3/31/2015 1 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 1 st 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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3/31/2015
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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
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
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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