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ACTN3 R577X Genotypes Associate with Class II and Deep Bite Malocclusions

Brian M. Zebrick, DDS Temple University Kornberg School of Dentistry

Department of Orthodontics May 16, 2015

Kornberg School of Dentistry

TEMPLE UNIVERSITY

>140 orthognathic surgeries/ year • Including mandibular bilateral sagittal split ramus

osteotomy “High Throughput” clinic for collection of clinical & biological samples

RT-PCR Genotyping Muscle Fiber Type Morpometrics

1. IGF1 Growth pathway

So far we have identified 28 significant genetic associations for malocclusion, including: HDAC4, KAT6B, MYO1H, MYO1C, ACTN2, ACTN3, ENPP1, ATP2A2, NUAK1, PPP1CC, RUNX2, GABRA6, CACNA2D1, IL1B, IL1R2, IL6, IL8, CCL2, CCL3, CCL3L3, CCL4, CXCR1 and Nodal Pathway genes Nodal, Lefty, Nodal-modulators Nomo-1, 2, 3, MiR15B/16 and Pitx2.

Genotyping & Masseter Gene expression using GWAS candidate genes

2. Nodal Pathway development of skeletal & muscular structures of 1st Brachial Arch

Asymmetric Nodal expression in the node, and signaling in the left Lateral Plate Mesoderm.

Medical Physiology & Pathophysiology Essentials & clinical problems 2004

Babu & Roy, 2013 Open Biol. 3(5):130052, 2013

577RR, 577RX= (+) α-actinin-3 577XX= (-) α-actinin-3

ACTN3 Associates with Athleticism and Muscle Size

A gene for Speed: The emerging role of α-Actinin-3 in muscle metabolism. Berman & North Physiol 25:2010.

Athleticism by R577X Genotype

ACTN3

What is the association of ACTN2 and ACTN3 expression and muscloskeletal malocclusion phenotypes in masseter

muscle of orthognathic surgery patients?

Lateral Cepholograms

Class II Deep Normal Open

Class III Deep Normal Open

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Genotyping

2 SNPs

rs1815739 (the 577X SNP)

rs678397 (no known functional consequences)

Fiber type and RT-PCR analysis of muscle

Immunostaining with antibodies specific for myosin heavy chain (MyHC) isoforms

Type I, type hybrid, type IIA and/or IIX, neonatal, and atrial

Quantification of Actinin mRNA

ACTN2 and ACTN3 were quantified by TaqMan® quantitative real time PCR (qRT-PCR)

Materials and Methods

A) Class III Open Bite (B) Class II Open Bite RR Genotype XX Genotype

ACTN3 Genotypes Have Significant Associations with Differences in Jaw Length and Face Height

Differences in mandibular length by R577X genotype

Zebrick B. et al. 2014:146:603-11

Masseter Muscle Fiber Types Change with ACTN3 Genotypes

(A) Anti-Fast MyHC; (B) Anti-IIA; (C) Anti-Type I; (D) Anti–ACTN3; Bar = 1000μm.

RR Genotype: α-Actinin-3 is in Type II and Hybrid, but not Type I Fibers

XX Genotype: No α-Actinin-3 is in Hybrid or Type II Fibers

Area and Occupancy of Fiber Types by ACTN3 Genotypes

0.043*

Actinin Expression in Masseter Muscle of Orthoganthic Surgery Subjects

ACTN2 ACTN3

Rela

tive Q

uan

tity

0.0

0.5

1.0

1.5

2.0

CC

TC

TT

*

*P = 0.001P = 0.780

TaqMan RT-PCR Quantification

1. ACTN3 expression varied more than ACTN2, with relative amounts of α-actinin-3 decreased in open bite and skeletal Class II.

2. Decreased α-actinin-3 associated with smaller type II fiber diameter.

3. ACTN3 mRNA expression decreased to almost undetectable with 577XX genotype, while ACTN2 expression levels remained unchanged, suggesting α-actinin-2 may not compensate for loss of α-actinin-3.

4. Actn3 knockout mice have significant decreases in bone mineral density. Further, absence of α-actinin-3 in bone resulted in increased expression of Enpp1, a negative regulator of mineralization. These relationships may be important to the development of Class II malocclusions in humans and merit further investigation.

Conclusions The Actn3 Knock Out Mouse: Effects on Bone and Enpp1 Expression

Bone Growth is Decreased In the Actn3-/- Mouse Model

Trabecular Architecture

WT Actn3-/-

Mineral Apposition Rate

WT Actn3-/-

Enpp1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase1)

• Inhibits Mineralization (ectonucleotide pyrophosphatase ephosphodiesterase family)

Enpp1 Expression is Significantly Up-Regulated in Actn3-/- Mice

Bone Volume & Trabecular Number are Reduced

Mineral Apposition Rate is Lower

From:Yang et al., Bone 2011;49:790-798.

Symbol Illumina* KO vs WT

TaqMan KO vs WT TaqMan p-value

Enpp1 1.45 1.66 0.02

*Osteoblast Microarrays

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Future Directions Actn3 KO Reductions in Mandibular Length

Skull Jaw Length Nasal Inter-orbital

ACTN3-/- Length Width Height Mandible

Maxilla Length Width

n=2 Mean 25.60 11.10 11.85 11.70 13.15 16.85 7.20

SD 0.42 0 0.21 0 0.21 0.78 0.57

ACTN3+/+ Mean 25.40 11.00 11.85 12.25 13.30 16.55 6.98

n=4 SD 0.69 0.67 0.43 0.33 0.55 0.70 0.17

Δ 0.20 0.10 0 -0.55 -0.15 0.30 0.22

p 0.73 0.85 1.00 0.09 0.55 0.65 0.44

KO WT

Future Directions: Gene microarray comparing mandibular gene expression in Actn3 KO, The GIANT Consortium GWAS as a priori genes of interest

Personalized Medicine Approach

Salivary Tests

More Personalized and Specific Treatment Plans

Optimized Results

Future Directions

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References

Acknowledgements

James Sciote

Michael Horton

Teesit Teeramongkolgul

James Cavalancia

Jeffrey Godel

Alex Vieira

Pf. Joel Ferri

Pf. Gwenael Raoul

Resident:

Romain Nicot

Pf. Kathryn North

THANK YOU!