Research Proposal Manos Emmanouil July 2017 I. INTRODUCTION Too many individual sentences and/or unnecessarily short paragraphs. Need to combine individual sentences into topical paragraphs. Example paragraph:First sentence = topic of paragraph. This is followed by details. You can conclude the paragraph using a sentence that provides a summary or conclusion of the paragraph.
Hypophosphatasia (HPP) is a rare, heritable form of rickets or osteomalacia that occurs due to a loss-of-function mutation in the gene (Alpl) that encodes tissue non-specific alkaline phosphatase (TNAP) (1). TNAP hydrolyzes pyrophosphate (PPi), to inorganic phosphate (Pi), which is essential for the growth of hydroxyapatite crystals (the mineral content of bone) (2-4). HPP directly disrupts the mineralization of the skeleton and dentition, with clinical consequences that include demineralized bones that are prone to bending and breakage, demineralized teeth that are prone to breakage and wear, seizures, and death.
The incidence of HPP is estimated to be between 1:6,370 and 1:300,000 live births depending on severity, with the most severe form being the least common (5). HPP presents clinically in varying severity, ages of onset, and involved tissues (6). 7 main clinical forms of HPP have been identified, depending on the age at diagnosis and severity of the clinical symptoms: perinatal, infantile, childhood, adult, with the rarer forms being pseudohypophosphatasia, odontohypophosphatasia, and benign prenatal hypophosphatasia.
The prognosis for HPP is a reflection oreflectsf the severity of the skeletal disease, which usually corresponds to the age at presentation. Severity is usually highest in individuals who are symptomatic at a very young age (7). Previous studies have shown that the delivery of a bone-targeted form of TNAP enzyme (asfotase-alpha) prevents the dental and skeletal manifestations of HPP in the genetically engineered mouse model of the disorder (Alpl-/- mice).
Clinical trials in juvenile HPP patients confirmed the success of this treatment for the correction of bone and tooth abnormalities in humans. Despite significant improvements in survival and quality of life after treatment with asfotase-alpha starting several months to years after birth, young hypophosphatasia patients still exhibit a high incidence of craniosynostosis (8).Craniosynostosis is a craniofacial anomaly characterized by the premature fusion of one or more of the cranial sutures, the fibrous joints that divide the cranial bones of the skull. Incidence of craniosynostosis is estimated to be 1 in 2500 live births (9). Craniosynostosis can occur in isolation or as part of a syndrome, including Apert, Saethre-Chotzen, Jackson-Weiss, Crouzon, Pfeiffer, Antley-Bixler and Carpenter syndromes (9). The infantile form of HPP is also associated with craniosynostosis, as the result of a biological process that is currently being further investigated (10,11).Development of the skull vault depends on interactions at the boundary between cranial neural crest cell-derived elements (frontal bones) and mesoderm-derived tissue (parietal bones). The skull vault and base comprise the neurocranium, which consists of bones that surround and protect the brain, including the temporal, parietal, frontal, sphenoid, ethmoid, and occipital bones.
The rest of the skull is formed by the viscerocranium, which includes the jaws and other pharyngeal derivatives (including nasal, lacrimal, palatine bones, mandible, maxilla, and zygoma). The viscerocranium is neural crest-derived and functions to form the face, and support feeding and breathing actions (10,11).Normal development of the cranial bones is crucial for accommodating growth of the brain. Disruption of normal suture development, can lead to re-direction of overall calvarial growth to accommodate the growing brain. Left untreated, craniosynostosis can have various harmful effects including abnormalities in skull shape, increased intracranial pressure, hydrocephalus, impaired cerebral blood flow, deafness, blindness, swallowing dysfuction, nasopharyngeal airway obstruction, speech impairment, developmental delay, seizures and/or death (12,13). Currently the main treatment for craniosynostosis involves significant surgical intervention to correct the craniofacial shape anomalies and relieve intracranial pressure, therefore preventing craniosynostosis by intervention at the cellular level, could lead to a dramatic improvement in quality of life for HPP patients.Infantile HPP presents by six months after birth with seemingly normal development until the onset of failure to thrive, hypotonia, and clinical signs of rickets.
Craniosynostosis occurs in 40% of patients with infantile HPP (14). Results of craniosynostosis in infants with HPP include bulging of the anterior fontanel, raised intracranial pressure, mild hypertelorism, proptosis, and brachycephaly (15, 16).Alpl-/- mice mimic the long bone, tooth, and neural abnormalities seen specifically in infantile HPP (17).
These mice are born with normal calcified skeletons, but begin to display skeletal hypomineralization at post-natal days 6-10 that continues to worsen until they die prior to weaning around post-natal day 20 (17,18). Extracellular TNAP ultimately encourages tissue mineralization by increasing phosphate levels and decreasing pyrophosphate (19,20). Without the presence of TNAP, tissue mineralization is greatly diminished.
ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase-1) is a membrane-bound, nucleoside triphosphate pyrophosphohydrolase that generates pyrophosphate through the hydrolysis of nucleotides and nucleotide sugars. ENPP1 can inhibit ectopic soft tissue mineralization, and also functions to promote eutopic bone mineralization.Pyrophosphate is a well established inhibitor of hydroxyapatite crystal deposition and growth, yet pyrophosphate can also serve as an essential source of phosphate to enhance mineralization when it is hydrolyzed by TNAP, which is co-expressed with ENPP1 in matrix vesicles and plasma membranes of mineralizing cells. Additionally, while physiologic inorganic pyrophosphate levels inhibit soft tissue hydroxyapatite calcification, excessive pyrophosphate can promote the pathologic calcification of non-bony tissues in the form of calcium pyrophosphate dihydrate crystals (21). In this study, it is hypothesized that a potential mechanism by which craniosynostosis is caused in the mouse model of HPP, involves locally increased extracellular pyrophosphate (PPi) due to unabated ENPP1 activity.
Pi to PPi tissue concentration ratios control tissue mineralization and can also directly alter cell behavior (32). If high PPi levels seen in Alpl-/- mice, can become normalized in Alpl-/-/ENPP1-/- double mutant mice, simultaneous deletion of TNAP and ENPP1 would be an ideal approach for establishing the degree to which ENPP1-generated PPi mediates the influence of TNAP on craniosynostosis. Results from this study can provide additional information regarding molecular mechanisms controlling cranial bone fusion, which will can lead to the future development of various biomedical therapeutics to prevent and/or treat craniosynostosis. II. SPECIFIC AIMSAIM 1: Determine if craniofacial anomaliescraniosynostosis seen in an Alpl-/- mouse model can be rescued by the elimination of ENPP1 enzyme activity through the generation of an Alpl-/-, ENPP1 double knockout mouse model.AIM 2: Determine if diminished bone mineralization seen in an Alpl-/- mouse model can be rescued by the elimination of ENPP1 enzyme activity through the generation of an Alpl-/-, ENPP1 double knockout mouse model.IV.
BACKGROUND AND SIGNIFICANCECraniofacial anomalies represent one third of all congenital birth defects. They are usually part of complex disorders that require a multi-disciplinary treatment approach, including early and possibly several surgical interventions. This can have long term effects on the patients’ psychological, and socio-economic status.
The prevalence and severity of such developmental disorders, highlights the importance of research into the role and pathway of craniofacial development and underlying mechanisms leading to craniofacial anomalies. Better understanding of the regulatory gene mechanisms involved in this process, can have major future therapeutic implications, as it helps anticipate future disease phenotypes and can provide a helpful tool to promote directed differentiation (22). If this study shows that craniosynostosis is rescued in the HPP mouse model by deletion of ENPP1, further investigation will be required to determine if the influence of ENPP1 on craniosynostosis occurs via changes in progenitor cell proliferation, differentiation or tissue mineralization.
This also has the potential to lead to future development of ENPP1 inhibitors for diminishing craniosynostosis. Such a finding would have a major impact for HPP patients, as it can eventually help treat or even prevent craniosynostosis at the molecular level, and dramatically improve the quality of life for these individuals.If deletion of ENPP1 does not diminish craniosynostosis, this is still an important finding and would indicate that ENPP1 and PPi levels are not central to the mechanism by which TNAP deficiency causes craniosynostosis.
Understanding whether controlling pyrophosphate levels has an effect on rescuing craniosynostosis or not, can provide important direction for future studies in order to identify the mechanism by which ENPP1 and/or PPi modulate craniofacial development and craniosynostosis.V. RESEARCH DESIGN AND METHODSAnimalsTo pursue this aim, PPi levels in global Alpl-/- mice will be decreased through genetic deletion of the PPi generating enzyme, ENPP1. Alpl-/- mice will be maintained on chow fortified with Vitamin B6 to avoid seizures due to defective pyridoxal-5′-phosphate metabolism. On this diet, the median age for survival of global Alpl-/- mice is approximately three weeks after birth. Coronal suture fusion is present in Alpl-/- mice at this age. Mice will be sacrificed at postnatal day 15.The efficacy of ENPP1 ablation on craniosynostosis in Alpl-/- mice can be evaluated on congenic 129Sv backgrounds.
This population will represent the varying severity of the phenotype of HPP. Four groups of eight (n=8) mice on a 129Sv genetic background:Wild typeAlpl-/- ENPP1-/- Alpl -/-/Enpp1-/- double-KOThe genotype of the animals will be confirmed by PCR using tail DNA obtained at the time of tissue collection as previously described (23).All animal procedures will be conducted according to the University of Michigan’s University Committee on Use and Care of Animals and federal guidelines.MicroCTWhole dissected skulls will be dissected and fixed in 100% ethanol overnight. Specimens will be scanned over the entire length of the skulls at the University of Michigan’s Orthopedic Research MicroCT Core. Scanning will occur on an eXplore Locus SP MicroCT system (?CT100 Scanco Medical, Bassersdorf, Switzerland). Scan settings will be as follows: voxel size 18 ?m, medium resolution, 70 kVp, 114 ?A, .5mm AL filter, and integration time 500 milliseconds.
Measurements will be taken at an operating voltage of 80 kV and 80 mA of current, with an exposure time of 1600 milliseconds. Scans will be calibrated to the manufacturer’s hydroxyapatite phantom. Craniosynostosis analysisFusion of coronal, lambdoid and sagittal sutures will initially be assessed using the micro-CT scans of mice dissected calvaria. Patency or fusion of craniofacial bones will be established by walking through serial scan layers perpendicular to the suture in question. Serial viewing of individual slices in the axial, sagittal, and coronal planes throughout the entire length of the suture in question can be accomplished using Microview Version 2.
2 (GE Healthcare PreClinical Imaging, London, ON). Sutures will be viewed using the 2D micro CT slices in orthogonal views across the entire length of the suture in question, in a manner similar to that previously described (24,25).Bone analysisCranial bone volume, density and mineral content will be measured using Microview software version 2.2 (GE Healthcare PreClinical Imaging, London, ON). Advanced regions of interest (ROIs) for parietal and frontal bones will be established as 1 mm in length, 1 mm in width, and 1 mm in depth equivalent to thickness of bone and position starting at a 0.5 mm distance from coronal, lambdoid and sagittal sutures.
Total volume (TV), bone volume (BV), bone mineral content (BMC), bone mineral density (BMD), tissue mineral content (TMC), tissue mineral density (TMD), and bone volume fraction (BV/TV) will be calculated using established algorithms.Serum AnalysisBlood will be collected before sacrifice by aortic puncture under surgical anesthesia to obtain measurements of Pi, Ca, FGF23, AP enzyme activity. Digital Caliper measurementsLinear digital caliper measurements will be made using previously established landmarks (23,24). Euclidian Distance Matrix Analysis (EMDA) will be used to quantify the form, size, and shape difference between samples. EDMA is a morphometric analysis created by Richstmeier and Lele, 2001 (26) that uses landmark coordinate data without using a fixed coordinate axis. The analysis calculates all the linear distances between all possible pairs of landmarks in each individual and compares these distances as ratios between groups. This method has been previously used in human (27-29) and non-human (24,25,30,31) studies and is currently one of the major methods for morphometric comparison across many biological disciplines.
Skull morphology analysisA principal component analysis (PCA) will be conducted to summarize all measurements by the most contributing variables, named principal components (PCs), and to identify differences across phenotypes in PCs. Rather than provide a p-value stating specific linear measurement or overall form/shape differences, the purpose of the PCA is to summarize multiple variables into multiple summary indexes and help visually display the differences among groups presented in those variables. PCs are calculated from all digital caliper measurements, both normalized and non-normalized, are created as a linear combination of measures, are independent of each other, and summarize information from original variables as much as possible, measured by the reduction in variance, or eigen value.
In other words, PCA tells us whether there are differences among all measures across phenotypes. The differences across phenotypes can be seen by differences in each measure (student’s T-test and mixed model comparison), but these differences more likely lie in the combination of a few measures. PCA summarizes measurements into two primary PCs. Clear visualization of the different phenotype groups identified by all analyzed measures can be obtained by plotting all subjects on the coordinate plane span of those two PCs.
The quality of the PCs is determined by the overall consistency among all measures and is represented by the Kaiser-Meyer-Olkin (KMO) value. If overall the measures are not consistent, the meaning of the summarized PCs is questionable. If the KMO value is less than 0.50 the analysis cannot be trusted, a KMO value of 0.50-0.
79 is considered questionable, a KMO value greater than 0.80 indicates a good description or fit, and a KMO of greater than 0.90 indicates an excellent description of the difference among all measures across phenotypes.Statistical analysisVarious statistical approaches can be taken to quantify the differences in measurements across all phenotypes. Initially, the differences can be estimated and evaluated separately for each measurement by a student’s T test.
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APPENDIX IIVII. TIMELINEJune-August 2018Literature review and development of research proposalEstablish a committee Submit final research proposal by end of AugustSeptember-December 2018Submit for fundingHold first committee meetingLearn and practice digital caliper measurements of mice skulls Practice microCT analysis technique Thesis proposal presentationJanuary-May 2019Meet with CSCAR to discuss appropriate statistical analysis of future results.Have methods for project finalized and perfectedCommittee meeting for updates and progressJune-August 2019Complete data acquisition Begin analysis of micro CT mice skullsCommittee meeting for updates and progressBegin writing Introduction and Methods section of written thesisSeptember-December 2019 Project presentation to orthodontic residents and facultyReview data, organize and analyze resultsPerform statistical analysis (meet with CSCAR)Committee meeting for updates and progressHave Introduction and Methods section of written thesis completedJanuary-May 2020Committee meeting for updates and progressFinishing writing and defend thesisAPPENDIX IIIVIII. BUDGET:ITEM ESTIMATED COST MicroCT Analysis $78/scan x 32 (8 mice per treatment group) $2,496Lab Materials/ Supplies ?TOTAL: ?