Objective: Genetic loci for ATP2A2 kinase, NUAK1, and phosphatase PPP1CC are associated with skeletal muscle strength phenotypes. ATP2A2 is a calcium ion transport ATPase in sarcoplasmic reticulum that is predominantly expressed in cardiac and slow-twitch fibers. NUAK1, an AMP-activated protein kinase, and PPP1CC, a subunit of protein phosphatase 1, are involved in glycogen metabolism during skeletal muscle contraction. The aim of this study is to investigate whether these genes are associated with masseter muscle composition and function in the development of skeletal malocclusion. Methods: A total of 56 orthognathic surgery patients were classified as having skeletal Class I, Class II, or III sagittal malocclusions with normal, open, or deep bites vertically, with or without facial asymmetry. Masseter muscle samples were collected during the mandibular osteotomies, frozen, and sent to the Kornberg School of Dentistry. Tissue from eleven patients was used for gene expression analysis on Affymetrix HT2.0 microarray chips and a principle components analysis. Then, these plus an additional 45 masseter samples were used for quantitative RT-PCR. Expression data for the three genes of interest were evaluated in the microarray and corroborated and expanded upon with RT-PCR data. ANOVA and unpaired t-tests were performed to determine correlations between ATP2A2, NUAK1, and PPP1CC expression levels and vertical and sagittal malocclusion classifications. Additional ANOVA and unpaired t-tests were performed to determine correlations between ATP2A2 expression level and signs and symptoms of temporomandibular disorder (TMD) with and without pain and facial asymmetry, and relative to the expression levels of a second gene associated with muscle strength phenotypes (ACTN3). Finally, Kendall Tau analyses were performed to compare ATP2A2 expression levels in subjects grouped by malocclusion classification to masseter muscle composition, including mean fiber area (MFA) and mean percent occupancy (MPO) of each fiber type. Results: Principle component analysis revealed two patients with genetic expression levels that deviated from the group. These were the only patients diagnosed with facial asymmetry. Microarray data showed that in these patients ATP2A2 and PPP1CC were significantly decreased. NUAK1 was decreased to a lesser extent. Also, among other genes in the same functional categories, ATP2A1 expression was -30.45 fold (P<6.11X10-6) and PPP3CC expression was -2.96 fold (P<2.03X10-5) in the patients with facial asymmetry. RT-PCR results showed NUAK1 and PPP1CC were differentially expressed at lower, but not statistically significant levels in subjects with craniofacial asymmetry. However, RT-PCR did verify that ATP2A2 expression is down regulated in subjects with mild to severe forms of asymmetry as compared to subjects with facial symmetry (p=0.022). ANOVA and unpaired t-test analyses illustrated that there was no significant differences in ATP2A2 expression in patients with different vertical, saggital, or combined vertical/sagittal malocclusion diagnoses. There was a significant association between the lateral differences in ATP2A2 expression, between right- and left-sided masseter biopsies within the same individual, in subjects with Class III malocclusions with different vertical diagnoses. Here, lateral differences were greatest in open bite, intermediate in deep bite, and lowest in normal Class III subjects. Kendall tau analyses were performed to compare ATP2A2 expression levels and masseter composition (MFA/MPO of type I, hybrid, and type II fibers) in all subjects, subjects with Class II malocclusions, and subjects with Class III malocclusions. Regardless of sagittal malocclusion, all subjects showed a negative correlation with type IIA MPO that was highly significant (r=-0.46; p=0.004). Also, ATP2A2 associations in Class II subjects were positive with type I MFA (r=0.36; p=0.04) and negative with type IIA MPO (r=-0.59; p=0.001). Correlations for Class III subjects were typically negative and not significant. Also, Kendall tau correlations were performed to compare ATP2A2 expression with the composition of each fiber type in patients grouped by both sagittal and vertical malocclusion classification. These found decreased type IIA fiber MPO correlated significantly (p = 0.024) with increased relative ATP2A2 expression in subjects with Class II, normal bite malocclusions (n = 6, R2 = 0.8127). Finally, ATP2A2 expression was not associated with most phenotypic traits exhibited by the surgery subjects such as presence of signs/symptoms of TMD with and without pain and facial asymmetry. However there was an association between decreased lateral differences in ATP2A2 expression in subjects with asymmetry with the TC ACTN3 genotype as compared to subjects with the TC ACTN3 genotype and facial symmetry. Conclusions: ATP2A2 promotes calcium transport in slow twitch and cardiac muscle contraction-relaxation cycling. Decreased expression of this gene in patients with asymmetries suggests that down regulation of the calcium handling capacities of muscle fibers may influence the development of abnormal craniofacial phenotypes. Both NUAK1 and PPP1CC are thought to play metabolic regulatory or responsive roles to muscle contraction. Decreased expression of these genes may accompany alterations of fiber-type form and metabolic properties to adversely affect jaw development. Additionally, ATP2A2 correlations indicate that this calcium channel protein may be important for type I fiber function, but not type IIA in masseter muscle from Class II subjects, suggesting a functional influence on malocclusions. ATP2A2 does not appear to function differentially in fiber types that influence development of Class III malocclusion. Further studies with more subjects are needed to increase experimental power. / Oral Biology

Alterações morfológicas e dentofaciais têm sido repetidamente atribuídas ao impedimento da função naso-respiratória devido à hipertrofia de tonsilas faríngea e palatinas. O objetivo da presente investigação foi avaliar o padrão esquelético e características oclusais de crianças respiradoras bucais antes (T1) e em média 28 meses após (T2) serem submetidas à cirurgia para remoção de tonsilas hipertrofiadas, comparando com crianças respiradoras nasais. O grupo experimental foi composto de 33 crianças respiradoras bucais (RB) e o grupo controle, de 32 crianças respiradoras nasais (RN). Os exames ortodônticos (radiografia cefalométrica e modelos de estudo) foram realizados em ambos os grupos nos tempos T1 e T2. Na comparação entre os grupos, os resultados permitiram concluir que os respiradores bucais apresentaram maior inclinação do plano mandibular em relação à base craniana e ao plano palatal (SN.GoGn; PP.PM); ângulo goníaco mais obtuso (ArGo.GoMe); tendência ao tipo morfológico dolicofacial (BaN.PtGn); altura do ramo da mandíbula (Ar-Go) e altura posterior da face (S-Go) diminuídas; maior número de mordidas cruzadas e menor distância intermolares. Em T2, o padrão morfológico predominante da face foi mesofacial no grupo RN e dolicofacial no grupo RB; o overbite foi normal nos RB e profundo nos RN e o overjet apresentou-se maior nos RB. Em relação à mordida aberta, mordida cruzada, relação de caninos, plano terminal dos segundos molares decíduos e distância intercaninos e intermolares houve semelhança entre os grupos RB e RN. Ao analisar cada grupo separadamente, verificou-se que, vinte e oito meses após a cirurgia, nos RB, houve alteração na direção do crescimento da face e inclinação do plano mandibular no sentido anti-horário, com diminuição dos valores de SN.GoGn, PP.PM, SNGn, ArGo.GoMe e aumento de BaN.PtGn. Em ambos os grupos houve crescimento vertical anterior e posterior da face, evidenciado pelo aumento das medidas verticais lineares (N-Me, N-ENA, ENA-Me, S-Go, S-Ar, Ar- Go). O plano terminal dos segundos molares decíduos modificou-se de reto para degrau mesial nos dois grupos; o overbite alterou de negativo para normal no grupo RB e tornou-se profundo, no grupo RN. No presente estudo, a desobstrução das vias aéreas através da remoção cirúrgica das tonsilas faríngea e/ou palatinas em crianças entre 3 e 6 anos de idade, resgatou o padrão de crescimento normal para esses pacientes respiradores bucais, mostrando resultados excelentes sobre as características oclusais e esqueléticas. / Morphological and dentofacial abnormalities have been attributed to respiratory obstruction caused by adenoid and tonsils hypertrophy. The objective of the present study was to evaluate the skeletal patterns and occlusal characteristics in mouthbreathing children before (T1) and a mean of 28 months after (T2) they have been were submitted to surgery for tonsils hypertrophy, compared with those of nosebreathing children. The experimental group was composed of 33 mouth-breathing children (MB) and the control group, 32 nose-breathing children (NB). Orthodontic examinations (cephalometric radiography and study models) were performed on both groups at the times T1 and T2. After comparisons between the groups, it could be concluded that the MB presented greater inclination of the mandibular plane in relation to the cranial base and palatal plane (SN.GoGn, PP.PM); more obtuse gonial angle (ArGo.GoMe); tendency towards the dolicofacial pattern (BaN.PtGn); reduced height of the mandibular ramus (Ar-Go) and lower posterior height of the face (S-Go); higher frequency of crossbite and smaller intermolar distance. After surgery (T2), the predominant morphological pattern of the face was mesofacial in NB and dolicofacial in MB; the overbite was normal in MB and deep in NB; the overjet was greater in MB; the two groups were similar in relation to open bite, crossbite, the antero-posterior canines relationship and the second deciduous molars terminal plane, and the intercanine and intermolar distances. Separate analysis on each group showed that, 28 months after surgery, the MB presented an counterclockwise rotation of the mandible, smaller SN.GoGn, PP.PM, SNGn and ArGo.GoMe values and larger BaN.PtGn value. In both groups, there was anterior and posterior vertical growth of the face, with increase in the linear vertical measurements (N-Me, N-ENA, ENA-Me, S-Go, S-Ar and Ar-Go). The second deciduous molars terminal plane changed from straight to mesial, in both groups; the overbite changed from negative to normal in MB and it became deep in NB. In the present study, the surgery for tonsils hypertrophy in children aged 3 to 6 years restored the normal growth pattern for these mouth-breathing patients, with excellent results regarding occlusal and skeletal characteristics.

Alterações morfológicas e dentofaciais têm sido repetidamente atribuídas ao impedimento da função naso-respiratória devido à hipertrofia de tonsilas faríngea e palatinas. O objetivo da presente investigação foi avaliar o padrão esquelético e características oclusais de crianças respiradoras bucais antes (T1) e em média 28 meses após (T2) serem submetidas à cirurgia para remoção de tonsilas hipertrofiadas, comparando com crianças respiradoras nasais. O grupo experimental foi composto de 33 crianças respiradoras bucais (RB) e o grupo controle, de 32 crianças respiradoras nasais (RN). Os exames ortodônticos (radiografia cefalométrica e modelos de estudo) foram realizados em ambos os grupos nos tempos T1 e T2. Na comparação entre os grupos, os resultados permitiram concluir que os respiradores bucais apresentaram maior inclinação do plano mandibular em relação à base craniana e ao plano palatal (SN.GoGn; PP.PM); ângulo goníaco mais obtuso (ArGo.GoMe); tendência ao tipo morfológico dolicofacial (BaN.PtGn); altura do ramo da mandíbula (Ar-Go) e altura posterior da face (S-Go) diminuídas; maior número de mordidas cruzadas e menor distância intermolares. Em T2, o padrão morfológico predominante da face foi mesofacial no grupo RN e dolicofacial no grupo RB; o overbite foi normal nos RB e profundo nos RN e o overjet apresentou-se maior nos RB. Em relação à mordida aberta, mordida cruzada, relação de caninos, plano terminal dos segundos molares decíduos e distância intercaninos e intermolares houve semelhança entre os grupos RB e RN. Ao analisar cada grupo separadamente, verificou-se que, vinte e oito meses após a cirurgia, nos RB, houve alteração na direção do crescimento da face e inclinação do plano mandibular no sentido anti-horário, com diminuição dos valores de SN.GoGn, PP.PM, SNGn, ArGo.GoMe e aumento de BaN.PtGn. Em ambos os grupos houve crescimento vertical anterior e posterior da face, evidenciado pelo aumento das medidas verticais lineares (N-Me, N-ENA, ENA-Me, S-Go, S-Ar, Ar- Go). O plano terminal dos segundos molares decíduos modificou-se de reto para degrau mesial nos dois grupos; o overbite alterou de negativo para normal no grupo RB e tornou-se profundo, no grupo RN. No presente estudo, a desobstrução das vias aéreas através da remoção cirúrgica das tonsilas faríngea e/ou palatinas em crianças entre 3 e 6 anos de idade, resgatou o padrão de crescimento normal para esses pacientes respiradores bucais, mostrando resultados excelentes sobre as características oclusais e esqueléticas. / Morphological and dentofacial abnormalities have been attributed to respiratory obstruction caused by adenoid and tonsils hypertrophy. The objective of the present study was to evaluate the skeletal patterns and occlusal characteristics in mouthbreathing children before (T1) and a mean of 28 months after (T2) they have been were submitted to surgery for tonsils hypertrophy, compared with those of nosebreathing children. The experimental group was composed of 33 mouth-breathing children (MB) and the control group, 32 nose-breathing children (NB). Orthodontic examinations (cephalometric radiography and study models) were performed on both groups at the times T1 and T2. After comparisons between the groups, it could be concluded that the MB presented greater inclination of the mandibular plane in relation to the cranial base and palatal plane (SN.GoGn, PP.PM); more obtuse gonial angle (ArGo.GoMe); tendency towards the dolicofacial pattern (BaN.PtGn); reduced height of the mandibular ramus (Ar-Go) and lower posterior height of the face (S-Go); higher frequency of crossbite and smaller intermolar distance. After surgery (T2), the predominant morphological pattern of the face was mesofacial in NB and dolicofacial in MB; the overbite was normal in MB and deep in NB; the overjet was greater in MB; the two groups were similar in relation to open bite, crossbite, the antero-posterior canines relationship and the second deciduous molars terminal plane, and the intercanine and intermolar distances. Separate analysis on each group showed that, 28 months after surgery, the MB presented an counterclockwise rotation of the mandible, smaller SN.GoGn, PP.PM, SNGn and ArGo.GoMe values and larger BaN.PtGn value. In both groups, there was anterior and posterior vertical growth of the face, with increase in the linear vertical measurements (N-Me, N-ENA, ENA-Me, S-Go, S-Ar and Ar-Go). The second deciduous molars terminal plane changed from straight to mesial, in both groups; the overbite changed from negative to normal in MB and it became deep in NB. In the present study, the surgery for tonsils hypertrophy in children aged 3 to 6 years restored the normal growth pattern for these mouth-breathing patients, with excellent results regarding occlusal and skeletal characteristics.