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Infant with craniosynostosis CRANIOSYNOSTOSIS

• Premature fusion of cranial bones ANALYSIS OF CRANIOFACIAL • 1/2500 live births MORPHOLOGY IN A TNAP KNOCKOUT • May involve one or multiple sutures • Isolated (85%) or Syndromic (15%) MOUSE MODEL • Biologic process unknown

• Elevated intracranial pressure John DuRussel MS, DDS, MS • Impaired cerebral blood flow

• Airway obstruction

• Deafness, blindness, seizures • Developmental delay and learning disabilities • Esthetic compromises

CURRENT TREAMENT: CRANIAL VAULT REMODELING SURGERY HYPOPHOSPHATASIA (HPP) • Deficiency of Tissue Non-specific Alkaline Phosphatase (TNAP) • Disrupted mineralization of the skeleton and dentition (weak bones and teeth) • Seizures due to poor Vitamin B metabolism • Craniosynostosis in the context of low bone density (40% of patients) • Death (respiratory dysfunction due to weak rib bones)

• Excise prematurely fused sutures and correct calvarial deformities • Initial surgery at 3-6 months old to allow for exponential brain growth

ENPP1 AND TNAP CONTROL MINERALIZATION ENPP1 AND TNAP CONTROL MINERALIZATION

• Ectonucleotide pyrophosphatase/phosphodiesterase-1 • Tissue non-specific alkaline phosphatase (TNAP)

(Enpp1) - the primary osteoblastic generator of hydrolyzes PPi to Pi which is essential to the growth of pyrophosphate (PPi) hydroxyapatite crystals.

ATP Enpp1 PPi ATP Enpp1 PPi TNAP Pi Matrix Matrix

Osteoblast Osteoblast

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Qualitative Craniofacial Phenotype of the ENPP1 AND TNAP CONTROL MINERALIZATION TNAP-/- Mouse Model of Infantile Hypoposphatasia

Deposition of calcium -/- WT TNAP WT WT pyrophosphate ? dihydrate crystals

Low bone density

KO KO

ATP Enpp1 PPi TNAP Pi Matrix • shape abnormalities • hypomineralization • craniosynostosis Open cranial suture in P20 wild type mouse * Fused/closed cranial suture in TNAP KO mouse Osteoblast

2D Shape Abnormalities TNAP Enzyme Replacement Therapy in TNAP-/- Mouse Model of Infantile Hypoposphatasia

Current regimen Revised earlier 2D linear craniofacial measurements (digital calipers) of TNAP enzyme regimen Hypophosphatasia replacement therapy of TNAP therapy

survival Age = P15 strong bones KO strong teeth • brachycephalic normal skull • acrocephalic • dome shaped

Age = P20

Surgeries to alleviate craniosynostosis

WT = white KO = black Whyte et. al., 2012

SPECIFIC AIMS HYPOTHESES 1. Determine if TNAP-/- mice exhibit craniofacial morphologic abnormalities similar to those seen in patients with infantile hypophosphatasia (TNAP • TNAP-/- mice have craniofacial skeletal shape anomalies deficiency) similar to those seen in human infants with • Genotype comparison hypophosphatasia • HPP long bone phenotype subset comparison • TNAP-/- mice have an increased incidence of 2. Establish the timing of onset of craniosynostosis in craniosynostosis the TNAP-/- mouse model of infantile hypophosphatasia

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SAMPLE Mouse Paw WTPhenotype Criteria WT

• Mixed genetic background • Age Slight Slight • 15 day old (P15) METHODS • 20 day old (P20) • Genotype Severe Severe • TNAP-/- • Wild type • TNAP-/- Clinical Phenotype • Normal, slight, moderate, severe

MICRO-COMPUTED TOMOGRAPHY (MICRO-CT) CORONAL SUTURE FUSION ASSESSMENT

• Whole dissected calvaria • Skulls scanned at 18 micron resolution • Coronal sutures viewed on two-dimensional slices of micro-CT scans • Verified by visualization under a dissecting microscope • Three dimensional images reconstructed at 18 cubic • Comparison between TNAP-/- and Wild Type mice micron effective voxel size • Craniosynostosis assessment • Craniofacial shape assessment TNAP-/- TNAP-/- P15 (n=24) P20 (n=32)

(n=48) Wild Type (n=71) Wild Type (n=24) (n=39)

3D MORPHOMETRIC CRANIOFACIAL CORONAL SUTURE FUSION ASSESSMENT ANALYSIS

• Pre-established 3D landmarks utilized to analyze differences in craniofacial form, shape, and growth with micro-CT imaging and Dolphin

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MORPHOMETRIC CRANIOFACIAL ANALYSIS MORPHOMETRIC CRANIOFACIAL ANALYSIS BY 3D LINEAR ANALYSIS BY EUCLIDEAN DISTANCE MATRIX ANALYSIS Linear Measurement Landmarks

• Linear Distance Analysis (Dolphin) Nasal Length 1-2 Frontal Bone 2-3 Parietal Bone 3-4 Interparietal Bone 4-5 Anterior Cranial Vault Width 18-19 Posterior Cranial Vault Width 24-25 Palatal Width 20-21 Posterior Palate to 33-29 Intersphenoidal Suture Basiphenoid Bone 29-30 • Allows for quantification and comparison of 3D form (size and Basioccipital Bone 30-31 shape), shape and growth differences between sample groups Foramen Magnum Diameter 31-32 Intersphenoidal Suture to 29-1 • Uses x, y, z landmark coordinate data (Dolphin) for statistical Nasale comparison of linear distances between every landmark placed as Cranial Vault Height 29-3 ratios between groups Cranial Vault Height 29-4 Cranial Vault Height 30-3 Cranial Vault Height 30-4

Richtsmeier and Lele, 2001 Cranial Vault Height 30-5

TNAP-/- AND TNAP+/+ MICE NORMALIZATION OF 3D LINEAR GROUPING BY PHENOTYPE MEASUREMENTS FOR 3D MORPHOLOGICAL ANALYSIS

Wild Type Wild Type (n=18) (n=18) Normal Normal (n=6) (n=6) P15 Slight P20 Slight (n=42) (n=6) (n=42) (n=10) Moderate Moderate (n=6) (n=1) Severe Severe (n=6) (n=7)

STATISTICS • Rater reliability tests for 3D landmark placement • Fischer’s exact test—coronal suture fusion assessment • EDMA – form, shape, growth RESULTS • Mixed model pairwise comparison with Tukey’s test— comparison between genotypes and across phenotypes with combined information from all linear distances (normalized measurements) • Principle Component Analysis—summarize all linear measurements by the most contributory variables (normalized measurements)

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Craniosynostosis Assessment with Micro-CT EUCLIDEAN DISTANCE MATRIX ANALYSIS: SIGNIFICANT FORM DIFFERENCES

Group Comparisons p values P15 Wild Type vs. TNAP-/- Mice < 0.001 P15 Wild Type vs. Severe TNAP-/- Phenotype < 0.001 TNAP+/+ TNAP-/- TNAP+/+ TNAP-/- P20 Wild Type vs. TNAP-/- Mice < 0.001 P20 Wild Type vs. Severe TNAP-/- Phenotype < 0.001 P15 Wild Type vs. Slight TNAP-/- Phenotype 0.013

α = 0.05

• Significant form difference between genotypes at both 2 and 3 weeks old

-/- • Significant form difference between phenotypes at both 2 and 3 weeks old Coronal suture synostosis in ~34% of 20 day TNAP mice • Severe phenotype vs. wild type – significant differences

• Other phenotypes vs. wild type – not statistically different, other than that shown

EUCLIDEAN DISTANCE MATRIX ANALYSIS: EUCLIDEAN DISTANCE MATRIX ANALYSIS— SIGNIFICANT SIZE AND SHAPE DIFFERENCES SIGNIFICANT GROWTH DIFFERENCES

Group Comparisons Confidence Interval— Confidence Interval— Size Shape Upper Lower Upper Lower 15-20 Day Group Comparisons p value -/- P15 WT vs. TNAP-/- 0.488 0.190 0.300 0.104 WT vs. TNAP < 0.001 -/- P15 WT vs. Severe TNAP-/- 1.155 0.801 0.671 0.483 WT vs. Severe TNAP < 0.001 α = 0.05 P20 WT vs. TNAP-/- 1.057 0.588 0.319 0.163 P20 WT vs. Severe TNAP-/- 1.791 1.635 0.660 0.589 *Statistically significant if confidence intervals do not cross zero; α = 0.01 or the 99% confidence interval • Significant growth pattern difference between genotypes from two to three weeks of age • Significant size and shape difference between genotypes at both 2 and 3 weeks • Significant growth pattern difference between wild type mice and • Significant size and shape difference between wild type mice and the severe -/- TNAP-/- phenotype at both 2 and 3 weeks old the severe TNAP phenotype from two to three weeks of age

MIXED MODEL PAIRWISE COMPARISON OF EDMA SUMMARY 3D CRANIOFACIAL LINEAR DISTANCES BY GENOTYPE • Significant difference in form, size, and shape between genotypes at both two and three weeks of age 15 day old Wild Type vs. TNAP-/- • Significant difference in form, size, and shape between Wild Type 3D Linear Measure Difference p value • Significantly shorter frontal -/- mice and the Severe TNAP subset at both two and three weeks bone length of age Frontal bone length -0.19 mm 0.04 • Significantly larger skull Skull width 0.19 mm 0.06 • Significant difference in the growth pattern from two to three width and height weeks of age between the genotypes Skull height 0.20 mm 0.02 α = 0.05 • Significant difference in the growth pattern from two to three weeks of age between the Wild Type mice and the Severe TNAP-/- subset 20 day old Wild Type vs. TNAP-/- 3D Linear Measure Difference p value • Significantly shorter frontal Frontal bone length -0.40 mm < 0.01 bone length

α = 0.05

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MIXED MODEL PAIRWISE COMPARISON OF CRANIOFACIAL LINEAR DISTANCES BY PHENOTYPE—15 DAYS OLD

15 day old Wild Type vs. Severe TNAP-/- Subset 3D Linear Measure Difference p value Nasale-anterior 0.67 mm < 0.01 frontal bone Frontal bone length -1.00 mm < 0.01 Skull height 0.31 mm 0.01 Skull height 0.31 mm < 0.01 Skull height 0.36 mm < 0.01

α = 0.05

• Significantly shorter frontal bone length • Significantly larger skull height dimensions

MIXED MODEL PAIRWISE COMPARISON OF MIXED MODEL COMPARISON OF 3D LINEAR CRANIOFACIAL LINEAR DISTANCES DISTANCES SUMMARY BY PHENOTYPE—20 DAYS OLD • Significantly shorter frontal bone length, larger skull height, and 20 day old Wild Type vs. Severe TNAP-/- Subset larger skull width between genotypes at two weeks of age 3D Linear Measure Difference p value • Two-fold decrease in average frontal bone length difference Nasale-anterior 0.94 mm < 0.01 between genotypes from two to three weeks of age frontal bone • Loss of significant difference for skull width and height measures Frontal bone length -1.34 mm < 0.01 between genotypes at three weeks of age Skull height 0.34 mm < 0.01 • Significantly shorter frontal bone length and larger skull height Skull height 0.38 mm < 0.01 measures between Wild Type and Severe TNAP-/- subset at two Skull height 0.41 mm < 0.01 weeks of age α = 0.05 • Same results, with even greater differences at three weeks of age • Significantly shorter frontal bone length • Significantly larger skull height dimensions • No significant differences found for other phenotypes

PRINCIPLE COMPONENT ANALYSIS: PRINCIPLE COMPONENT ANALYSIS: 15 DAY OLD MICE COMPONENT LOADING—15 DAY OLD MICE

• Greatest component loading of the following measures: • Frontal bone length • Interparietal bone length • Posterior skull width 15 day old • Posterior skull height • Suggests that these measures exhibit a greater contribution to the variance among all measures in the severe phenotype subset

• 2 principle components covered 77% of the variance • Severe TNAP-/- phenotype subset significantly different from other subsets and from wild type mice

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PRINCIPLE COMPONENT ANALYSIS: PRINCIPLE COMPONENT ANALYSIS: 20 DAY OLD MICE COMPONENT LOADING—20 DAY OLD MICE

• Greatest component loading of the following measures: • Frontal bone length • Posterior skull width measures • Posterior skull height measures 20 day old • Suggests that these measures exhibit a greater contribution to the variance among all measures in the severe phenotype subset • This is the dome-shaped appearance that is expected to occur with premature coronal suture fusion

• 2 principle components covered 81% of the variance • Severe TNAP-/- phenotype subset significantly different from other subsets and from wild type mice

DISCUSSION

STUDY LIMITATIONS CONCLUSIONS

• Inability to verify landmarks on micro CT scans due to poor tissue mineralization • TNAP-/- mouse model of infantile HPP does phenocopy the • This limited the number of landmarks available for the study craniofacial morphology of infantile HPP in humans • Limited ages of samples available for analysis • This study reports a 34% incidence of coronal craniosynostosis in TNAP-/- mice at 3 weeks of age • Analysis of different ages (prenatal, P1, P7, etc.) would allow for delineation of when the morphological changes seen in the mutant mice begin to occur • This study showed a difference in form, shape, and growth • This would help to establish an altered treatment regimen with enzyme patterns between genotypes replacement • Long bone clinical severe TNAP-/- phenotype displays significantly • Significant damage of samples during preparation greater amount of abnormal craniofacial form, shape, and growth • Use of micro CT for coronal suture analysis than other phenotypic subsets • Histological analysis is the gold standard • This study showed the timing of onset of craniosynostosis and described the morphology of the TNAP-/- mouse model of human • Could only analyze the coronal suture by micro-CT due to hypomineralization infantile HPP, which has yet to be done

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FUTURE STUDIES PARTING THOUGHTS… • Studies of younger post-natal and pre-natal mice would serve to • If diminished TNAP activity does have a role in further describe development at an earlier age the etiology of craniosynostosis, TNAP enzyme • Continue with my pilot study efforts to stain, visualize, and place landmarks on embryonic mice replacement therapy could be the first • Optimize staining protocol successful, non-surgical treatment for craniosynostosis • Difficult to see tissues due to poor mineralization • Histo-morphogenic studies of both the endochondral and • If the timing of onset of the craniofacial intramembranous bones in the infantile HPP mouse model abnormalities seen in infantile HPP patients is • Cell type specific TNAP knockout mice: osteoblasts, chondrocytes determined, the TNAP replacement regimen • Double mutant mice  identify additional contributors to can be altered accordingly abnormal growth and development of the craniofacial skeleton

REFERENCES REFERENCES

• Whyte MP. Hypophosphatasia: Nature’s window on alkaline phosphatase function in humans. In: Bilezikian JP, Raisz LG, Martin • Kjellman M, Oldfelt V, Nordenram A, Olow-Nordenram M. Five cases of hypophosphatasia with dental findings. Int J Oral Surg TJ, editors. Principles of Bone Biology. 3rd ed. San Diego: Academic Press; 2008. p. 1573-98. 1973;2:152-8.

• Johnson K, Moffa A, Chen Y, Pritzker K, Goding J, Terkeltaub R. Matrix vesicle plasma cell membrane glycoprotein-1 regulates • Bixler D. Heritable disorders affecting cementum and the periodontal structure. In: Stewart RE, Prescott GH, editors. Oral Facial mineralization by murine osteoblastic MC3T3 cells. J Bone Miner Res 1999 Jun;14:883-92. Genetics. Saint Louis: Mosby; 1976. p.262. • Mornet E, Yvard A, Taillandier A, Fauvert D, Simon-Bouy B. A molecular-based estimation of the prevalence of hypo- • phosphatasia in the European population. Ann Hum Genet 2011. E-Pub. Whyte MP, Murphy WA, Fallon MD. Adult hypophosphatasia with chondrocalcinosis and arthropathy. Variable penetrance of hypophosphatasemia in a large Oklahoma kindred. Am J Med 1982;72:631-41. • Whyte MP, et al. Enzyme-replacement therapy in life-threatening hypophosphatasia. N Engl J Med. 2012; 366:904-13. • Whyte MP. Atypical femoral fractures, bisphosphonates, and adult hypophosphatasia. J Bone Miner Res 2009;24:1132-4. • Renier D, Lajeunie E, Arnaud E, Marchac D. Management of craniosynostosis. Childs Nerv Syst. 2000; 16:645-58 • Wenkert D, McAlister WH, Coburn SP et al. Hypophosphatasia: skeletal presentation in utero of non-lethal disease (seventeen • Mornet E. Hypophosphatasia. Orphanet J Rare Dis 2007;2:40. new cases and literature review). J Bone Miner Res 2011;26:2389-98

• Anderson HC. Molecular biology of matrix vesicles. Clin Orthop Relat Res 1995 May(314):266-80. • Whyte MP, et al. Enzyme-replacement therapy in life-threatening hypophosphatasia. N Engl J Med. 2012; 366:904-13.

• Terkeltaub R, Rosenbach M, Fong F, Goding J. Causal link between nucleotide pyrophosphohydrolase overactivity and increased • Renier D, Lajeunie E, Arnaud E, Marchac D. Management of craniosynostosis. Childs Nerv Syst. 2000; 16:645-58. intracellular inorganic pyrophosphate generation demonstrated by transfection of cultured fibroblasts and osteoblasts with plasma cell membrane glycoprotein-1. Relevance to calcium pyrophosphate dihydrate deposition disease. Arthritis Rheum 1994;37:934-41. • Anderson HC, Sipe JB, Hessle L, Dhanyamraju R, Atti E, Camacho NP, Millan JL. Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am J Pathol 2004;164:841-7. • Terkeltaub RA. Inorganic pyrophosphate generation and disposition in pathophysiology. Am J Physiol Cell Physiol 2001;281:C1- C11. • Cohen MM, Jr. Editorial: perspectives on craniosynostosis. Am J Med Genet A 2005;136A:313- 26. • Shohat M, Rimoin DL, Gruber HE, Lachman RS. Perinatal lethal hypophosphatasia; clinical, radiologic and morphologic find- ings. Pediatr Radiol 1991;21:421-7. • Kozlowski K, Sutcliffe J, Barylak A, et al. Hypophosphatasia. Review of 24 cases. Pediatr Radiol 1976;5:103-17. • Collmann H, Mornet E, Gattenlohner S, Beck C, Girschick H. Neurosurgical aspects of childhood hypophosphatasia. Childs Nerv Syst 2009;25:217-23. • Brenner RL, Smith JL, Cleveland WW, Bejar RL, Lockhart Jr WS. Eye signs of hypophosphatasia. Arch Ophthalmol 1969;81:614- 7.

ACKNOWLEDGEMENTS THANK YOU! QUESTIONS?

• Chair, Dr. Nan E. Hatch • Committee members • Dr. Katherine Kelly • Dr. Yuji Mishina • Dr. Christopher Roberts • Jin Liu • Hwa-Kyung Nam • Cassandra Campbell • Nicole Pentis • Funding: Le Gro, Delta Dental Foundation, University of Michigan Department of Orthodontics

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