolding secondary to MDB dysfunction in the equine HERDA and human EDS populations, which could be conducted by measuring the subarachnoid space with the head and neck positioned in neutral, flexion, and extension. Additionally, CSF was banked for future analysis to allow comparison of equine HERDA and human EDS CSF for markers of inflammation and collagen breakdown products, such as pyridinoline and deoxypyridinoline42. Additionally, horses with HERDA can be a difficult population of horses to obtain and maintain.A limitation of this study was our sample size. Although our sample size of 5 horses and 5 controls is admittedly small, it is in keeping with many of the recent referred publications on HERDAet al. (2006) found that sex differences in people do not impact the MDB36.Although our controls and HERDA cases are not perfectly sex matched we do have representative horses in each group. In the control horses there were four mares and one gelding while in the HERDA horse population there were three mares and two geldings. Further support for a lack of impact due to this discrepancy comes from the literature where Zumpano 43. Radiographs of the cervical spine were not obtained because we were able to directly assess the spine at necropsy. A single negative western blot on CSF in this horse is within the standard of practice to rule out EPM in veterinary medicine. The sensitivity and specificity for the western blot on CSF both approach 89%45.One true limitation of our study is our inability to accurately assess horses, and for that matter most species, for headaches. Unfortunately, there are no tests for head pain in the horse. Also, the significance of the mildly abnormal neurological exam in one HERDA horse is unclear. Osteoarthritis in this animal was observed throughout the cervical spine during ultrasonographic examination, which is a clinically appropriate imaging modality to address this issueAll horses had the same procedures performed  with few exceptions. The initial horse did not receive a neurologic or ultrasonographic examination but was not noticeably neurologic upon enrollment. This horse presented with a phalangeal fracture and could not be subjected to additional testing prior to euthanasia. We felt it was still appropriate to use this horse to establish necropsy and histopathologic techniques before commencing the larger study. One horse did not undergo histopathology or transmission electron microscopy as it was client owned and not euthanized. The necropsy of one control horse occurred more than 1 hour after being euthanized but did not display histological disintegration so it was included in the study. The high fat content in some of the tissues and lack of enzymatic activity in all tissues studied make them more resistant to degradation when compared to tissues such as the pancreas or stomach. Also, ultrasonographic studies were only added once we were confident about the structure of the equine MDBs after several horses were euthanized.We feel that the strength of our study lies in its thorough descriptive nature. Evidence for the presence of abnormalities in the HERDA horse MDB by virtue of anatomic and dynamic validation via necropsy, ultrasound and ultrastructural techniques indicate that MDBs are abnormal in HERDA horses when compared to controls.The ultrastructural findings observed in the HERDA MDB, such as decreased collagen fibril packing density and abnormal collagen fibril orientation, suggest decreased integrity of connective tissue. This study is a step towards better understanding the role of MDBs in human disease(s). Further ultrasonographic studies in horses and people with connective tissue disease might determine the impact of MDB action on subarachnoid space width and correlation with clinical symptoms, i.e. headaches and craniocervical neurological symptoms.Equine Atlantoaxial Ultrasonographic Examination


Tobacco harm reduction, substituting less harmful tobacco products for combustible cigarettes, is a complementary approach for smokers who would otherwise continue to smoke. The Philip Morris International (PMI) Tobacco Heating System (THS) 2.2 is a novel tobacco product with the potential to reduce the risk of harm in smokers compared to continued smoking of combustible cigarettes. It heats tobacco electrically in a controlled manner, never allowing the temperature to exceed 350°C, thereby preventing the combustion process from taking place and producing substantially lower levels of toxicants while providing nicotine, taste, ritual, and a sensory experience that closely parallels combustible cigarettes. Previous clinical studies have demonstrated reduced exposure to the toxicants (approaching the levels observed after quitting) for smokers who switched to THS 2.2, for three months. For adult smokers who would otherwise continue smoking combustible cigarettes, switching to THS 2.2 may represent an alternative way to reduce the risk of tobacco-related diseases.This study aimed to further substantiate the harm reduction potential of THS 2.2 by demonstrating favorable changes in a set of 8 coprimary endpoints, representative of pathomechanistic pathways , linked to smoking-related diseases, in smokers switching from combustible cigarettes to THS 2.2.This study was a randomized, controlled, two-arm parallel group, multicenter ambulatory US study conducted in healthy adult smokers switching from combustible cigarettes to THS 2.2 compared with smokers continuing to smoke combustible cigarettes for six months. Subjects had a smoking history of at least ten years and did not intend to quit within the next six months.Enrollment started in March 2015 and the trial was completed in September 2016. In total, 984 subjects were randomized , and 803 completed the study. The results are expected to be available in a subsequent publication in 2019.In this paper, we describe the rationale and design for this clinical study that focused on the evaluation of THS 2.2’s potential to reduce the risk of smoking-related diseases compared with that of combustible cigarettes. This study will provide insights regarding favorable changes in biological and functional endpoints informed by effects known to be seen upon smoking cessation.ClinicalTrials.gov NCT02396381; http://clinicaltrials.gov/ct2/show/NCT02396381 (Archived by WebCite at http://www.webcitation.org/71PCRdagP)RR1-10.2196/11294 Cigarette smoking is the leading cause of preventable disease in the US, accounting for more than 480,000 smoking-related deaths every year. More than 16 million Americans live with a smoking-related disease , and oneIn addition to the prevention of smoking initiation and the promotion of smoking cessation, tobacco harm reduction is being recognized as a valuable and promising approach to further accelerate the decline in smoking prevalence and smoking-related population harm . TobaccoImportantly, to improve health at the population level, these substitutes for cigarettes must be acceptable for smokers, providing adequate nicotine delivery and satisfaction to prevent relapse to cigarette smoking. In this context, Philip Morris International (PMI) has developed Tobacco Heating System (THS) 2.2, as a candidate MRTP that has been designed to provide nicotine to smokers, who otherwise would have continued to smoke, offering sensory and ritual aspects like cigarettes while reducing the exposure to harmful and potentially harmful constituents (HPHC) found in cigarette smoke -8. AdditTHS 2.2 uses a precisely controlled heating device into which a specially designed tobacco product, the Tobacco Stick, is inserted and heated to generate an aerosol. The THS 2.2 heater starts heating the Tobacco Stick in a controlled and gradual manner, with the temperature set between 320°C and 350°C. Heating the Tobacco Stick in a controlled manner and not allowing the temperature to exceed 350°C prevents the combustion process from taking place. The elimination of combustion results in a significant reduction in the production and exposure to HPHCs  while th−/− mouse model, which is commonly used to study atherosclerosis and emphysema, exposure to THS 2.2 aerosol did not induce a change in the lipid profile or enlargements of aortic plaque area, nor lung inflammation or emphysema, unlike cigarette smoke. Additionally, switching from cigarette smoke to THS 2.2 aerosol exposure reversed inflammation, and halted aortic plaque growth and the progression of emphysema in a manner that mimics smoking cessation . Su. Su33]. Collection of 24-hour urine started at the subject’s home on the morning of the day before the scheduled visit and ended the morning of the day of the study visit. Blood was collected after at least 10 hours of fasting except for carboxyhemoglobin measurement. Exhaled breath was measured for CO using a Smokerlyzer device  as another biomarker of exposure to CO. Cough assessment by visual analog scale and Likert scales  were conducted at baseline (V3), at month three, and at month six.This study enrolled current adult smokers of nonmenthol cigarettes who did not intend to quit smoking within the next six months. At least 950 participants were to be randomized. Once 950 subjects had been randomized, no additional subjects were enrolled; however, all subjects who were already enrolled and started the run-in period were still randomized.The main inclusion and exclusion criteria are listed in Approval for the study was granted by one central Institutional Review Board for each of the participating sites. All participants provided written informed consent before the start of the study. The study was conducted following Good Clinical Practice guidelines and the ethical principles of the Declaration of Helsinki -40. StudA sample size of 950 subjects (randomized 1:1) was calculated to be enough to attain a statistical power of >99% to show statistically significant favorable changes in at least five out of eight CREs of the smokers’ health profile at six months. The Hailperin-Rüger approach will be used to adjust for test multiplicity ,42.Although this was an open-label study, and the subjects and the investigators or their designees were unblinded to the subject’s study arm after randomization, a limited degree of blinding was implemented during the conduct of the study, including the data review and data analysis process. The study statisticians and clinical scientists involved with the definition of the analyses were blinded to the actual values of primary CREs from the time of randomization until database lock.The primary analysis will be run on the full analysis set (FAS) of subjects based on their actual product exposure  accordinInclusion criteriaHealthy smokerAt least 30 years oldSmoking history of at least 10 yearsSmoking history of at least 10 nonmenthol cigarettes per day on average in the 12 months preceding the screeningNo intention to quit smoking within the next six monthsExclusion criteriaClinically relevant gastrointestinal, renal, hepatic, neurological, hematological, endocrine, oncological, urological, pulmonary, immunological, psychiatric, or cardiovascular disorders or any other conditions that would jeopardize the safety of the participant or affect the validity of the study resultsAbnormal findings on physical examination, in the medical history, or in clinical laboratory results deemed clinically relevant by investigators (as per the common terminology criteria for adverse events)Acute illness  requiring treatment within 30 days before enrollment in the studyUse of any prescribed or over-the-counter systemic medications with an impact on the clinical risk endpoints of the smokers’ health profile within five half-lives of the medication before study enrollment, except over-the-counter vitamin supplements, hormonal contraceptives, and hormone replacement therapy1/FVC below 0.7 and FEV1 below 80% predicted value at postbronchodilator spirometry FEVPregnancy or breastfeedingUnwilling to use an acceptable method of effective contraception For the primary analysis, substantiation that switching to THS 2.2 from cigarettes modifies the risk of smoking-related diseases will rely on the following criteria. First, all CREs in the smokers’ health profile must shift in the same direction as they would upon smoking cessation. Second, switching to THS 2.2 must show a statistically significant improvement in at least five of the 8 components of the smokers’ health profile, with each CRE evaluated using a one-sided alpha of 1.5625%, corresponding to half of the Hailperin-Rüger adjusted type I error (.031). The Hailperin-Rüger approach calculates the statistical significance that is required for each test when at least five of the 8 primary CREs in the smokers’ health profiles are required to be significant to maintain the overall study-wise alpha-level of 5%. Effect estimates will be presented accompanied by 2-sided 96.875%  confidence intervals. It will be finally evaluated if most of the effect of smoking cessation is preserved in subjects switching to THS 2.2, based on the results of an integrated analysis pooling data from a separate smoking cessation study (NCT02432729) designed to benchmark the clinical, biological, and functional changes in smokers who are continuously abstinent from smoking for 1 year. The methods and results will be reported in a separate manuscript. There were no interim analyses planned.For the study, 2,556 subjects were screened, 1,039 were enrolled, 984 were randomized (483 to CC arm and 477 to THS 2.2 arm), and 803 completed the study . The datEnrollment started in March 2015 and the trial was completed in September 2016. The results of this paper are expected in 2019.The diseases attributed to smoking are complex. Continuous exposure to HPHCs affects multiple organ systems, disease pathways, and mechanisms, such as inflammation, oxidative stress, platelet activation, and lipid metabolism, which coincide, leading gradually to the development of smoking-related diseases over the course of years. This study examined changes in biological and functional CREs in adult smokers switching to THS 2.2 in an ambulatory, near real-life setting. Because no single CRE is validated as a surrogate measure for any smoking-related disease, the primary endpoint of this study was a selection of equally important, nonhierarchical, co-primary CREs defined as the smokers’ health profile.The analysis of this study uses a robust approach in the field of tobacco harm reduction. All co-primary CREs of the smokers’ health profile must shift in the same direction as they would upon smoking cessation, and at least five of the 8 components of the smokers’ health profile must be significantly improved statistically in THS 2.2 users compared with those who continued smoking cigarettes. Significance will be evaluated using a one-sided test with the Hailperin-Rüger adjusted alpha-level.Furthermore, the planned analysis approach considers baseline comparability of confounding factors that can potentially influence study results, such as exercise, diet, alcohol intake, and potential exposure to passive smoking. Additional CREs that are representative of various mechanistic or pathological pathways will be evaluated in the secondary objectives to support the analysis of the primary objective. Because smoking alters multiple pathways, tissues, and organs, which together contribute to disease risk, this approach will provide coherent and multifaceted scientific evidence of the reduced-risk potential of THS 2.2.The ambulatory setting will provide information not only on product consumption and combined or dual-use (smoking of cigarettes in addition to using THS 2.2) but also on user satisfaction and acceptance of the product, as assessed by the proportion of product used, the “intent to use” questionnaire, and the modified cigarette evaluation questionnaire.In summary, results from this study will be a noteworthy addition to the growing body of data from the assessment program to scientifically substantiate that THS 2.2 can potentially reduce the risk of smoking-related diseases . The desThis study is part of a multilayered assessment program designed to evaluate whether THS 2.2 can potentially reduce the risk of smoking-related diseases relative to continued smoking. The results of this study will confirm whether the reduction in exposure to HPHCs when switching from cigarettes to THS 2.2 leads to statistically significant favorable changes in CREs linked to smoking disease and following the direction expected upon smoking cessation. Detailed information on product use, product satisfaction, and acceptance will also emerge from this study. This study will provide evidence to substantiate the reduced-risk potential of THS 2.2. Longer term duration of exposure is needed to evaluate these changes in biological and functional biomarkers further.


Localization of RNAs to various subcellular destinations is a widely used mechanism that regulates a large proportion of transcripts in polarized cells. In many cases, such localized transcripts mediate spatial control of gene expression by being translationally silent while in transit and locally activated at their destination. Here, we investigate the translation of RNAs localized at dynamic cellular protrusions of human and mouse, migrating, mesenchymal cells. In contrast to the model described above, we find that protrusion-localized RNAs are not locally activated solely at protrusions, but can be translated with similar efficiency in both internal and peripheral locations. Interestingly, protrusion-localized RNAs are translated at extending protrusions, they become translationally silenced in retracting protrusions and this silencing is accompanied by coalescence of single RNAs into larger heterogeneous RNA clusters. This work describes a distinct mode of translational regulation of localized RNAs, which we propose is used to regulate protein activities during dynamic cellular responses. Targeting of RNA molecules to distinct subcellular destinations has emerged as a widely-used mechanism that controls the cytoplasmic distribution of a large proportion of expressed transcripts in a range of organisms . Such RNThis model is supported by several lines of evidence. Localized RNAs are associated during transport with RNA-binding proteins (RBPs) that function as translational repressors, which block the initiation or elongation steps of translation. For example, the RBPs Khd1 and Puf6 repress the translation of the yeast ASH1 mRNA until it reaches the tip of the emerging yeast bud. Puf6 inhibits translation by binding to the initiation factor eIF5B, thus preventing ribosome subunit binding to the mRNA . SupportOnce the RNA reaches its final destination, spatial control of gene expression is achieved by local de-repression of translation. Exemplifying this notion, at the yeast bud tip, the Puf6 and Kdh1 RBPs are phosphorylated by the Yck1 kinase, leading to release of eIF5B and translation activation . Similarnanos mRNA is deadenylated and translationally repressed in the bulk cytoplasm of Drosophila embryos through the action of the RBP Smaug and the CCR4/NOT deadenylase. At the posterior pole, the Oskar protein relieves this inhibition and leads to de-repression of nanos translation  constructs leads to a preferential mislocalization of APC-dependent RNAs from protrusions see . Brief tTo test whether mislocalization from protrusions is accompanied by changes in translation, polysome gradient fractions were isolated from control and cUTR- or parthenolide-treated cells and analyzed for the presence of various RNA species. In both cases, cUTR overexpression or parthenolide treatment did not affect the overall gradient profile, suggesting that no overt changes in translation resulted from either treatment  and 2B. We note that both cUTR overexpression and parthenolide treatment resulted in a small apparent reduction in the amount of APC-dependent RNAs sedimenting in the heavy polysome fraction (fraction 4) and an apparently corresponding increase of RNA amounts in polysome fraction 3. These changes are small, they are not statistically significant and do not alter the conclusion that APC-dependent RNAs remain translationally active. Nevertheless, we make a note of this observation because it is manifested quite consistently by APC-dependent RNAs under conditions that disrupt their peripheral localization. In light of data presented below, we believe that this might reflect a real change in the organization of a small fraction of these RNPs (see discussion).While the above results suggest that transport and translation of APC-dependent RNAs are not coordinated, they also raise the possibility that assessing the translation status in a whole-cell extract derived from heterogeneous cell populations might not offer the required sensitivity to detect local changes occurring on a single-cell level. To address this, we took advantage of the recently developed SunTag-based reporters that allow imaging of translation of single RNA molecules in live cells  Figure . These rWe first validated the RNA and translation signals detected with our implementation of the assay. To assess the specificity of the detected RNA spots, we compared the signal observed upon expression of the control PP7-containing reporter RNA to that of a similar reporter carrying a deletion of the PP7-binding sites (−PP7) . mCherryWe further imaged cells expressing either control or localized reporters (carrying the Rab13 or Pkp4 UTRs) –4. ImagiTo address whether transport to the periphery is accompanied by changes in the translation state of the RNAs, we determined the translation efficiency of single RNA molecules as a function of their distance from the periphery. Consistent with the stochastic translation bursts reported in other systems , for allWe point out that our analysis is focused on less mobile molecules. We cannot currently assess if translation persists, or not, during periods of active movement. In this regard, we have observed single RNAs that undergo short-range directed movements while being translationally active  and 6. TThe data mentioned above have focused on single RNA molecules. However, in the course of our studies we have observed that endogenous APC-dependent RNAs exist in two states: they are found either as single molecules or as clusters made up of multiple RNAs in close spatial proximity. We have observed these clusters for virtually all APC-dependent RNAs we have detected by in situ hybridization . These RThis spatially-defined clustering behavior is not observed by RNAs that show a more uniform distribution in the cell body and are not targeted to protrusions . With imTo investigate the translation status of RNAs within these clusters we employed the localized translation reporters. Consistent with the data described above, translation reporters carrying the Rab13 or Pkp4 UTRs could be found at the tips of protrusions in clusters containing multiple RNA molecules, as indicated by the increased mCherry intensity compared to the intensity exhibited by the single molecules in more internal regions . TranslaOur initial expectation was that RNAs at the periphery would be locally translated. While the above data show that this indeed happens, the additional observation of silencing at the periphery was rather counterintuitive. We thus first sought to validate that the above findings indeed reflect the regulation of endogenous localized transcripts. For this, we focused on Rab13 and employed the puro-PLA assay (puromycylation followed by proximity ligation amplification) to detect nascent Rab13 protein molecules in situ . Puro-PLConsistent with our observations in mouse NIH/3T3 cells, primary human fibroblasts also significantly localize the Rab13 RNA to the periphery compared to the overall distribution of polyadenylated RNAs in the cytoplasm . Visual To try to understand this paradox we looked at the spreading rate of cells around the time points used for translation site imaging. We noticed that the reduction in peripherally translated Rab13 is associated with a decrease in spreading rate . In otheFor this reason, we turned to MDA-MB-231 breast cancer cells. These cells, when migrating on collagen-coated glass, exhibit a characteristic tail which retracts over a few minutes  and 12. Importantly, in MDA-MB-231 cells, the Rab13  are also peripherally localized and can be found both in front lamellipodia as well as in retracting protrusions . Rab13-pIn mouse fibroblasts we had observed an association of translationally silent RNAs with peripheral heterogeneous clusters  and 6. TTo determine whether RNA clusters in retracting MDA-MB-231 protrusions were composed of heterogeneous RNA species, we tested for the co-localization of Rab13 with polyA RNA. As mentioned above, in fibroblast protrusions, the majority of individual RNA clusters overlap with areas of visible concentration of polyA RNA . We obseTo probe more into the assembly of these peripheral clusters, we tested how their formation is affected upon translational inhibition or disruption of RNA transport to the periphery. We performed these experiments in mouse fibroblast cells, in which peripheral clusters can be readily detected through polyA accumulation . Indeed,Scoring of cell populations for the presence of polyA granules at protrusions revealed that 25–30% of protrusions contain peripheral polyA granules . InteresWe further disrupted the microtubule network with nocodazole, or specifically disrupted detyrosinated microtubules using the tubulin carboxypeptidase inhibitor parthenolide . Of noteHere, we investigate the translational regulation of APC-dependent RNAs which are targeted to cell protrusions. We find that the cytoplasmic position of APC-dependent RNAs does not affect their translation, since they can be translated similarly in both internal and peripheral locations. Rather, translation of APC-dependent RNAs is coordinated with specific peripheral cellular processes, being activated at extending protrusions/lamellipodia and suppressed upon protrusion retraction. Silencing is coupled to a change in the physical state of the RNAs manifested as single RNAs clustering into heterogenous granules at the tips of protrusions.This mode of regulation is distinct from the one proposed for several other localized transcripts, whereby RNAs are transported in a silenced state and are translationally activated only upon reaching the final destination or upon receipt of specific signals . A reasoOur observation of translation regardless of cytoplasmic location suggests that the proteins encoded by APC-dependent RNAs can be produced in internal regions without deleterious effects. We propose that this mode of regulation could additionally have functional implications for the encoded proteins. Specifically, translation in local environments can attribute proteins with different properties. This could result from differential protein modifications or through proximity to protein partners, which during co-translational assembly could affect the type or efficiency of multimeric complex formation . In lighWe additionally show that translation of APC-dependent RNAs is specifically suppressed in retracting protrusions and this silencing is associated with the formation of multimeric heterogeneous clusters. Formation of these clusters is reduced by treatments that prevent RNA transport to the periphery and is increased upon global translational inhibition, suggesting that silencing is a limiting step in their formation. Nevertheless, silencing can occur outside of clusters, since a large proportion of RNAs are observed as single particles in retracting, translationally-silent protrusions. The appearance of these heterogenous RNA clusters at the tips of protrusions, and their relation to translation, are reminiscent of other types of RNA granules formed by liquid-liquid phase separation . For exaThe silencing of Rab13, and likely of other APC-dependent RNAs, at retracting protrusions is contrasted by their translational activation in extending lamellipodia. Given that localization of APC-dependent RNAs to the periphery is important for cell migration  these daA potential underlying mechanism for local silencing and granule formation could rely on spatially restricted phosphorylation/dephosphorylation events, which have been shown to affect the propensity of RNA-binding proteins to form phase-separated granules or to bind to RNAs . In thisWith regards to the assembly/disassembly dynamics of peripheral clusters and the fate of the sequestered RNAs, limitations in the duration of live-imaging we can accomplish do not permit concrete conclusions. However, our current data indicate that the extent of cluster formation might be influenced by the rate of retraction. Specifically, in NIH/3T3 cells, the majority of peripheral clusters are characterized by a visible accumulation of polyA signal, indicative of a high concentration of heterogeneous RNA species. By contrast, a substantial fraction of retracting MDA-MB-231 protrusions do not exhibit obvious polyA accumulation . A promiThe existence of translationally silent multimeric RNA clusters also offers a potential explanation for the slight decrease of APC-dependent RNAs found in the heavy RNP fraction under conditions that disrupt transport to the periphery  and 2B. We had previously reported that APC-dependent RNAs can also be found in internal cytoplasmic granules induced by expression of FUS mutants carrying ALS-associated mutations. Intriguingly, in these FUS-granules APC-dependent RNAs are translationally active . Taken tWe note that the above study describes the regulation of APC-dependent RNAs in migrating mesenchymal cells. It would be interesting to explore how translation and transport of this RNA group is carried out in larger and more stably polarized cells such as neurons.Overall, we describe here a distinct mode of translational regulation of localized RNAs. These findings provide a different perspective towards understanding how local translation can influence protein activities and how these regulatory mechanisms could be integrated with dynamic cellular behaviors.Plasmids for translation reporters: pHR-tdPP7-3x-mCherry, pcDNA4TO-24xGCN4_v4-kif18b-24xPP7 and pcDNA4TO-24xGCN4_v4-kif18b were gifts from Marvin Tanenbaum . pcDNA4TO-24xGCN4_v4-kif18b-24xPP7 was used to introduce different mouse UTR sequences at EcoRI/AscI sites after the PP7 repeats, and the inserts encompassing the coding sequence and UTRs were transferred into pInducer 20 lentivector , using the Gateway LR clonase II Enzyme mix  according to the manufacturer’s instructions, to generate plasmids: pInducer20-24xGCN4_v4-kif18b-24xPP7-Rab13 UTR and pInducer20-24xGCN4_v4-kif18b-24xPP7-Pkp4 UTR. pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE was a gift from Ron Vale (Addgene plasmid # 60906). The NLS sequence in this construct was deleted and replaced with a stop codon, to generate pHR-scFv-GCN4-sfGFP-GB1-deltaNLS-dWPRE.Plasmids for MS2-GFP-reporters: Phage-ubc-nls-ha-tdMCP-gfp was a gift from Robert Singer (Addgene plasmid # 40649). pcDNA3-based plasmids expressing the β-globin genomic sequence followed by 24xMS2 binding sites and different 3’UTRs, were previously described . InsertsLentiviruses were produced in HEK293T cells cultured in DMEM containing 10% FBS and Penicillin/Streptomycin. HEK293T cells were transfected with lentivectors, together with packaging plasmids pMD2.G and psPAX2 using PolyJet In Vitro DNA transfection Reagent (SignaGen) for 48 hr. Harvested virus was precipitated with Polyethylene Glycol at 4 °C overnight.For Western blot detection the following antibodies were used: anti-Rab13 rabbit polyclonal  and anti-GAPDH rabbit monoclonal 14C10 .2. The stable NIH/3T3 cell line that inducibly expresses the Pkp4 cUTR has been described before (2. MDA-MB-231 breast cancer cells (ATCC) were grown in Leibovitz’s L15 media supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C in atmospheric air. Cell lines have been tested for mycoplasma and are free of contamination. To generate cell lines expressing translation reporters, NIH/3T3 cells were sequentially infected with lentiviruses expressing tdPP7-3x-mCherry and scFv-GFP; a subpopulation was isolated through fluorescence activated cell sorting (FACS) and clonal lines were derived. A line expressing uniformly low levels of mCherry and GFP was used to introduce the various pInducer 20-based reporter constructs, and stably expressing cells were selected with geneticin (Thermo Fisher Scientific). The derived cell lines are: PCFvGdelta1-PP7 (control translation reporter); PCFvGdelta1-deltaPP7 (control translation reporter with deletion of the PP7 repeats); PCFvGdelta1-Rab13U (translation reporter with Rab13 UTR); PCFvGdelta1-Pkp4U (translation reporter with Pkp4 UTR). Expression of the reporters was induced by addition of 1 μg/ml Doxycycline (Fisher Scientific) approximately 2–3 hr before imaging.NIH/3T3 mouse fibroblast cells (ATCC) were grown in DMEM supplemented with 10% calf serum, sodium pyruvate and penicillin/streptomycin (Invitrogen) at 37°C, 5% COd before . PrimaryTo generate cell lines expressing MS2-reporters, NIH/3T3 cells were infected with lentivirus expressing tdMCP-GFP and GFP-expressing cells with low level of GFP expression were sorted by FACS. This stable population was infected with pInducer20-based reporter constructs carrying 24xMS2 binding sites, and stable lines were selected with geneticin (Thermo Fisher Scientific). Derived cell lines are: tdMCP-GFP_pIND20-b24bs/Net1 (MS2 reporter/Net1 UTR) and tdMCP-GFP_pIND20-b24bs/Rab13 (MS2 reporter/Rab13 UTR). Expression of the reporters was induced by addition of 1 μg/ml Doxycycline (Fisher Scientific) approximately 2–3 hr before imaging.For translation inhibition, cells were treated with 100 μg/ml cycloheximide , 2 μg/ml harringtonine , 1 μM lactimidomycin , 50 μg/ml anisomycin , 100 μg/ml puromycin . For knockdown experiments, 40 pmoles of siRNAs were transfected into cells with Lipofectamine RNAiMAX  according to the manufacturer’s instructions. Cells were assayed after three days. siRNAs used were: AllStars Negative control siRNA (cat# 1027281) and si-Rab13 #8  from Qiagen.Protrusions and cell bodies were isolated from serum-starved cells plated for 2 hr on Transwell inserts equipped with 3.0 μm porous polycarbonate membrane (Corning) as previously described . BrieflyFor nanoString analysis, RNA samples were analyzed using a custom-made codeset and the nCounter analysis system according to the manufacturer’s instructions.Cells were plated the day before so that they were actively growing on the day of the assay. Cells were treated with 50 μg/ml cycloheximide for 30 min at 37°C and cytoplasmic extract was collected essentially as described in Net1 cat# VB1-3034209, Cyb5r3 Cat# VB1-18647, Cenpb cat# VB1-18648, mouse Rab13 cat# VB1-14374, human Rab13 cat# VA1-12225, Pkp4 Cat# VB4-600264, Kif18b cat# VA6-3170686, Ddr2 cat# VB1-14375, Arpc3 cat# VB1-14507, P4hb cat# VB6-15898. To detect polyA RNAs, LNA modified oligodT probes (30 nucleotides) labeled with ATTO 655 were added during hybridization, pre-amplification, amplification and last hybridization steps of ViewRNA ISH Cell Assay. Cell mask stain (Thermo Fisher Scientific) was used to identify the cell outlines. Samples were mounted with ProLong Gold antifade reagent (Thermo scientific).FISH was performed with ViewRNA ISH Cell Assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following Affymetrix probe sets were used: 2, 25 mM KCl, 100 μg/ml cycloheximide, 0.15 mg/ml digitonin, 0.5 U/μl RNasin, and Halt protease inhibitor cocktail), washed twice with ice-cold permeabilization buffer and fixed with 4% formaldehyde in PBS for 15 min at room temperature. Cells were subsequently blocked in blocking buffer (5% goat serum in PBS) for 1 hr at 37C and incubated with a pair of primary antibodies diluted in blocking buffer (in a humidified chamber for 1.5 hr at room temperature). Antibodies used were anti-Rab13  and anti-puromycin 3RH11 . After washing, PLA probes were applied in 1:10 dilution using the diluent buffer provided in the Duolink In Situ Red kit . Incubations and subsequent ligation and amplification steps were performed according to the manufacturer’s instructions. After the final washes, cells were post-fixed for 10 min at room temperature with 4% formaldehyde in PBS, stained with Alexa Fluor-488 phalloidin  in blocking buffer for 30 min and mounted using Duolink PLA Mounting medium with DAPI.Cells plated on Fibronectin - or Collagen IV -coated coverslips were pre-treated (or not) with the indicated translation inhibitors for 15 min and incubated for 5 min with 100 μg/ml puromycin at 37C. Subsequent steps were based on the protocol by FISH and puro-PLA images were obtained using a Leica SP8 confocal microscope, equipped with a HC PL APO 63x oil CS2 objective. Z-stacks through the cell volume were obtained and maximum intensity projections were used for subsequent analysis. Surface plot profiles, number and intensity of detected particles, were derived using the ‘Surface plot’ and ‘Analyze particles’ functions of ImageJ software (version 2.0.0-rc-69/1.52 n). Calculation of PDI index was performed using a custom Matlab script. The code is described and is available in For live imaging of Lifeact-expressing cells, cells were plated on LabTek chambered coverglass, in phenol red-free media and were imaged on a Leica SP8 confocal microscope equipped with HC PL APO 63x oil CS2 objective, at constant 37C temperature and 5% CO2  or atmospheric air (for MDA-MB-231 cells). The 488 nm laser line was used for illumination, z-stacks through the cell volume were acquired over time and maximal intensity projections were produced.2. 488 nm and 561 nm laser lines were used for illumination and single z-plane images were acquired over time. Two channel imaging was performed with sequential line scanning. Acquisition speed was approximately 500 ms/frame. No detectable photobleaching was observed under these conditions during a 20–40 s acquisition period. Diffraction-limited spots were identified using a custom IDL software provided by Dr. Daniel Larson . Second, they do not consider alternative interpretations, for example that the translation is stalled until the mRNAs reach their destination, and the proteins are released there. More quantitation is needed before conclusions can be drawn as to polysome loading, for instance using qPCR. There is a noticeable lack of free protein, this seems to be a conundrum, along with the observation that translation is silenced exactly where it is needed. Discussion of the significance of this counter-intuitive observation is lacking. Finally, no consideration is given to the nature of the cells analyzed, and whether these results are an artifact of the cells or reporter chosen, The cells used, 3T3, are not good examples of cells with protrusive activity, or of polarized cells in general where localization is essential for motility , and the reporter is not an endogenous gene with an endogenous promoter. They should test their system on primary cells to see if their observations hold there. So while the observations are potentially interesting they must be tempered by these specific constraints. The Discussion either needs to be softened, or the authors must provide additional data to address the generality of their observations. Finally, what conclusions the authors do want to draw on their specific system needs to be backed up by quantitation.Reviewer #1:This manuscript by Yasuda et al. have employed single molecule imaging of mRNA reporters to uncover a novel and very interesting mechanism for translation regulation of protrusion-localized mRNAs in cultured fibroblasts. In contrast to other reports that indicate transported mRNAs are translationally repressed during transport and unsilenced at the destination, here the data show that translational repression occurs after transport and involves the clustering of multiple mRNAs into heterogenous granules. The present experiments using fluorescent reporters is complemented by a biochemical approach to perturb mRNA localization which does not seem to alter translation. The strength of this paper is the use of SunTag reporters for live cell imaging of mRNA localization and translation, which does support a model for the formation of translationally repressed mRNPs within protrusions. While this finding does advance the state of knowledge for the field, there are concerns with some of data and resulting interpretations that suggest lack of translational repression during active mRNA transport. It does seem that new experiments could provide stronger evidence of mRNA silencing during active transport.Specific comments:Figure 1 shows that disruption of endogenous APC-dependent mRNA localization to protrusions using a competitive Pkp4 3'UTR reporter does not result in a shift of mRNA levels from polysome to mRNP fractions on sucrose gradients, in contrast to what was observed with puromycin treatment. The data need to be quantitated by real time PCR for several localized mRNAs. Specifically, the authors should report the actual values for% mRNA in each of the four fractions for several examples of localized mRNAs. While puromycin treatment resulted in marked mRNA shifts from heavy to light fractions, the competitive 3'UTR does appear to accumulate mRNA in an intermediate fraction #3. Another interpretation is that these are stalled ribosomes. This possibility has not been considered or discussed.Figure 2 – the same comment as stated above also relates to use of pharmacologic perturbation of detyrosinated microtubules.Figure 4 shows data obtained with the Sun-Tag reporter implying that the majority of single mRNAs have comparable translation efficiencies regardless of localization. The authors conclude that "RNAs targeted to protrusions are not silenced during transport." One major concern with this strong interpretation is that the quantitative analysis was not done on "directed" particles but rather on the majority of particles that were stationary or oscillatory. A qualitative example shown does suggest translation can occur during transport. Can an experiment be done to stimulate mRNA transport and increase the% of directed RNAs, for example, serum starvations followed by stimulation as done by Singer and colleagues for b-actin? Quantitative analysis of SunTag reporter dynamics can also be done following depolymerization of detyrosinated MTs. Without evidence on quantitation of mRNAs that are actively being transported, the authors will need to soften their conclusions. Such RNAs that are not moving and not localized may be translated, but this does not imply a similar fate for those that are actively being transported to the cell protrusion.In the Discussion, the authors should not generalize their findings in fibroblasts to all polarized cells, but rather restrict their interpretation to cells that localize mRNAs as single mRNPs. In neurons, for example, some types of mRNA granules contain multiple mRNAs which do appear to be silenced during transport. These different models should be compared.Reviewer #2:Many localized mRNAs are translationally silenced during transport and become activated only upon reaching their cellular destination. In their manuscript, Yasuda et al. present evidence that a specific set of mRNAs, previously characterized as APC-dependent and associated with detyrosinated microtubules, is translationally active both during transport and at the cellular periphery. However, once at the periphery, a subset of APC-dependent RNAs coalesce into translationally inactive clusters which the authors speculate are needed for dynamic regulation during cell migration. The authors use translation reporters containing the SunTag peptide array and PP7 hairpins coupled with live cell imaging to reach the conclusion that the 3′ UTRs of several APC-dependent RNAs confer translation during transport and silencing in large clusters near the periphery. The clustering effect is also seen by FISH against endogenous APC-dependent RNAs. This paper provides an interesting example that runs counter to the dominant paradigm of translationally silent mRNA transport. However, some figures seem incomplete, for example lacking comparison to the control reporter. The discussion could also be made much stronger by further explaining the authors' hypothesis about the function of the peripheral RNA clusters. Are the clusters an intermediate in an RNA degradation pathway or are they RNA storage granules with translationally-competent RNAs that can be dispersed in a regulated fashion? These points are discussed in more detail below.Major points:1) Figure 5 presents one of the most interesting findings of the paper – that APC-dependent RNAs form clusters at protrusions. However, the presentation does not come across as rigorous and should be improved by: 1) quantification of cluster sizes and statement of number of cells analyzed and 2) comparison to the control reporter in the same figure. It seems that Figure S4 makes this argument in a more rigorous way. Perhaps part of Figure S4 could be moved to Figure 5 and part of Figure 5 could be moved to S4 . It is additionally confusing that Figure S4 suggests that most RNAs are not in clusters but Figure 5 shows examples where almost no non-clustered RNAs can be seen. It seems like the images in Figure 5 must be the more exceptional examples. Therefore, some statement or quantification of the frequency of clusters from the live cell imaging is needed.2) Similar to the previous point, Figure 6 should state how many cells were analyzed and include the control reporter as a comparison.3) It would be interesting to see more analysis of polyA RNA clusters vs. APC-dependent RNA clusters. It is shown in one example for Pkp4 RNA in Figure 5E, but not quantified and not shown for other RNAs. I believe the authors' hypothesis is that the RNA clusters are translationally repressed, but still translationally competent (i.e. not degradation products). If the authors could show that most APC-dependent RNA clusters are polyA+, this finding would support that hypothesis.4) Similar to point 4, the discussion does not explain the authors' hypothesis about the function of the RNA clusters clearly and why they are translationally silenced. There is only one sentence suggesting that they reflect 'a local regulatory mechanism used to silence APC-dependent RNAs'. It would be good to elaborate on this point and maybe offer a brief comparison to other types of translationally silent RNA granules. The following significant concerns must be addressed in a revised manuscript, should you wish to resubmit:The reviewers agreed that the authors make generalizations that are not supported by the data. For instance, that they see mRNAs being translated while moving, these appear to be rare cases, and do not apply to all the mRNAs without necessary statistical analysis. The non-moving mRNAs may be more likely to translate, but there is a lack of quantification to evaluate this possibility. .while engaged in active transport, but rather that they can be translated similarly in both internal and peripheral locations. We have changed the text throughout the manuscript to clarify this. We have also specifically pointed out that the translation reporter imaging assay does not allow us to make accurate conclusions about the behavior of fast-moving molecules  and have referenced the papers mentioned above. Furthermore, we have now used puro-PLA to detect in situ nascent Rab13 protein distribution  . This approach, apart from looking at the translation of an endogenous RNA, also circumvents the problem of fast-moving molecules by allowing us to look at translation in situ, in fixed samples. The results are fully consistent and support the conclusion that protrusion-localized RNAs can be translated in both peripheral and internal locations.We completely agree with these comments and realized that some of the conclusions in the original manuscript were awkwardly stated. Our intent was not to conclude that RNAs are being translated Second, they do not consider alternative interpretations, for example that the translation is stalled until the mRNAs reach their destination, and the proteins are released there.To address this possibility, we have performed run-off experiments, by brief treatment (15min) with harringtonine or lactimidomycin, and now show that the detected translation signals largely reflect active translation. Specifically, the new Figure 3—figure supplement 1  shows that the signal detected by the translation imaging reporters is reduced almost to background levels upon harringtonine (or lactimidomycin), but not upon cycloheximide treatment, showing that it reflects active translation. Additionally, detection of Rab13-puro PLA signal is reduced to background level upon harringtonine treatment (compare harringtonine with anisomycin treatment)  showing that endogenous Rab13 is also mostly translated by actively scanning ribosomes  . We have also seen that RNAs fractionating in polysomal fractions of sucrose gradients are shifted towards the non-translating fractions of the gradient upon harringtonine treatment. We have seen this result for a few RNAs we have detected by RT-PCR in human breast cancer cells . These results are shown in Unfortunately, due to technical issues we have been unable to perform this experiment in mouse fibroblasts with the full panel of RNAs analyzed in Figures 1 and 2. However, given the effect of harringtonine seen in all other assays, we believe that even without this experiment we provide strong support for the conclusion that stalled ribosomes are not a significant part of the described mechanism.More quantitation is needed before conclusions can be drawn as to polysome loading, for instance using qPCR.We believe that we might have not clearly conveyed that the heatmaps shown in Figures 1 and 2 are indeed averages of independent experiments. The associated p-values were meant to reflect whether the underlying distributions were different or not. We have now supplied, as source data, files with the exact values of all replicates used for the generation of the heatmaps and statistics in those figures.With regards to the use of qPCR, we have opted to quantify RNA abundance using nanoString analysis instead of RT-qPCR because it allows for direct RNA counting and thus avoids biases introduced by reverse transcription and amplification. This is now stated in subsection “Disrupting the localization of APC-dependent RNAs at protrusions does not affect their translation”. Indeed, when we detect the spike RNA  we find that it is much more consistently and uniformly detected by nanoString analysis. RT-PCR detection shows much larger variations in the detected spike (even though the same amount was added). This underscores the variability and biases that can result from reverse transcription and amplification, which we think are exaggerated in samples with very different RNA complexities, such as those isolated from sucrose gradients.There is a noticeable lack of free protein, this seems to be a conundrum, along with the observation that translation is silenced exactly where it is needed. Discussion of the significance of this counter-intuitive observation is lacking.With regards to the lack of free protein, with think that this is probably a perception caused by the contrast of some images, which makes the free protein not so evident. However, this is not a real issue with the assay. Even though we specifically induce the expression of the reporters for only a short period of time (2-4hrs), precisely to avoid accumulation of both the RNA and protein to high levels, we nevertheless always detect a significant amount of free protein, seen as fast-moving GFP particles that are brighter compared to the diffuse background of the single chain GFP-labeled antibody. We think this is evident, for example, in Figures 3, figure 3—figure supplement 1 and Figure 4.With regards to the conundrum of silencing translation at the periphery, we had tried to argue before that this reflects regulation of translation during dynamic processes, however we fully understand that more and better evidence was needed. Therefore, we have now provided substantial new evidence that we believe convincingly shows that silencing occurs at retracting protrusions. Thus, the resolution of the conundrum that we propose is that RNAs are translated in extending protrusions/lamellipodia and are silenced in retracting protrusions. These data are based on the use of puro-PLA to detect nascent Rab13 protein in situ and are detailed in the new Figures 7 and 8 .Finally, no consideration is given to the nature of the cells analyzed, and whether these results are an artifact of the cells or reporter chosen, The cells used, 3T3, are not good examples of cells with protrusive activity, or of polarized cells in general where localization is essential for motility …We have now provided data in primary human dermal fibroblasts as well as in breast cancer cells (MDA-MB-231). We note that for the group of APC-dependent RNAs we are studying, we see robust localization in all cell types we have tested . We specifically show that in all cell types tested we see very similar behaviors in terms of the localization of the studied RNAs at protrusions; their translation in both internal and peripheral regions; their silencing at the periphery; and their coalescence in heterogeneous granules . We therefore believe that we provide strong evidence that the reported mechanism is not an artifact of the cell line used but is generally exhibited by mesenchymal migrating cells.3T3s are indeed different than the other two cell types used, in that they tend to form more contractile stable protrusions whose dynamics are much slower . As we briefly discuss (Discussion paragraph seven), we believe these slower dynamics might be the reason for the more pronounced formation of peripheral granules in 3T3 cells compared to the granules formed in the much more dynamic MDA-MB-231s. We believe that all cell types can provide valid information and that each one, through accentuating certain behaviors, can offer a valuable model to facilitate the study of different aspects of these structures.… and the reporter is not an endogenous gene with an endogenous promoter.The point of how much any reporter reflects endogenous regulation is of course very valid. Even if a reporter recapitulates certain behaviors of endogenous RNAs , there is no guarantee that other aspects are also accurately reflecting the endogenous regulation. We find however, that it would be very difficult to ever provide unequivocal evidence that an exogenous reporter accurately reflects endogenous regulation, unless the regulation of the endogenous RNAs is first known. Therefore, instead of testing the effect of adding further elements  to our translation reporters, or of engineering aptamers into the endogenous genes, we rather chose to directly assess in situ the translation of an endogenous APC-dependent RNA. For this we used the puro-PLA method (puromycylation followed by proximity ligation amplification), which allows visualization of nascent proteins and translation sites of specific endogenous transcripts. In the new Figures 7-8 we provide controls for the validation of the assay in different cell types and use it to corroborate the findings obtained with the translation imaging reporters, as well as to extend them, showing that silencing occurs at retracting protrusions.We are aware that some of the conclusions we have reached with these experiments could be further extended by live imaging of translation reporters in the more dynamic MDA-MB-231 cells. While these are certainly very interesting experiments that we intend to pursue in the future, we hope that you appreciate that generation of such stable lines, appropriate for high quality imaging, are a lengthy endeavor. We believe that the current data fully support the conclusions of the manuscript and hope you will find that the information contained in it is warranting publication at this stage.They should test their system on primary cells to see if their observations hold there.As mentioned above we have provided data in both primary cells and transformed cancer cells that are fully consistent with our previous conclusions.So while the observations are potentially interesting they must be tempered by these specific constraints. The Discussion either needs to be softened, or the authors must provide additional data to address the generality of their observations. Finally, what conclusions the authors do want to draw on their specific system needs to be backed up by quantitation.We have added quantitations that we believe support all the claims made.Given the new data added, we have modified sections of the Abstract, Results and Discussion to reflect the new information included in the revised version. Please see manuscript text with tracked changes for exact modifications.Reviewer #1:[…] The strength of this paper is the use of SunTag reporters for live cell imaging of mRNA localization and translation, which does support a model for the formation of translationally repressed mRNPs within protrusions. While this finding does advance the state of knowledge for the field, there are concerns with some of data and resulting interpretations that suggest lack of translational repression during active mRNA transport. It does seem that new experiments could provide stronger evidence of mRNA silencing during active transport.Specific comments:Figure 1 shows that disruption of endogenous APC-dependent mRNA localization to protrusions using a competitive Pkp4 3'UTR reporter does not result in a shift of mRNA levels from polysome to mRNP fractions on sucrose gradients, in contrast to what was observed with puromycin treatment. The data need to be quantitated by real time PCR for several localized mRNAs. Specifically, the authors should report the actual values for% mRNA in each of the four fractions for several examples of localized mRNAs. While puromycin treatment resulted in marked mRNA shifts from heavy to light fractions, the competitive 3'UTR does appear to accumulate mRNA in an intermediate fraction #3. Another interpretation is that these are stalled ribosomes. This possibility has not been considered or discussed.We believe that we might have not clearly conveyed that the heatmaps shown in Figures 1 and 2 are indeed averages of independent experiments expressing the% mRNA in each fraction. The associated p-values were meant to reflect whether the underlying distributions were different or not. We have now supplied, as source data, files with the exact values of all replicates used for the generation of the heatmaps in those figures.We have detailed in point 3 our rationale for using nanoString instead of RT-qPCR for quantitation of RNA levels.We have performed ribosome run-off experiments with harringtonine to show that stalled ribosomes are not a significant part of the described mechanism. Please see response to point 2 for details.Figure 2 – the same comment as stated above also relates to use of pharmacologic perturbation of detyrosinated microtubules.The heatmaps in this figure are also showing the averages of independent experiments expressing the% mRNA in each fraction. We have now supplied the source data with the exact values of all replicates used for the generation of the heatmaps in this figure.Figure 4 shows data obtained with the Sun-Tag reporter implying that the majority of single mRNAs have comparable translation efficiencies regardless of localization. The authors conclude that "RNAs targeted to protrusions are not silenced during transport." One major concern with this strong interpretation is that the quantitative analysis was not done on "directed" particles but rather on the majority of particles that were stationary or oscillatory. A qualitative example shown does suggest translation can occur during transport. Can an experiment be done to stimulate mRNA transport and increase the% of directed RNAs, for example, serum starvations followed by stimulation as done by Singer and colleagues for b-actin? Quantitative analysis of SunTag reporter dynamics can also be done following depolymerization of detyrosinated MTs. Without evidence on quantitation of mRNAs that are actively being transported, the authors will need to soften their conclusions. Such RNAs that are not moving and not localized may be translated, but this does not imply a similar fate for those that are actively being transported to the cell protrusion.We agree with this point and acknowledge that our conclusions were awkwardly stated. Please see response to point 1 for details.In the Discussion, the authors should not generalize their findings in fibroblasts to all polarized cells, but rather restrict their interpretation to cells that localize mRNAs as single mRNPs. In neurons, for example, some types of mRNA granules contain multiple mRNAs which do appear to be silenced during transport. These different models should be compared.It would indeed be interesting to explore how these RNAs are regulated in neuronal cells. We have added in the Discussion a specific mention to that effect and a statement emphasizing that our findings are describing behaviors of mesenchymal migrating cells (Discussion penultimate paragraph).Reviewer #2:[…] The clustering effect is also seen by FISH against endogenous APC-dependent RNAs. This paper provides an interesting example that runs counter to the dominant paradigm of translationally silent mRNA transport. However, some figures seem incomplete, for example lacking comparison to the control reporter. The discussion could also be made much stronger by further explaining the authors' hypothesis about the function of the peripheral RNA clusters. Are the clusters an intermediate in an RNA degradation pathway or are they RNA storage granules with translationally-competent RNAs that can be dispersed in a regulated fashion? These points are discussed in more detail below.Major points:1) Figure 5 presents one of the most interesting findings of the paper – that APC-dependent RNAs form clusters at protrusions. However, the presentation does not come across as rigorous and should be improved by: 1) quantification of cluster sizes and statement of number of cells analyzed and 2) comparison to the control reporter in the same figure. It seems that Figure S4 makes this argument in a more rigorous way. Perhaps part of Figure S4 could be moved to Figure 5 and part of Figure 5 could be moved to S4 . It is additionally confusing that Figure S4 suggests that most RNAs are not in clusters but Figure 5 shows examples where almost no non-clustered RNAs can be seen. It seems like the images in Figure 5 must be the more exceptional examples. Therefore, some statement or quantification of the frequency of clusters from the live cell imaging is needed.The confusion about the prevalence of clusters probably stems from the fact that Figure 5 showed enlargements of protrusive regions only, while Figure S4 showed whole-cell images and the associated quantifications. We have now noted this more obviously in both the text, figure legends and the figures themselvesWe have also reorganized the data of Figure 5 and Figure S4  and added new panels in order to present quantitations and statistical comparisons in the main figure  (subsection “APC-dependent RNAs associate with heterogeneous clusters at the tips of protrusions”). We have tried throughout to mention sample sizes used for analyses.2) Similar to the previous point, Figure 6 should state how many cells were analyzed and include the control reporter as a comparison.We have included in the legend the number of cells imaged. We had not included the control reporter because we don’t see any clusters. In any case, the point of this figure is not to provide a quantitative assessment of cluster formation between control and localized reporters, but to compare the translation status of RNAs within clusters to single RNAs within the same protrusions.3) It would be interesting to see more analysis of polyA RNA clusters vs. APC-dependent RNA clusters. It is shown in one example for Pkp4 RNA in Figure 5E, but not quantified and not shown for other RNAs. I believe the authors' hypothesis is that the RNA clusters are translationally repressed, but still translationally competent (i.e. not degradation products). If the authors could show that most APC-dependent RNA clusters are polyA+, this finding would support that hypothesis.We have added further quantitation regarding the coincidence of polyA signal with APC-dependent RNA clusters  to support the use of polyA staining as an identifier of peripheral clusters in 3T3 cells. Indeed, the majority of APC-dependent RNA clusters are polyA+, and polyA staining can even reveal clusters that don’t contain the particular APC-dependent RNA being imaged.While it is of interest, we cannot currently make any assessment regarding the fate of RNA within granules. As mentioned in point 6, we are aware that information relevant to these questions could be provided by live imaging of translation reporters in the more dynamic MDA-MB-231 cells. While these are certainly very interesting experiments that we intend to pursue in the future, we hope that you appreciate that they are too involved to pursue in the context of the current manuscript. For now, we have added a mention to these issues in the Discussion (paragraph seven).4) Similar to point 4, the discussion does not explain the authors' hypothesis about the function of the RNA clusters clearly and why they are translationally silenced. There is only one sentence suggesting that they reflect 'a local regulatory mechanism used to silence APC-dependent RNAs'. It would be good to elaborate on this point and maybe offer a brief comparison to other types of translationally silent RNA granules.This point is now addressed more deeply with the addition of new data showing that silencing occurs at retracting protrusions .


GUCA1A) is associated with a dominant retinal dystrophy?Is there evidence that the Pro50Leu substitution in guanylate cyclase activating protein-1  variant in  As genetic and genomic screening is becoming more widely accessed, correctly distinguishing pathogenic from nonpathogenic variants is of increasing relevance.GUCA1A was associated with a dominant retinal dystrophy, in light of new examination findings in the proband’s daughter.To reevaluate a previously reported family in whom the p.(Pro50Leu) variant in the gene A genetic study relating to a family with an inherited retinal dystrophy was performed at the retinal genetics service of Moorfields Eye Hospital from October 27, 2009, to May 23, 2019, after the proband’s daughter underwent fundus examination.RPGR gene.Results of sequencing of X chromosome–linked retinitis pigmentosa genes in the proband and specific analysis of the repetitive ORF15 region of the RPGR , which may be associated with the loss of 121 amino acid residues at the carboxyl terminus of the protein. The p.(Pro50Leu) variant in GUCA1A was also found to be too common in a publicly available genome database to be a fully penetrant cause of a dominant retinal dystrophy.A frame-shifting single-nucleotide deletion was found in the ORF15 exon of RPGR. The findings suggest that the p.(Pro50Leu) variant in GUCA1A should not be regarded as pathogenic. This report also highlights the relevance of examining relatives, of reevaluating diagnoses in light of new data, and of considering X chromosome–linked inheritance in apparently autosomal dominant pedigrees unless there is clear male-to-male transmission.The phenotype in the family is now associated with the variant in  GUCA1A was associated with a dominant retinal dystrophy, in light of new examination findings in the proband’s daughter.This cross-sectional study reevaluates a previously reported family in whom the p.(Pro50Leu) variant in the gene  GUCA1A (OMIM *600364) encodes the protein guanylate cyclase activating protein-1, a calcium-sensitive protein integral to maintaining cyclic guanosine monophosphate levels in the outer segment of photoreceptors. Variants in this gene may be associated with a dominantly inherited cone dystrophy, largely by affecting calcium sensitivity.4 In 2001, 3 families with variants in GUCA1A were reported in this journal.2 Two families had the p.(Tyr99Cys) variant, with a relatively consistent phenotype. The third family had the p.(Pro50Leu) variant and exhibited a more variable phenotype . Since that publication, this family has undergone more detailed investigation, the results of which are described in the present report; we now associate their phenotype with a disease-causing variant in RPGR  and not GUCA1A. Two of us (S.M.D. and A.B.) were also contributing authors of the original report.2The gene 5 Participants provided written informed consent.This study was performed from October 27, 2009, to May 23, 2019, after the original evaluation of the family, because the proband’s daughter underwent a fundus examination (see Results). A fundal examination was performed at the slitlamp after mydriasis, and color fundus images and red-free images were taken from both eyes. The study was approved by Moorfields Eye Hospital and Northwest London Research Ethics Committee, and conformed to the tenets of the Declaration of Helsinki.RPGR and RP2 (OMIM *300757) genes (Manchester Genomic Diagnostics Laboratory). The publicly available gnomAD data set  was used to estimate the prevalence of the p.(Pro50Leu) variant in GUCA1A.Sanger sequencing of DNA from the proband was performed to screen for the 2). The proband exhibited a cone dystrophy, whereas his relative (IV 3) had features consistent with RP. One of the proband’s daughters was examined subsequently in our service and found to have high myopia and a tapetal reflex (RPGR and RP2 (2 X chromosome–linked genes in which variants can give rise to RP) using Sanger sequencing were negative. A subsequent reanalysis of the original sequencing data of the ORF15 exon of RPGR demonstrated a frame-shifting single-nucleotide deletion ), which may be associated with the loss of 121 amino acid residues at the carboxyl terminus of the protein. This variant has been previously reported in association with X chromosome–linked retinal disease.6 Interrogation of the gnomAD data set revealed an allele frequency for the p.(Pro50Leu) variant in GUCA1A of 0.12% , reaching a frequency in Northwest European alleles of 0.22% .Results of initial screening of RPGR and not the p.(Pro50Leu) variant in GUCA1A. The latter is too common to be a pathogenic, fully penetrant, dominantly acting variant. In addition, reported pathogenic changes in the gene are clustered in regions affecting calcium sensitivity3; this substitution occurs in an earlier part of the protein, and biochemical studies have shown that the p.(Pro50Leu) variant is not associated with calcium sensitivity .7 Pathogenic variants in GUCA1A also are not associated with the RP phenotype, which was exhibited by an affected relative of the proband. Taken together with our updated findings of a variant in RPGR in the reported family, we conclude that there is little evidence for the pathogenicity of the GUCA1A variant.Based on our findings, we now believe that the phenotype in this family is due to the pathogenic variant in RPGR is the gene most frequently associated with X chromosome–linked retinal dystrophy. Male patients with pathogenic variants in this gene usually have RP , but affected individuals can display a cone or cone-rod dystrophy.8 Female carriers can show a tapetal reflex or disease phenotypes of varying severity, including a phenotype as severe as that seen in affected males.9 Screening of the ORF15 exon, where many pathogenic variants lie,6 can be challenging owing to a repetitive nucleotide sequence; hence, variants can be missed, even in the context of whole-genome sequencing. Variants in this exon also are more often associated with a cone or cone-rod dystrophy phenotype (compared with changes in other parts of the gene) and have been shown occasionally to be associated with both cone dystrophy and RP phenotypes in the same family,8 as in this pedigree. Discordant phenotypes have been reported in fraternal twins with the same pathogenic variant as that found in the family we studied.1011 Such reports occasionally are false positives, as is the case here, and its correction is therefore necessary.As genetic and genomic testing become more common, distinguishing nonpathogenic and pathogenic variants is increasingly relevant. One key piece of evidence is a report of the variant cosegregating with the disorder in question.RPGR variant in the family, as other family members were no longer able to be contacted. However, as noted above, the variant has been associated with both cone-rod dystrophy and RP phenotypes, and so would be consistent with the phenotype previously reported in affected family members.A limitation of the study was that we were unable to fully check segregation of the RPGR-related disease likely is of increasing relevance because this is now the subject of gene replacement trials.12Our report also highlights a number of points of wider relevance. Investigators should be wary of prior reports of pathogenic variants in publicly available databases if they are based on 1 family, especially when testing was performed with earlier methods. Second, it is advisable, when feasible, for genetic investigators to reevaluate their published reports on families and prior diagnoses in light of new findings, such as a high prevalence in the gnomAD data set, or new examination findings of family members. Third, families with X chromosome–linked disease can be misinterpreted as having autosomal dominant inheritance owing to female family members being affected; X chromosome–linked inheritance should be considered in all families for which multiple generations of family members are affected unless there is clear male-to-male transmission. Correctly identifying