Proposed thesis project - AAO John -- Bone...of age • Significant difference in the growth pattern from two to three weeks of age between the genotypes Skull height • Significant

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John DuRussel MS, DDS, MS

ANALYSIS OF CRANIOFACIAL MORPHOLOGY IN A TNAP KNOCKOUT

MOUSE MODEL

CRANIOSYNOSTOSIS

• Premature fusion of cranial bones

• 1/2500 live births

• May involve one or multiple sutures

• Isolated (85%) or Syndromic (15%)

• Biologic process unknown

Infant with craniosynostosis

• Elevated intracranial pressure

• Impaired cerebral blood flow

• Airway obstruction

• Deafness, blindness, seizures

• Developmental delay and learning disabilities

• Esthetic compromises

CURRENT TREAMENT: CRANIAL VAULT REMODELING SURGERY

• Excise prematurely fused sutures and correct calvarial deformities

• Initial surgery at 3-6 months old to allow for exponential brain growth

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)

ATP Enpp1 PPi

Osteoblast

ENPP1 AND TNAP CONTROL MINERALIZATION

• Ectonucleotide pyrophosphatase/phosphodiesterase-1 (Enpp1) - the primary osteoblastic generator of pyrophosphate (PPi)

Matrix

ATP Enpp1 TNAP PPi Pi

Osteoblast

Matrix


• Tissue non-specific alkaline phosphatase (TNAP) hydrolyzes PPi to Pi which is essential to the growth of hydroxyapatite crystals.

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ATP Enpp1 TNAP PPi Pi

Osteoblast

Matrix


Deposition of calcium pyrophosphate dihydrate crystals

? Low bone density

Qualitative Craniofacial Phenotype of the

TNAP-/- Mouse Model of Infantile Hypoposphatasia

Open cranial suture in P20 wild type mouse

Fused/closed cranial suture in TNAP KO mouse *

• shape abnormalities • hypomineralization • craniosynostosis

WT TNAP-/- WT WT

KO KO

2D linear craniofacial measurements (digital calipers)

Age = P20

KO

WT = white KO = black

• brachycephalic

• acrocephalic

• dome shaped

2D Shape Abnormalities

in TNAP-/- Mouse Model of Infantile Hypoposphatasia

Age = P15

TNAP Enzyme Replacement Therapy

Hypophosphatasia

Current regimen of TNAP enzyme

replacement therapy

survival strong bones strong teeth normal skull

Revised earlier regimen

of TNAP therapy

Surgeries to alleviate craniosynostosis

Whyte et. al., 2012

SPECIFIC AIMS

1. Determine if TNAP-/- mice exhibit craniofacial morphologic abnormalities similar to those seen in patients with infantile hypophosphatasia (TNAP deficiency)

• Genotype comparison

• HPP long bone phenotype subset comparison

2. Establish the timing of onset of craniosynostosis in the TNAP-/- mouse model of infantile hypophosphatasia

HYPOTHESES

• TNAP-/- mice have craniofacial skeletal shape anomalies similar to those seen in human infants with hypophosphatasia

• TNAP-/- mice have an increased incidence of craniosynostosis

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METHODS

SAMPLE

• Mixed genetic background

• Age

• 15 day old (P15)

• 20 day old (P20)

• Genotype

• TNAP-/-

• Wild type

• TNAP-/- Clinical Phenotype

• Normal, slight, moderate, severe

Mouse Paw Phenotype Criteria WT WT

Slight Slight

Severe Severe

MICRO-COMPUTED TOMOGRAPHY (MICRO-CT)

• Whole dissected calvaria

• Skulls scanned at 18 micron resolution

• Three dimensional images reconstructed at 18 cubic micron effective voxel size

• Craniosynostosis assessment

• Craniofacial shape assessment

CORONAL SUTURE FUSION ASSESSMENT

P15

(n=48)

TNAP-/-

(n=24)

Wild Type

(n=24)

P20

(n=71)

TNAP-/-

(n=32)

Wild Type

(n=39)

• Coronal sutures viewed on two-dimensional slices of micro-CT scans

• Verified by visualization under a dissecting microscope

• Comparison between TNAP-/- and Wild Type mice

CORONAL SUTURE FUSION ASSESSMENT

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

3D MORPHOMETRIC CRANIOFACIAL ANALYSIS

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MORPHOMETRIC CRANIOFACIAL ANALYSIS BY EUCLIDEAN DISTANCE MATRIX ANALYSIS

• Allows for quantification and comparison of 3D form (size and shape), shape and growth differences between sample groups

• Uses x, y, z landmark coordinate data (Dolphin) for statistical comparison of linear distances between every landmark placed as ratios between groups

Richtsmeier and Lele, 2001

• Linear Distance Analysis (Dolphin)

Linear Measurement Landmarks

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 Intersphenoidal Suture

33-29

Basiphenoid Bone 29-30

Basioccipital Bone 30-31

Foramen Magnum Diameter 31-32

Intersphenoidal Suture to Nasale

29-1

Cranial Vault Height 29-3





MORPHOMETRIC CRANIOFACIAL ANALYSIS BY 3D LINEAR ANALYSIS

NORMALIZATION OF 3D LINEAR MEASUREMENTS

TNAP-/- AND TNAP+/+ MICE GROUPING BY PHENOTYPE

FOR 3D MORPHOLOGICAL ANALYSIS

P15

(n=42)

Wild Type

(n=18)

Normal

(n=6)

Slight

(n=6)

Moderate

(n=6)

Severe

(n=6)

P20

(n=42)

Wild Type

(n=18)

Normal

(n=6)

Slight

(n=10)

Moderate

(n=1)

Severe

(n=7)

STATISTICS

• Rater reliability tests for 3D landmark placement

• Fischer’s exact test—coronal suture fusion assessment

• EDMA – form, shape, growth

• 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)

RESULTS

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Craniosynostosis Assessment with Micro-CT

Coronal suture synostosis in ~34% of 20 day TNAP-/- mice

TNAP+/+ TNAP-/- TNAP+/+ TNAP-/-

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

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

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

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

• Severe phenotype vs. wild type – significant differences

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

α = 0.05

Group Comparisons Confidence Interval—Size

Confidence Interval—Shape

Upper Lower Upper Lower

P15 WT vs. TNAP-/- 0.488 0.190 0.300 0.104

P15 WT vs. Severe TNAP-/- 1.155 0.801 0.671 0.483

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

EUCLIDEAN DISTANCE MATRIX ANALYSIS: SIGNIFICANT SIZE AND SHAPE DIFFERENCES

*Statistically significant if confidence intervals do not cross zero; α = 0.01 or the 99% confidence interval

• Significant size and shape difference between genotypes at both 2 and 3 weeks

• Significant size and shape difference between wild type mice and the severe TNAP-/- phenotype at both 2 and 3 weeks old

15-20 Day Group Comparisons p value

WT vs. TNAP-/- < 0.001

WT vs. Severe TNAP-/- < 0.001

EUCLIDEAN DISTANCE MATRIX ANALYSIS—SIGNIFICANT GROWTH DIFFERENCES

• Significant growth pattern difference between genotypes from two to three weeks of age

• Significant growth pattern difference between wild type mice and the severe TNAP-/- phenotype from two to three weeks of age

α = 0.05

EDMA SUMMARY

• Significant difference in form, size, and shape between genotypes at both two and three weeks of age

• Significant difference in form, size, and shape between Wild Type mice and the Severe TNAP-/- subset at both two and three weeks of age

• Significant difference in the growth pattern from two to three weeks of age between the genotypes

• Significant difference in the growth pattern from two to three weeks of age between the Wild Type mice and the Severe TNAP-/- subset

3D Linear Measure Difference p value

Frontal bone length -0.19 mm 0.04

Skull width 0.19 mm 0.06

Skull height 0.20 mm 0.02

MIXED MODEL PAIRWISE COMPARISON OF 3D CRANIOFACIAL LINEAR DISTANCES

BY GENOTYPE

15 day old Wild Type vs. TNAP-/-


Frontal bone length -0.40 mm < 0.01

α = 0.05

α = 0.05

20 day old Wild Type vs. TNAP-/-

• Significantly shorter frontal bone length

• Significantly larger skull width and height


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Nasale-anterior frontal bone

0.67 mm < 0.01


Skull height 0.31 mm 0.01

Skull height 0.31 mm < 0.01



• Significantly larger skull height dimensions

15 day old Wild Type vs. Severe TNAP-/- Subset

α = 0.05

MIXED MODEL PAIRWISE COMPARISON OF CRANIOFACIAL LINEAR DISTANCES

BY PHENOTYPE—15 DAYS OLD


Nasale-anterior frontal bone

0.94 mm < 0.01






• Significantly larger skull height dimensions

• No significant differences found for other phenotypes

20 day old Wild Type vs. Severe TNAP-/- Subset

α = 0.05

MIXED MODEL PAIRWISE COMPARISON OF CRANIOFACIAL LINEAR DISTANCES

BY PHENOTYPE—20 DAYS OLD

MIXED MODEL COMPARISON OF 3D LINEAR DISTANCES SUMMARY

• Significantly shorter frontal bone length, larger skull height, and larger skull width between genotypes at two weeks of age

• Two-fold decrease in average frontal bone length difference between genotypes from two to three weeks of age

• Loss of significant difference for skull width and height measures between genotypes at three weeks of age

• Significantly shorter frontal bone length and larger skull height measures between Wild Type and Severe TNAP -/- subset at two weeks of age

• Same results, with even greater differences at three weeks of age

PRINCIPLE COMPONENT ANALYSIS: 15 DAY OLD MICE

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

subsets and from wild type mice

15 day old

• Greatest component loading of the following measures:

• Frontal bone length

• Interparietal bone length

• Posterior skull width

• Posterior skull height

• Suggests that these measures exhibit a greater contribution to the variance among all measures in the severe phenotype subset

PRINCIPLE COMPONENT ANALYSIS: COMPONENT LOADING—15 DAY OLD MICE

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PRINCIPLE COMPONENT ANALYSIS: 20 DAY OLD MICE

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

subsets and from wild type mice

20 day old

• Greatest component loading of the following measures:

• Frontal bone length

• Posterior skull width measures

• Posterior skull height measures

• 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

PRINCIPLE COMPONENT ANALYSIS: COMPONENT LOADING—20 DAY OLD MICE

DISCUSSION

STUDY LIMITATIONS

• Inability to verify landmarks on micro CT scans due to poor tissue mineralization

• This limited the number of landmarks available for the study

• Limited ages of samples available for analysis

• 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 would help to establish an altered treatment regimen with enzyme replacement

• Significant damage of samples during preparation

• Use of micro CT for coronal suture analysis

• Histological analysis is the gold standard

• Could only analyze the coronal suture by micro-CT due to hypomineralization

CONCLUSIONS

• TNAP-/- mouse model of infantile HPP does phenocopy the craniofacial morphology of infantile HPP in humans

• This study reports a 34% incidence of coronal craniosynostosis in TNAP-/- mice at 3 weeks of age

• This study showed a difference in form, shape, and growth patterns between genotypes

• Long bone clinical severe TNAP-/- phenotype displays significantly greater amount of abnormal craniofacial form, shape, and growth than other phenotypic subsets

• This study showed the timing of onset of craniosynostosis and described the morphology of the TNAP-/- mouse model of human infantile HPP, which has yet to be done

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FUTURE STUDIES

• Studies of younger post-natal and pre-natal mice would serve to further describe development at an earlier age

• Continue with my pilot study efforts to stain, visualize, and place landmarks on embryonic mice

• Optimize staining protocol

• Difficult to see tissues due to poor mineralization

• Histo-morphogenic studies of both the endochondral and intramembranous bones in the infantile HPP mouse model

• Cell type specific TNAP knockout mice: osteoblasts, chondrocytes

• Double mutant mice identify additional contributors to abnormal growth and development of the craniofacial skeleton

PARTING THOUGHTS…

• If diminished TNAP activity does have a role in the etiology of craniosynostosis, TNAP enzyme replacement therapy could be the first successful, non-surgical treatment for craniosynostosis

• If the timing of onset of the craniofacial abnormalities seen in infantile HPP patients is determined, the TNAP replacement regimen can be altered accordingly

REFERENCES • Whyte MP. Hypophosphatasia: Nature’s window on alkaline phosphatase function in humans. In: Bilezikian JP, Raisz LG, Martin

TJ, editors. Principles of Bone Biology. 3rd ed. San Diego: Academic Press; 2008. p. 1573 -98.

• Johnson K, Moffa A, Chen Y, Pritzker K, Goding J, Terkeltaub R. Matrix vesicle plasma cell membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells. J Bone Miner Res 1999 Jun;14:883-92.

• Mornet E, Yvard A, Taillandier A, Fauvert D, Simon-Bouy B. A molecular-based estimation of the prevalence of hypophosphatasia in the European population. Ann Hum Genet 2011. E-Pub.

• Whyte MP, et al. Enzyme-replacement therapy in life-threatening hypophosphatasia. N Engl J Med. 2012; 366:904-13.

• Renier D, Lajeunie E, Arnaud E, Marchac D. Management of craniosynostosis. Childs Nerv Syst. 2000; 16:645-58

• Mornet E. Hypophosphatasia. Orphanet J Rare Dis 2007;2:40.

• Anderson HC. Molecular biology of matrix vesicles. Clin Orthop Relat Res 1995 May(314):266-80.

• Terkeltaub R, Rosenbach M, Fong F, Goding J. Causal link between nucleotide pyrophosphohydrolase overactivity and increased 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 findings. 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.

REFERENCES

• Kjellman M, Oldfelt V, Nordenram A, Olow-Nordenram M. Five cases of hypophosphatasia with dental findings. Int J Oral Surg 1973;2:152-8.

• Bixler D. Heritable disorders affecting cementum and the periodontal structure. In: Stewart RE, Prescott GH, editors. Oral Facial Genetics. Saint Louis: Mosby; 1976. p.262.

• 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. Atypical femoral fractures, bisphosphonates, and adult hypophosphatasia. J Bone Miner Res 2009;24:1132-4.

• Wenkert D, McAlister WH, Coburn SP et al. Hypophosphatasia: skeletal presentation in utero of non-lethal disease (seventeen

new cases and literature review). J Bone Miner Res 2011;26:2389-98

• Whyte MP, et al. Enzyme-replacement therapy in life-threatening hypophosphatasia. N Engl J Med. 2012; 366:904-13.

• Renier D, Lajeunie E, Arnaud E, Marchac D. Management of craniosynostosis. Childs Nerv Syst. 2000; 16:645-58.

ACKNOWLEDGEMENTS

• 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

THANK YOU! QUESTIONS?

Proposed thesis project - AAO John -- Bone...of age • Significant difference in the growth pattern from two to three weeks of age between the genotypes Skull height • Significant

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