Phenotypic Characterization of Class II Malocclusion PDF

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Theses and Dissertations
Spring 2012
Phenotypic characterization of Class II

malocclusion
Sara Christine Howe
University of Iowa
Copyright 2012 Sara Howe
This thesis is available at Iowa Research Online: https://ir.uiowa.edu/etd/2896
Recommended Citation
Howe, Sara Christine. "Phenotypic characterization of Class II malocclusion." MS (Master of Science) thesis, University of Iowa, 2012.
https://doi.org/10.17077/etd.v30ksz3g
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Part of the Orthodontics and Orthodontology Commons

PHENOTYPIC CHARACTERIZATION OF CLASS II MALOCCLUSION
by
Sara Christine Howe
A thesis submitted in partial fulfillment

of the requirements for the Master of
Science degree in Orthodontics
in the Graduate College of
The University of Iowa
May 2012
Thesis Supervisor: Assistant Professor Lina M. Moreno Uribe

Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
MASTER'S THESIS
_______________
This is to certify that the Master's thesis of
Sara Christine Howe
has been approved by the Examining Committee

for the thesis requirement for the Master of Science
degree in Orthodontics at the May 2012 graduation.
Thesis Committee:
Lina Moreno, Thesis Supervisor
Deborah V. Dawson
James Wefel
Robert N. Staley
To my supportive parents, Scott and Julie
ii
ACKNOWLEDGMENTS
I would like to thank Dr. Lina Moreno for all of her dedication and guidance with
this research study. This project would not have been possible without her and the rest of
my thesis committee, Drs. Deborah Dawson, Robert Staley, and James Wefel. A special
thanks to Colleen Kummet for all her hard work and time dedicated to this research, as
well as Patricia Hancock and Chicka Takeuchi for gathering the sample for this study.
I am very grateful to Dr. Tom Southard and the rest of the faculty at the
University of Iowa, Department of Orthodontics, for giving me the opportunity to further
my education and pursue a career in orthodontics.
Finally, I would like to thank my wonderful family for all the support and love
they have given me.
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TABLE OF CONTENTS
LIST OF TABLES .............................................................................................................. v
LIST OF FIGURES ........................................................................................................... vi
INTRODUCTION .............................................................................................................. 1
Purpose of this Study ...................................................................................................... 2
LITERATURE REVIEW ................................................................................................... 3
Etiology of Class II Malocclusion .................................................................................. 3

Treatment of Malocclusion ............................................................................................. 6
Facial Morphology Studies ............................................................................................. 7
Phenotypic Characterization of Class II Malocclusion Using Multivariate Reduction
Methods: ....................................................................................................................... 11
Heritability .................................................................................................................... 16
Importance of Genetic Studies ...................................................................................... 17
MATERIALS AND METHODS:..................................................................................... 23
Sample........................................................................................................................... 23
Patient Inclusion/ Exclusion Criteria ............................................................................ 23
Procedure ...................................................................................................................... 24
Inter-rater and Intra-rater Reliability ............................................................................ 29
Statistical Analysis ........................................................................................................ 30
RESULTS ......................................................................................................................... 37
DISCUSSION ................................................................................................................... 66
Limitations of the Study................................................................................................ 69

Future Projects .............................................................................................................. 70
SUMMARY AND CONCLUSIONS ............................................................................... 71
REFERENCES ................................................................................................................. 72
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LIST OF TABLES
Table 1 Eligibility Criteria ..............................................................................................24
Table 2 Cephalometric Variables ....................................................................................26
Table 3 Cephalometric Landmarks .................................................................................27
Table 4 CII Intra Rater Ceph Variables ..........................................................................34
Table 5 CIII Intra Rater Ceph Variables .........................................................................35
Table 6 CIII Inter Rater Ceph Variables Howe vs. Vela ................................................36
Table 7 Eigenvalues of the Correlation Matrix ...............................................................42

Table 8 Summary of Class II Malocclusion Cluster Analysis ........................................44
Table 9 PC1 Cephalometric Variables ............................................................................46
Table 10 PC2 Cephalometric Variables ...........................................................................48
v
LIST OF FIGURES
Figure 1 CII division 1 Malocclusion.............................................................................5
Figure 2 CII division 2 Malocclusion.............................................................................5
Figure 3 Six Horizontal Subgroups from Moyers 1980 ...............................................14
Figure 4 Five Vertical Types from Moyers 1980 .........................................................15
Figure 5 Scree Plot .......................................................................................................41
Figure 6 Component Scores 95% Prediction Ellipse ...................................................42
Figure 7 Pseudo F and CCC Values by Cluster Number .............................................43

Figure 8 Plots of Clusters of Class II Malocclusion Subjects- Centroids Labeled ......44
Figure 9 3D Plot of 5 Clusters of Class II Malocclusion .............................................45
Figure 10 PC1 Extremes.................................................................................................47
Figure 11 PC1 Quartiles .................................................................................................47

Figure 21 PC6 Quartiles. ................................................................................................57
Figure 23 PC7 Quartiles. ................................................................................................59
Figure 24 Cluster Centroids with Descriptions ..............................................................60

Figure 25 Cluster 1 Centroid and Core with Description ...............................................61
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1
INTRODUCTION
Malocclusion, which may involve misalignment of the teeth, mal-positioning of
the jaws, or a combination of both, can create detrimental effects to a person‟s overall
facial esthetics, depending on the severity. In 1890, Edward Angle described three types
of malocclusion based on molar positioning. Class I malocclusion occurs when molars
are in proper position (mesiobuccal cusp of upper 1st permanent molar aligns with the
buccal groove of the lower 1st permanent molar) and is present in approximately 50%-
55% of the U.S population. Class II malocclusion is present in 15% of the U.S.
population and clinically appears with the maxillary molar ahead of the mandibular
molar. Patients usually have a convex profile with a retrusive mandible. Occurring least
commonly (1%), Class III malocclusion is seen when the maxillary molar is distal to the
mandibular molar and patients tend to have a concave profile.
There is a wide range of severity amongst malocclusion cases. The more severe
the case, the greater the psychosocial and functional problems present (Proffit, Fields, &
Sarver, 2007). To correct large jaw discrepancies and improve esthetics and function,
sometimes both orthodontic and surgical treatments are needed (Capelozza, de Araaujo
Almeida, Mazzottini, & Cardoso Neto, 1989). The exact etiology of these malocclusions
remains unclear. Both genetic and environmental factors may affect craniofacial
development, creating an intricate and elaborate multifactorial etiology for malocclusion
(Mossey, 1999a). Due to the significant genetic complexity in the formation of the face
and jaws, it is difficult to ascertain what genes are affecting various features in a
particular malocclusion case. Further research in this field can improve treatment
modalities and help in the prevention of moderate to severe malocclusion.

2
Purpose of this Study
The purpose of this study is to characterize the skeletal and dental variation
present in Class II malocclusion into distinct phenotypes to reduce heterogeneity and
empower studies aimed at identifying the etiology of malocclusion. This will specifically
be done using cephalometric radiographic landmarks and statistical data reduction
methods to find the most common phenotypic groupings of Class II patients. Having
Class II patients separated into homogenous phenotypic groups will provide the basis for
future studies to more precisely look at the genetic variation and environmental influence
on each subgroup of Class II malocclusion.

3
LITERATURE REVIEW
Etiology of Class II Malocclusion
It is important to understand the etiology of malocclusion so that orthodontic
treatments can focus more on the prevention of these conditions and their related
craniofacial deformities. The interaction of various genes has been shown to be the
primary cause of an unbalanced and malformed craniofacial complex (Ionescu,
Teodorescu, Badarau, Grigore, & Popa, 2008). An estimated two-thirds of the 25,000
human genes contribute to craniofacial development (Proffit, Fields, & Sarver, 2007).
Craniofacial structures are developed from complex processes of tissue
interactions, cell migrations, and coordinated growth (Kouskoura et al., 2011; Nieminen
et al., 2011). Neural crest cells are thought to be controlled by homeobox genes and their
derivations include the maxilla, mandible, zygomatic, nasal bones and bones of the
cranial vault. Homeobox genes, specifically Msx-1 and Msx-2, regulate expression
through proteins in the growth factor family and steroid/thyroid/retinoic acid superfamily.
Disruption and poor control in the migration of the neural crest cells can produce dento-
alveolar abnormalities and many craniofacial anomalies such as Treacher Collins
syndrome and hemifacial microsomia (Mossey, 1999a; Proffit, Fields, & Sarver, 2007).
Other abnormalities in the embryologic developmental stages can elicit many more
craniofacial malformations (Proffit, Fields, & Sarver, 2007). Disruption of the
embryologic process may result in missing or malformed dentition, cleft lips and palates,
craniosynostosis and more serious conditions like holoprosencephaly (Kouskoura et al.,
2011; Wilkie & Morriss-Kay, 2001).
Ionescu et al. proposed that the environment plays a large role in the development
of the craniofacial structure. The three main contributing factors include changes in
mastication affecting masticatory muscle development, abnormalities in the normal

functions like breathing, deglutition and speaking, and damaging habits including thumb-
4
sucking, lip-sucking or resting tongue (Ionescu, Teodorescu, Badarau, Grigore, & Popa,
2008). Teratogens like drugs, alcohol and viruses can also affect the craniofacial
development. Abnormal pressures during intrauterine molding or trauma during
development may also result in abnormal jaw growth (Proffit, Fields, & Sarver, 2007).
Chronic illness, prolonged starvation and excessive stress are other factors that can hinder
growth and development (Mossey, 1999a).
Both genetic and environmental influences play a role in the development of
Class II malocclusion. Studies of Class II division 1 patients have shown that this
condition is heritable and is consistent with a polygenic mode of inheritance. A polygenic
model implies that a number of genes with small additive effects provide genetic
predisposition to the phenotypic expression observed in the class II division 2
malocclusion. There is a definite environmental contribution to this malocclusion as
well. Disparate muscular pressures, including the tongue and lips, can enhance
proclination of maxillary incisors or retrocline lower incisors, creating a larger horizontal
distance between the maxillary and mandibular incisors, also known as increased overjet
(Mossey, 1999b).
Class II division 2 patients have more definable characteristics commonly
occurring together, which helps elicit a more obvious genetic component than Class II
division 1 patients (Mossey, 1999b). Thicker upper and lower lips were found in Class II
division 2 patients compared to Class I controls (McIntyre & Millett, 2006). In addition,
the Class II division 2 subjects had greater lower lip contact and thus increased resting
pressure on the maxillary incisors than the controls. This could be a causal effect in
producing maxillary incisor retrusion and can be a concern with post-treatment stability
(Lapatki, Klatt, Schulte-Monting, & Jonas, 2007; McIntyre & Millett, 2006).
Habits such as mouth breathing, thumb-sucking, lip sucking, or an aberrant

swallowing pattern are local factors often associated with the initiation of a Class II
5
division 1 malocclusion or that may exacerbate the already existing malocclusion. To
successfully treat these cases and prevent relapse these local factors must be removed
(Smith, 1938). In addition, any factor disrupting the nasopharyngeal pathway, including
allergies or enlarged adenoids can possibly affect the occlusion adversely. To aid in the
prevention of malocclusion it is crucial to begin identifying and correcting the
environmental factors that contribute to a disharmony in the face and jaws (Ionescu,
Teodorescu, Badarau, Grigore, & Popa, 2008).
Figure 1 CII division 1 Malocclusion
Figure 2 CII division 2 Malocclusion

6
Treatment of Malocclusion
Treatment of patients with Class II malocclusion depends on the severity, the
growth potential, and the patient‟s desires. The options for correction of mild to
moderate jaw discrepancies are a combination of growth modification and orthodontic
treatment (Bailey, Proffit, & White, 1999). Although the growth potential of patients is
unknown, the presence of a distal step (Class II) in the primary dentition almost always
elicits a Class II malocclusion in the permanent dentition (Bishara, 1981). If there are
inadequate amounts of growth remaining, orthodontic camouflage of the underlying
skeletal discrepancy could possibly be an option. The effect, however, of extractions or
camouflage treatment on a person‟s facial esthetics must be considered (Bailey, Proffit, &
White, 1999).
An innovative treatment methodology for orthodontic-only correction of
malocclusion is the temporary anchorage device or TAD (Shu, Huang, & Bai, 2011).
These specialized bone screws or mini-plates are usually placed with local anesthetic and
provide skeletal anchorage to move teeth and may possibly eliminate the need for
surgical procedures in certain cases. Anterior open bites, which are commonly seen in
class II cases that would generally require surgical correction, have been treated with
intrusion of the molars using TADs as anchorage (Costello, Ruiz, Petrone, & Sohn, 2010;
Sandler, Madahar, & Murray, 2011). This technique can also provide benefit with the
anterior-posterior correction needed in Class II patients as the control of the vertical
dimension can result in autorotation of the mandible creating an improved chin projection
and profile (Upadhyay, Yadav, & Nanda, 2010). With proper protocol and treatment
planning, temporary anchorage devices may possibly eliminate the need for surgical
treatment in moderate class II cases. However, more research and long-term clinical
studies are needed to determine the limitations and benefits of their use (Rossouw &
Buschang, 2009).
7
Patients with severe underlying skeletal discrepancies may not be corrected with
orthodontics alone and may require orthognathic surgery. The prevalence of severe Class
II malocclusions (using 7mm or greater of overjet as an estimator), possibly requiring
orthognathic surgery, is estimated at 1 million individuals in the United States with
approximately 24,000 new cases added annually (Bailey, Proffit, & White, 1999).
Facial Morphology Studies
Much research has been done to study the specific craniofacial morphology of
Class II patients. Most studies have analyzed and compared cephalometric landmarks to
determine commonly occurring skeletal and dental characteristics of this malocclusion.
In a McNamara study (1981) of Class II malocclusion cases, 277 cephalometric
radiographs of children age 8 to 10 years old were examined. All children were dentally
Class II division 1 or Class II division 2. One vertical and four horizontal components
were measured to determine the distribution of these components amongst the Class II
patients. There were many combinations of skeletal and dental measurements revealed.
The most common component made up approximately 10% of the total sample and
included a retrusive mandible, normal position of the maxilla, normal position of the
maxillary and mandibular dentition and excess vertical face height. In this particular
study, approximately half of the sample presented with anterior vertical excess. The
second most common grouping involved the maxilla in a more retrusive position and the
same findings in all other features as the first component.
This study, along with other studies, demonstrates that there are various
components, both horizontal and vertical, that occur in a multitude of combinations
resulting in Class II malocclusion (Moyers, Riolo, Guire, Wainright, & Bookstein, 1980;
Sassouni, 1969; Sassouni, 1970). Yet, a retrusive mandible and anterior vertical excess
are common findings amongst Class II cases. Unlike McNamara, maxillary dental
8
protrusion was also a common feature. The facial verticality of Class II patients was
studied based on amount of overjet patients presented with. In those Class II patients
with normal overjet, a hypodivergent face, or shorter anterior facial height was found.
The increased overjet group (3-6mm) had normal vertical measurements and the extreme
overjet group (>6mm) presented with a hyperdivergent face, or anterior vertical excess
(Saltaji, Flores-Mir, Major, & Youssef, 2011). The maxillary arch widths of both
subgroups (division 1 and 2) of Class II malocclusion have been shown to have more
constriction than those of Class I malocclusion (Ingervall & Lennartsson, 1973;
Marinelli, Mariotti, & Defraia, 2011).
In a study of Class II division 1 non-growing females, many dental and skeletal
differences existed between these subjects and the Class I controls. The maxilla, in the
Class II division 1 group, was in proper antero-posterior position but the mandible was
undersized and more retrognathic. There was also a tendency for the mandible to be
rotated down and back, due to a shorter ramus height and thus an increase in the
mandibular plane angle. Dentally, maxillary incisors were found to be at a normal
inclination, however the lower incisors were more proclined than the Class I controls.
The cranial base angle was also greater for Class II division 1 patients (Sayin &
Turkkahraman, 2005).
While studies have examined Class II division 1 characteristics, there has also
been significant focus on Class II division 2 malocclusion. Based on the many skeletal
and dental findings that differ from both the Class I and Class II division 1
malocclusions, Class II division 2 patients should be delineated as a separate
malocclusion category (E. A. Al-Khateeb & Al-Khateeb, 2009). Angle first described
Class II division 2 malocclusion based only on the dento-alveolar characteristics. Many
studies have shown that significant skeletal components occur within the division 2
subtype (Brezniak et al., 2002; E. A. Al-Khateeb et al., 2009; S. Peck et al., 1998).
9
Through cephalometric landmarks, a normal positioned maxilla and retruded mandible
were common findings, similar to previous morphologic studies of Class II malocclusion,
in general. Other notable skeletal characteristics of this malocclusion include an acute
gonial angle, increased posterior facial height, decreased anterior facial height and an
effective pogonion. Dentally, the maxillary incisors appeared retroclined and the
mandibular incisors were either retroclined or normal. Patients of this subgroup also
presented with a deep overbite possibly due to the skeletal vertical deficiencies, creating a
forward rotation of the mandible rather than overeruption of the mandibular incisors
(Brezniak et al., 2002). It appears from some studies that morphologically and clinically,
the Class II division 2 malocclusion patients tend to be more similar to those with Class I
malocclusion than to those with Class II division 1 malocclusion (Fisk, Culbert, Grainger,
Hemred, & Moyers, 1953).
“Coverbite” is a term to describe 100% or greater overbite of incisors. Overbite is
the term used to describe the vertical overlap of the maxillary incisor over the mandibular
incisor during occlusion of the teeth. An overbite of 2mm is considered ideal. Through
the cephalometric and dental exam of the „coverbite‟ malocclusion, several distinct
dento-skeletal features were found. The Class II division 2 deep bite population in this
study, representing 17% of the total, was shown to have increased vertical posterior
growth with anterior forward rotation of the mandible, thus creating a greater overbite.
Although both the maxilla and mandible appeared normal in size, the anterior dentition in
both males and females were smaller than in the controls. In addition, the mandibular
basal bone formation was increased creating a stronger chin projection (S. Peck, Peck, &
Kataja, 1998).
Other research has focused on discerning the skeletal and dental differences
between the subtypes of Class II malocclusion. Pancherz et al. (1997) compared

cephalometric radiographic measurements between Class II division 1 and Class II
10
division 2 subjects and found no distinct skeletal or dental differences between the two
subgroups besides position of the maxillary incisors. In this study, approximately 50% of
the Class II division 1 patients also had proclination of the mandibular incisors, which
was not a common finding in the division 2 patients. Unlike the study by McNamara
(1981) and others, a common feature was a shorter lower face height. Another
comparison to note was that the ANB difference lessened in Class II division 1 patients
as their age increased. This did not occur in Class II division 2 patients and was thought
to be contributed to maxillary incisor retrusion restricting forward growth of the
mandible. Other studies, however, have shown that incisor positioning had no effect on
mandibular growth. For instance, Demisch et al. (1992) concluded that there was no
forward positioning or additional forward growth of the mandible in Class II division 2
patients once the maxillary incisors were uprighted and the bite was opened with
orthodontic appliances. This was indicative that any such growth restriction of the
mandible was not caused by the retroclined upper incisors (Demisch, Ingervall, & Thuer,
1992).
In summary, many morphological studies have been done to determine distinct
structural characteristics of Class II malocclusion along with differentiating the Class II
subtypes. Many of these previous studies have performed such morphological

characterization by utilizing descriptive analyses and testing correlations in
cephalometric variables derived from different developmental stages, malocclusion
severities and across global populations (McNamara, 1981; Battagel, 1993; Pancherz et
al., 1997; Lau and Hagg, 1999; Brezniack et al., 2002; Sayin and Turkkahraman, 2005).
More recently, more sophisticated multivariate methods for cephalometric data
analysis such as cluster procedures, principal components analysis (PCA), and
discriminant function analysis (DFA) have also been utilized in the characterization of
malocclusion components for both class II (Moyers et al., 1980) and class III (Finkelstein
11
et al., 1989; MacKay et al., 1992; Lu et al., 1993; Tahmina et al., 2000; Stellzig-
Eisenhauer et al., 2002; Bui et al., 2006) patients. In addition, facial form analysis derived
from lateral cephalograms using different methods such as elliptical fourier functions,
thin-plate spline analysis, and finite elements analysis have been performed in class I
(Lowe et al., 1994; Franchi et al., 2001), II (Lowe et al., 1994; Franchi et al., 2007) and
III patients (Lowe et al., 1994; Singh et al., 1997a, 1997b, 1998, 1999; Baccetti et al.,
1999; Alkhamrah et al., 2001) offering additional possibilities for classification of facial
shapes beyond those that conventional cephalometric studies are able to explain. A
thorough review of such methods for facial form analyses (i.e. elliptical fourier functions,
thin-plate spline and finite element analysis) is beyond the scope of this work, and
therefore our next section will only summarize results from studies that utilized
multivariate reduction methods for conventional cephalometric data that are directly
relevant to the methods used in this thesis (Moyers et al., 1980).
Phenotypic Characterization of Class II Malocclusion
Using Multivariate Reduction Methods:
One of the most complete descriptions of phenotypic characterizations of the

Class II malocclusion is that presented in Moyers et al. (1980) using cephalometric data
of Class II patients and data reduction methods. A cluster analysis produced subgroups
based on horizontal and vertical characteristics present in Class II malocclusion in a
sample of 610 patients. A total of 6 horizontal subgroups (A-F) and 5 vertical subgroups
(1-5) were defined based on common phenotypic characteristics. Four of the horizontal
groups (B, C, D, and E) were “syndromic types” and represented truly defined and
distinct Class II groups, both skeletally and dentally. The other two subgroups (A and F)
presented with less severe characteristics of Class II malocclusion and thus were less
well-defined Class II entities.
12
Amongst the horizontal subgroups, Group A represented the “pseudo Class II”
which had normal skeletal relations but had maxillary dental protrusion. Group B
displayed maxillary skeletal and dental protrusion and normal mandibular positioning and
size. Group C had bimaxillary retrusion, maxillary dental protrusion and mandibular
dental protrusion. Both the maxilla and mandible were smaller in size. Group D was
similar to Group C, but did not include mandibular dental protrusion. Group E was
maxillary prognathic with bimaxillary dental protrusion. Group F was the largest and
least well-defined group and displayed mandibular retrusion. Figure 3 illustrates the
horizontal subgroups as shown in Moyers (1980).
The vertical components were less well defined and certain types usually occurred
within one particular horizontal subgroup more frequently. The specific features of each
vertical subgroup are described below and illustrated in Figure 4 (With broken lines
representing features of that group, and solid lines depicting normal vertical). The Type
1 group presented with a steeper mandibular plane, creating a much greater anterior
vertical face height. A flatter mandibular plane, occlusal plane and palatal plane were
associated with the Type 2 group. In Type 3, the palatal plane tipped upward in the
anterior. Vertical Type 4 had an anterior downward tipping palatal and occlusal plane,
possibly creating an excess gingival display at rest. This vertical grouping was always
associated with the horizontal group B. Type 5 presented with a downward tipped palatal
plane and skeletal deep bite (Moyers, Riolo, Guire, Wainright, & Bookstein, 1980).
In summary, studies of phenotypic characterization utilizing single (McNamara,
1981; Pancherz et al., 1997; Sayin and Turkkahraman, 2005) and multivariate analyses
(Moyers et al., 1980) have found that a large amount of variation in skeletal and dental
measurements exist for both types of Class II malocclusion, yet the causes of such
skeletal and dental variation still remains elusive (Pancherz, Zieber, & Hoyer, 1997).
These various unfavorable combinations of morphological characteristics result in a Class
13
II malocclusion and should be carefully characterized and considered for diagnosis and
treatment purposes (Fisk, Culbert, Grainger, Hemred, & Moyers, 1953).
Through phenotypic characterization of a particular malocclusion, similar growth
patterns and treatment approaches for individuals with the same horizontal and vertical
components would be expected. Analysis of this information can improve treatment
modalities and allow for more appropriate treatment timing (Moyers, Riolo, Guire,
Wainright, & Bookstein, 1980). Moreover, this distinct characterization will be crucial in
determining the etiology of Class II malocclusion as having well-defined phenotypes will
reduce heterogeneity and empower future genetic and environmental studies of
malocclusion.
14
Figure 3 Six Horizontal Subgroups from Moyers 1980

15
Figure 4 Five Vertical Types from Moyers 1980

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Heritability
The extent to which a particular trait is due to genetics is termed heritability
(Mossey, 1999a). Twin studies, especially those of monozygotic twins, are important and
useful in determining the effects of both genetic and environmental interaction and also
environmental factors alone on specific traits. Any difference found in the morphological
structure of monozygotic twins, who have identical genetic makeup, would be due to the
environment, whereas dizygotic twins, who have approximately 50% of genetic
correlation, will have differences due to the environment and genetics (Watnick, 1972).
Twin studies, along with parent-offspring genetic correlation studies, which show the
effects of additive genes through the variation between parent and offspring, help
determine the hereditary nature of the craniofacial complex (N. Nakata, Yu, Davis, &
Nance, 1973).
The extent to which genetics versus environmental factors affect the development
of Class II malocclusion remains unclear (Ruf & Pancherz, 1999). Class II div. 2
malocclusion, as discussed before, has specific morphologic features and has been
documented to have a strong hereditary component (S. Peck, Peck, & Kataja, 1998).
Studies in twins with class II div. 2 showed that monozygotic twins displayed high
concordance within malocclusion features, while dizygotic twins showed 90%

discordance in these features (Mossey, 1999b).
Research studying bony contours, which represented areas of bone deposition and
resorption, or potential growth sites, were compared in twin studies to determine
structures of greater heritability (Watnick, 1972). In these twin studies by Watnick, the
following defined areas were shown to be more under genetic control: the lingual
symphysis, lateral surface of the ramus, and frontal curvature of the mandible whereas
environmental components have shown to effect areas like the antegonial notch (Mossey,
1999b). Other twin studies have shown strong heritability (greater than 70%) in dental
17
features including number of third molars present, tooth crown size, arch width, and
maxillary lateral incisor malformation (Liu, Deng, Cao, & Ono, 1998).
Many studies have shown a strong familial segregation for the Class II division 2
„coverbite‟, which may occur through an autosomal dominant mode of inheritance with
incomplete penetrance (i.e. the percentage of patients in the population who present the
disease causing mutation and expressed the specific phenotype). However, another
accepted mode of inheritance is that of a polygenic inheritance with many genes acting
additively (S. Peck, Peck, & Kataja, 1998).
Nakasima et al (1982) studied lateral and frontal cephalometric variables of Class
II and Class III Japanese patients and their parents. Stronger correlation coefficients
existed amongst skeletal measurements compared to dental measurements. All skeletal
measurements between parent and offspring were highly correlated (highest correlation
coefficient found was 0.502) in both the Class II and Class III groups consistent with a
polygenic mode of inheritance (Nakasima, Ichinose, Nakata, & Takahama, 1982).
Importance of Genetic Studies
Discrete traits, like cystic fibrosis, are more easily defined and can be explained
through a simple Mendelian mode of inheritance (Borecki & Rice, 2010). More
complex traits, like craniofacial anomalies, are continuous and multifactorial in nature,
thus creating a more complex mode of inheritance (Mossey, 1999). Identification of
chromosomal regions involved in a particular genetic trait requires familial studies to
compare genomic regions between affected members in a pedigree, an approach called
linkage analysis. However, it can also be accomplished by means of association analysis
which requires the comparison of genomic regions between unrelated individuals sharing
similar phenotypes (Ellsworth & Manolio, 1999; Townsend, Hughes, Luciano,
Bockmann, & Brook, 2009).

18
A linkage or an association analysis is utilized depending on the etiologic
architecture of the disease, followed by fine mapping of that chromosomal segment to
isolate a gene of interest (Ellsworth & Manolio, 1999b). Linkage analyses relate
phenotypes of genetically related individuals to specific similar locations on their
genomes and are more suited for the identification of rare variants with high impact on
the particular trait. Once more defined chromosomal regions have been identified
through a linkage analysis, candidate genes influencing a trait may be isolated.
Regression-based methods and association analyses are applied to unrelated individuals
(i.e. case-control designs) and have more power in the identification of common variants
with a smaller impact on the trait in question. These methods are used to determine the
variation within the gene as it relates to the variation in phenotypic expression of a trait
(Townsend, Hughes, Luciano, Bockmann, & Brook, 2009).
Genetic susceptibility to a disease is most commonly due to single nucleotide
polymorphisms (SNPS) (Wang & Moult, 2001), where a mutation presents with a single
base substitution and creates one nucleotide difference in two strands of DNA (Ellsworth
& Manolio, 1999a). Genomic association studies, which occur through correlating SNPS
with a certain complex trait, have elicited major advances in determining genetic
influence of a many complex traits (Manolio, 2010). One gene that accounts for multiple
phenotypes is said to be pleiotropic. Pleiotropy occurs in complex traits approximately
17% in genes and 5% in SNPs (Sivakumaran et al., 2011). Because of the pleiotropic
effect, a deleterious mutation in one gene can account for multiple morphologic
abnormalities (Mossey, 1999a) complicating our ability to discern etiologic mutations
that can explain malocclusion.
Very few human genetic studies to identify genes conferring susceptibility to
malocclusion have been performed. There have been more studies to identify the genetic
19
causes for class III than for class II malocclusion. This is likely due to the notion that
class III has a much stronger genetic component than the class II malocclusion.
Class III malocclusion mode of inheritance has been studied through family
pedigrees and the results suggest an autosomal mode of inheritance with a multifactorial
component. This possible Mendelian transmission includes a major gene with
incomplete penetrance (Cruz et al., 2008; Frazier-Bowers, Rincon-Rodriguez, Zhou,
Alexander, & Lange, 2009). A genome-wide linkage study was performed on Korean
and Japanese families with mandibular prognathism. Evidence of linkage was apparent
on 3 chromosomal loci: 1p36, 6q25, and 19p13. Loci 1p36 returned the highest linkage
result indicating the most likely genomic location for genes contributing to mandibular
prognathism (Yamaguchi, Park, Narita, Maki, & Inoue, 2005, Xue, Wong, & Rabie,
2010). In addition, the gene EPB41 was also determined to be associated with
mandibular prognathism (Xue, Wong, & Rabie, 2010). In a study of Hispanic families, 5
loci were found for linkage to mandibular prognathism or maxillary retrusion, which are
common features present in Class III malocclusion. They are 1p22, 3q26.2, 11q22,
12q13.13, and 12q23. One loci was found on chromosome 1, similar to the finding by
Yamaguchi, suggesting a common upstream genetic element regulating craniofacial
growth and influencing Class III tendency. Differences may lie in the racial variation of
each study and in the phenotypic differences of Class III malocclusion (Frazier-Bowers,
Rincon-Rodriguez, Zhou, Alexander, & Lange, 2009).
Genetic association has been found between mandibular ramus height and the
Growth hormone receptor gene (GHR). Two independent studies in Japanese and
Chinese Han demonstrated that single nucleotide polymorphisms (SNPs) within GHR
contributed to the differences in the mandibular ramus morphology (Tomoyasu et al.,
2009; Zhou et al., 2005).

20
Studies relating genetic predisposition to mandibular hypoplasia in humans are
limited. Most often in the literature, genetic descriptions of mandibular ramal deficiency
associated with craniofacial anomalies, like hemifacial microsomia or Pierre Robin
syndrome, are prevalent, as one of many clinical manifestations in these syndromes is
mandibular micrognathia and subsequential retrognathia (Pirttiniemi, Peltomaki, Muller,
& Luder, 2009). Other research has demonstrated the genetic effects that can account for
mandibular deficiencies in animals, however, the link to humans has not been made yet.
Quantitative trait loci (QTL) analyses have been beneficial in determining gene
identification for specific phenotypes (Nadeau & Frankel, 2000). This has been done in
mice by measuring the mandibular length, using gonion to menton as reference points,
and finding significant genetic linkage (2 significant QTLs) to areas on chromosome 10
and 11 that account for the differences in length. In addition, more sophisticated methods
of characterization of the mandible in mice have been performed utilizing Geometric-
morphometric approaches which use linear and angular measurements and identification
of specific landmarks to determine how they influence size and shape. This multivariate
analysis has been used to determine the linkage of QTLs and their effect on the size and
shape of mice mandibles using the Procrustes superimposition and 5 morphological
landmarks. Twelve QTLs were identified for size and 25 QTLs for shape, however there
was no morphologic clustering into distinct groups (Klingenberg, Leamy, Routman, &
Cheverud, 2001). This information can help perpetuate the study of gene regulation in the
size of the human mandibles as the chromosomal regions in mice correspond to particular
regions in human chromosomes (Dohmoto, Shimizu, Asada, & Maeda, 2002).
Other studies have suggested that Bone Morphogenic Proteins (BMPs), which
induce bone and cartilage formation, play a role in the development of the mandible, but
the extent of their involvement is unclear. The signal of the BMPs is strongly regulated
and proteins, such as Chordin (Chd) and Noggin (Nog), are BMP antagonists required for
21
normal mandibular development. Studies of mutant mice missing one or both copies of
the Chordin and Noggin gene elicited phenotypes displaying mandibular hypoplasia,
micrognathia and even agnathia. Mandibular malformations occurred most frequently
with mice missing both copies of the noggin gene (Stottmann, Berrong, Matta, Choi, &
Klingensmith, 2006). In a study of Colombian families with Class II tendency all
affected individuals were found to be homozygous for the rare allele in SNP rs1348322
on the Nog gene, but the exact effect of the polymorphism is uncertain (S. J. Gutierrez et
al., 2010).
The genetic involvement in the etiology of mandibular deficiency has been
studied as well as the ability to genetically alter this preexisting condition in animal
models. As many Class II malocclusion patients present with mandibular hypoplasia,
another avenue of treatment is using gene therapy to help stimulate condylar growth.
Injection of recombinant adeno-associated virus mediated vascular endothelial growth
factor (rAAV-VEGF) of rat condyles has stimulated condylar growth and induced bone
formation, increasing the length of the condyle as well as the mandibular length. By
either stimulating growth or being an alternate source to defective genes, gene therapy
could be beneficial in eliciting increased mandibular growth (Dai & Rabie, 2008).
Elucidating the etiology of malocclusion is important, as it will provide more avenues of

treatment to aid in the prevention of malocclusion.
Heritability not only accounts for skeletal features but also for most dental
features. For example, the eruption of primary teeth is estimated to be over 70%
heritable (Pillas et al., 2010). Tooth size and shape are genetically linked, as seen in
numerous twin studies. More recently, genome-wide association studies (GWAS) have
been used to isolate specific genes causing particular dental phenotypes and anomalies
(Townsend, Bockmann, Hughes, & Brook, 2012). Two separate GWAS studies isolated
4 loci that are associated with the eruption of permanent teeth in children, and 5 loci that
22
were significant in the eruption of primary teeth. Two of the loci were common in both
groups (Geller et al., 2011; Pillas et al., 2010). Moderate to severe crowding is a
common dental finding and has been significantly associated with 5 SNPs and the EDA
and XEDAR genes in a Chinese population (Ting, Wong, & Rabie, 2011). Determining
the genetic component of both dental and skeletal characteristics will be beneficial in
eliciting the best orthodontic treatment methods.
Orthodontic treatment attempts to change the phenotypic expression of a
malocclusion problem. If most pathological features present within a malocclusion case
are greatly determined by genetics, the more difficult it will be to try to achieve growth
modification or orthodontic correction. In cases with severe malocclusion, surgical or
palliative limited treatments are usually the only options. Elucidating the cause and
aiding in prevention of malocclusion will come from a better understanding of the genetic
role in the manifestation of particular malocclusion phenotypes (Mossey, 1999).
However, such malocclusion phenotypes need to be well characterized to decrease
heterogeneity, avoid misclassification of affected individuals, and empower genetic and
environmental studies.
With recent technological advances that allow the simultaneous characterization
of entire genomes via high throughput genotyping of SNPS or sequencing of the genome
to evaluate human genetic variation, future gene and gene-environment studies of
malocclusion can be performed on precisely defined phenotypes. This will provide
valuable insights into the etio-pathogenesis underlying malocclusion (Cantor, Lange, &
Sinsheimer, 2010; Manolio et al., 2009; Thomas, 2010).

23
MATERIALS AND METHODS:
The study protocol was reviewed and approved by the institutional review board
at the University of Iowa.
Sample
The sample consisted of 309 healthy Caucasian subjects (227 female, 82 male;
age range: 16-60 years) who met specific inclusion criteria. 2-D pre-treatment records
were used from the University of Iowa College of Dentistry and Hospital Dentistry at the
University of Iowa. All had a full set of pre-orthodontic treatment records including
lateral cephalographs, intra and extra-oral photos, and models. The original sample
included 346 individuals; 37 of non-Caucasian race were excluded. Data on the
remaining 309 subjects were used for this analysis.
Patient Inclusion/ Exclusion Criteria
To qualify for our study the subjects must have been of age 16 years or older for
females and 18 years or older for males. Two or more of the following criteria were also
required: ANB ≥ 4, overjet ≥ 4; Angle Class II molar or canine relationship on at least
one side and the determination of a convex profile. Profile convexity or concavity was
determined by measuring the internal angle between a line from the nose bridge to the
base of the upper lip and a line from the base of the upper lip to the chin. A smaller angle
and a forward-positioned upper jaw relative to the chin were indicative of a convex
profile. A concave profile was indicated by a larger angle and a backward-positioned
upper jaw. Exclusion criteria were any of the following: History of facial trauma;
previous orthodontic treatment; presence of facial syndromes; missing or poor quality
records; missing teeth other than third molars; impacted teeth or completely blocked
teeth. The following Table 1 illustrates all the inclusion and exclusion criteria for Class
24
II subjects. These stringent eligibility criteria were chosen to minimize heterogeneity and
increase our explanatory power of the variation observed.
Table 1 Eligibility Criteria

CII Inclusion Criteria Exclusion Criteria
*at least 2 or more
required
Adult (female > 16, male History of severe facial

> 18) trauma
Previous orthodontic
ANB > 4
treatment
Presence of facial
Overjet > 4
syndromes
Angle Class II molar or

Missing or poor quality
canine relationship on
records
at least one side
Missing teeth other than
Convex profile rd
3 molars
Impacted teeth
Procedure
346 Class II patients were selected based on the inclusion/exclusion criteria.
Lateral cephalometric radiographs of all patients were acquired and any film radiographs
were scanned and imported into Dolphin Imaging with a 100mm ruler. All analog
25
cephalographs were calibrated to eliminate any magnification difference between digital
and film radiographs (Cohen, 2005). Distance measures for film radiographs were scaled
(multiplied by 0.8929 for 12% magnified cephs from the College of Dentistry and 0.8850
for 13% magnified cephs from Hospital Dentistry) to match the digital radiographs in
size.
A total of 63 lateral cephalometric variables were derived from the 33
cephalometric landmarks traced (Table 2 and 3). The variables are both linear and
angular and are the most commonly used in lateral cephalometric analyses (Bishara,
1981; Bui, King, Proffit, & Frazier-Bowers, 2006; McNamara, 1984).
The first column provides all cranial base measurements. These include the
position of the maxilla and mandible, respectively, to the cranial base landmarks, such as
sella and nasion. The second column represents all intermaxillary measurements. Any
dental related and soft tissue measurements are noted in the third column.
Inter-rater and Intra-rater reliability of cephalometric landmark location were
achieved prior to the Class II cephalometric data collection. More than half of the
subjects had film cephalometric radiographs and because of the less accurate detail in
many of these scanned in x-rays, all film radiographs were measured twice and averaged
for better accuracy. Digital radiographs were measured once.

26
Table 2 Cephalometric Variables

27
Table 3 Cephalometric Landmarks

28
Table 3. Continued
29
Inter-rater and Intra-rater Reliability
To achieve inter-rater and intra-rater reliability on the location of cephalometric
landmarks, 15 cephalometric radiographs were randomly selected from both the Class II
and Class III subjects and measured twice by two examiners. The first measurements for
examiner one were compared to the same first measurements of examiner two. For the
comparison of the data sets for the two examiners, a one-way Anova test was created for
each variable with groups defined by participant ID. The residuals from each model were
examined for normality using the Shapiro-Wilk test. After normality of the sample was
validated, the parametric Intraclass Correlation test by Shrout and Fleiss was used to
assess inter-rater agreement. The intraclass correlation is a parametric procedure used
when data are paired but it is impossible to assign one variable independent and the other
dependent. The intraclass correlation coefficient gives the proportion of variance
attributable to between group differences, and the null hypothesis for significance testing
is that this coefficient is equal to zero (H0=0) (Fleiss, Paik, & Levin, 2003; Zar, 2010).
The difference was calculated for each pair of measurements (first minus second
for intra-rater, and Vela minus Howe for inter rater) using a Wilcoxon Signed-Rank test
to determine if the median difference between the measurements from the two measures
was equal to zero (assuming symmetry). All analysis was done using SAS 9.2, and type
1 error of 0.05 was assumed.
For the 63 continuous variables, the intra class correlation showed excellent
agreement (listed in Table 4). The parametric ICC ranged from 0.7140 to 0.9988 with
ICC>0.80 for all but 1 variable indicating excellent intra-rater agreement between the two
measurements. The variables with ICC below 0.80 are highlighted in the table. Nine
variables had significant differences between the measures and the greatest mean
difference was 2.06mm.

30
The intra class correlation for the 63 Class III cephalometric variables are listed in
Table 5 and all variables had ICC>0.80 ranging from 0.9180 to 0.9991. Nine variables
had significant differences in the first and second measures, however, the greatest mean
difference was -1.13mm.
The ICC estimates of inter rater reliability for the 63 Class III cephalometric
variables ranged from 0.8435 to 0.9957, with all variables ICC>0.80 indicating excellent
reliability for all measures (Table 6). There were several variables with significant
differences in the dual measure, but all mean differences were less than 1.87mm.
Intra rater comparisons seem to have good to excellent for cephalometric
variables; this was true for the Class II and Class III individuals. The inter rater
comparison showed excellent agreements on all cephalometric measures. Any variables
with significant differences were identified and examined to improve accuracy and
reliabililty for landmark identification.
Statistical Analysis
Data were standardized using a linear model for age, gender and appropriate
interactions. A separate model was fit for each of the 63 cephalometric measures using
standard regression multiple regression methods. In all, four different configurations of

covariate adjustment were used among the 63 models: all included an adjustment for
gender, and some also required age adjustment, as well as an additional consideration of
gender by age interaction, i.e., different age adjustment for each gender. Model
diagnostic procedures were performed on all standardization models and assumptions
were validated. The studentized (normalized) residuals were extracted from these models
and used as the standardized data for the principal component analysis.
Data reduction methods including the Principal Component Analysis (PCA) and
the Cluster Analysis (CA) were used to determine the most homogenous phenotypic
groups. Principal component analysis is a multivariate technique often used for
31
quantitative data reduction prior to regression or cluster analysis. Components are the
eigenvectors of the correlation matrix and the 63 components were sorted in descending
order by eigenvalues which represent the variances of the components (Kleinbaum et.al,
1998). This simplification method allows easier analysis of the data and more defined
clustering of individuals. Principal component 1 will describe the phenotypic
characteristics that account for the most variance. By determining more principal
components (PC2, PC3, etc.), progressively less of the variance in the data is represented.
Most often PCs that describe less than 5% of the variance are questioned as being
biologically meaningful (Zelditch, 2004). Calculated using weights of all 63 original
variables, the standardized principal component scores were extracted for each subject
ensuring that the variances are standardized prior to employing the clustering algorithm.
SAS 9.3 statistical software was used to perform the partitional cluster analysis with
methods based on the leader (Hartigan, 1975) and the k-means (MacQueen, 1967)
algorithms using the method of Anderberg (1973) called nearest centroid sorting. The
process initiates with the selection of cluster seeds based on an estimate of cluster means
or centroids, each subject is then placed in the cluster of the nearest centroid according to
Euclidean distance measures. Recalculation of centroids and iterative assignments of
subjects to nearest clusters continues until the minimum of the sum of squared Euclidean
distances between subjects and cluster means is accomplished. The final cluster
assignments are achieved when the algorithm converges (i.e. no further changes occur in
cluster centroids).
To visualize the cluster analysis results, a canonical discriminant analysis was
performed and scored canonical variables were computed. The purpose of canonical
discriminant analysis is to identify axes (i.e., in this case, the (k-1) axes for k clusters)
that best separate the clusters. The canonical discriminant procedures result in linear
combinations of the standardized principal components that summarize between-cluster
32
variation similar to the way in which principal components summarize total variation.
These linear functions are uncorrelated and define a (k-1) space that best separates the
projections of the k groups into that space.
Canonical discriminant analysis (due to Hotelling, 1936) transforms the
standardized principal component scores used in the cluster analysis so that the pooled
within-cluster covariance matrix is an identity matrix. Cluster means are then computed
for the transformed variables. Finally, a principal component analysis is performed on
the means, weighting each mean by the number of observations in the class (SAS 9.3
Proc FASTCLUS documentation). The eigenvalues are equal to the ratio of between-
cluster variation to within-cluster variation in the direction of each resulting principal
component; the analogy to principal components analysis is evident.
The first canonical variable, or canonical component, is the linear combination of
the variables that has the highest possible multiple correlation with the groups (clusters).
This maximal multiple correlation is called the first canonical correlation, and the
coefficients of the linear combination are the canonical coefficients or canonical weights.
The second canonical variable is obtained by finding the linear combination uncorrelated
with the first canonical variable that has the highest possible multiple correlation with the
groups. This process can be repeated until the number of canonical variables is equal to
the original number of variables, or the number of classes minus one, whichever is
smaller. The scored canonical variables are used in this study to plot pairs or triads of
canonical variables in order to aid visual interpretation of cluster differences. R statistical
program was used in conjunction with the rgl package to produce 3 dimensional graphs
of the data.
The k-means clustering algorithm is sensitive to extreme values as a consequence
of the least squares condition; however no subjects in this dataset appeared to be extreme
observations. The clustering algorithm was performed separately for a range of number
33
of clusters, from 2 to 7 clusters. The criterion based methods of pseudo F statistic,
approximate expected over-all R2, and cubic clustering criterion (valid because of the
uncorrelated nature of principal components); as well as data visualization techniques of
scored canonical variables were used to determine the appropriate number of clusters.
Cluster validation was performed by locating subjects closest to the final cluster means
and examining the subject‟s cephalometric data and profile to ensure that clusters
represented distinct clinical phenotypes. All analysis used SAS 9.3 with a 0.05 level of
significance.
34
Table 4 CII Intra Rater Ceph Variables

35
Table 5 CIII Intra Rater Ceph Variables

36
Table 6 CIII Inter Rater Ceph Variables Howe vs. Vela

37
RESULTS
The goal of this project was to explore the structure of cephalometric
measurement data using clustering techniques in order to identify subphenotypes within
Class II malocclusion subjects. Results indicated that 7 principal components account for
80.95% of the total variance in the data. The 7 eigenvectors are orthogonal and
uncorrelated (these are distinct properties), representing perpendicular directions in
space. The most heavily weighted components are visible in descending order in the
Scree plot (Figure 5 and Table 7) and the components before the shoulder of the curve
were deemed to be the most meaningful and explain the most variation in the data set.
There were no outliers seen in the data set as determined by the 95% prediction ellipse
for all components (one example shown in Figure 6). Specific cephalometric variables
are represented with each component (Tables 9, 10, 11, 13, 14, 15). Principal component
1 (PC1) refers to the vertical dimension in regards to the angulation of the mandibular
plane and explains 25.2% of the variation (Figure 10 and 11). Two patients were
identified as extremes for this component; the low extreme presented with a flat
mandibular plane while the high extreme had a very steep mandibular plane. The second
principal component (PC2) explains 14.8% of the variation and refers to the maxillary
incisor angulation (Figure 12 and 13). The low extreme of this component presented
with very upright incisors, typical to a Class II division 2 patient. The high extreme
presented with very proclined incisors. The subjects representing each quartile also
demonstrated progressively increasing incisor proclination. PC3 refers to the mandibular
horizontal and vertical lengths as well as the posterior facial height and explains 12.2% of
the variation (Figure 14 and 15). PC4 references the position of the maxilla, especially
in regards to the maxillary incisor angulation and accounts for 9.5% of the variation
(Figure 16 and 17). Mandibular incisor position relative to the mandibular plane and the
degree of facial taper are two features that represent PC5 and explain 8.3% of the
38
variation (Figure 18 and 19). In the low extreme, the mandibular incisors are more
upright and there is significant facial taper (the N-Gn-Go angle is acute). The high
extreme of this component has very proclined mandibular incisors and less facial taper.
The progressive proclination of the mandibular incisors is apparent in the patients who
represent each quartile. PC6 refers to the angulation of the cranial base and the position
of the maxilla and explains 6.1% of the variation (Figure 20 and 21). As the position of
sella to nasion becomes steeper, the angle S-N to F-H becomes greater. A very small
angulation of the cranial base describes the low extreme and a very steep anterior cranial
base is demonstrated in the high extreme. The final component, PC7, explains 5.0%, or
the least amount of variation of all the components thus far (Figure 22 and 23). It refers
to the WITS analysis (A-O to B-O) and the amount of overjet a patient presents with.
With all of the numerical and graphical information as well the best the clinical
depiction of distinct groups, it was concluded that the Class II malocclusion sub-
phenotypes, in our sample, is best represented by the 5 cluster model (Table 8 and
Figures 8, 9). Based on the Pseudo F and CCC data (Figure 7), 2, 3, or 4 clusters may
also seem reasonable, however, the two and three cluster models were too simplistic. The
four cluster model was compared to the five cluster model based on a clinically
meaningful interpretation of cephalometric features of the centroid and core. The 4

cluster model did not include the open-bite group seen in the 5 cluster model, and
because this was deemed clinically relevant, the 4 cluster model was not selected.
Although the most well defined clusters would be separate entities, some overlap
between groups is expected and was observed. Cluster 2 was the central cluster and
contained the most observations (n=85), however cluster 4 had the largest standard
deviation (spread of observations). Cluster 4 also had the fewest observations (n=53).
The centroid of each cluster is the numerical average of that cluster and the individual
39
closest to the centroid was drawn to represent the characteristics of that group. All
centroid individuals for the 5 clusters are illustrated in Figure 24.
Cluster 1, representing approximately 18% of total (n=56), depict distinct dental
and skeletal characteristics (Figure 25). The anterior cranial base was longer, with a
slightly decreased saddle angle. The maxilla was mildly retrusive and the mandible was
larger but mildly retropositioned with a normal mandibular plane angle. The longer
anterior cranial base, extending more forward than normal, is a contributing factor of the
retropositioned maxilla and mandible. The maxillary incisors were upright and the
mandibular incisors were normal with a mildly increased overjet and normal overbite.
This cluster would represent the milder skeletal Class II with a normal vertical
component.
The largest group, representing 27.5% of the total (n=85), was Cluster 2 (Figure
26). The cranial base and maxillary position and size were normal. The mandible was
moderately retrusive with a mildly decreased mandibular plane angle. The profile was
moderately convex and the centroid representation had the least facial taper of all the
centroids. The maxillary incisors were upright and the mandibular incisors were
protrusive, with normal overjet and overbite. Overall, this group represents a more
skeletal Class II patient due to a retrusive mandible, with dental compensations present,
and a normal vertical component.
Cluster 3 represents 18% (n=57) of the total and has a shorter anterior cranial base
and normal saddle angle (Figure 27). The maxilla is mildly protrusive and the mandible
is retrusive with a smaller unit length and shorter posterior facial height. The profile is
moderately convex and the patient has upright maxillary incisors and normal mandibular
incisors, with increased overjet and overbite. Vertically, there is a shorter anterior facial
height with some lip redundancy. In summary, both jaws are involved in producing the
Class II malocclusion, the patient has a deep bite and is slightly over-closed.
40
Cluster 4 represents approximately 17% of the total sample and has a shorter
anterior cranial base and slightly decreased saddle angle (Figure 28). The maxilla is
mildly protrusive and the mandible is retrusive with smaller unit length, smallest ramus
height and very steep mandibular plane angle. There is a severely convex profile with
increased facial taper. Dentally, the maxillary incisors are normal and the mandibular
incisors are protrusive. Increased overjet and an anterior open-bite are present in this
centroid. Vertically, there is a mildly increased anterior face height. Soft tissue findings
include a larger interlabial gap. This cluster is defined by the steepness of the mandibular
plane due to the shortness of the ramus, which creates an open-bite tendency and a more
severe maxillo-mandibular discrepancy, caused by both jaws.
Cluster 5 accounts for approximately 19% of the individuals and clinically
features a normal cranial base, mildly protrusive maxilla and mildly retrusive mandible
(Figure 29). This group presents with a severely decreased mandibular plane angle. The
maxillary incisors are protrusive and the mandibular incisors are normal with increased
overjet and normal overbite. Vertically, there is a shorter anterior facial height with lip
redundancy. Overall, the flat mandibular plane, protrusive maxillary incisors and
increased overjet are common findings in this cluster.

41
Figure 5 Scree Plot: Heaviest loaded components charted in descending order; Seven
principal components selected.

42
Figure 6 Component Scores 95% Prediction Ellipse: No outliers present
Table 7 Eigenvalues of the Correlation Matrix

43
Pseudo F and CCC Values by Cluster Number

35 0
-1
34
-2
33
-3
32
Pseudo F
-4
CCC
-5 Pseudo F
31
CCC
-6
30
-7
29
-8
28 -9
2 3 4 5 6 7
Number of Clusters
Figure 7 Pseudo F and CCC Values by Cluster Number: Five clusters selected
44
Table 8 Summary of Class II Malocclusion Cluster Analysis

Cluster Summary - 5 Clusters
Root Mean Squares Nearest Distance Between
Frequency
(St. Dev) Cluster Centroids
1 56 0.7527 2 2.13
2 85 0.7685 5 2.10
3 57 0.9007 2 2.26
4 53 0.9419 2 2.19
5 58 0.8739 2 2.10
*n=309 Caucasian. Data age, gender adjusted and normalized PCA
Figure 8 Plots of Clusters of Class II Malocclusion Subjects: Centroids Labeled

45
Figure 9 3D Plot of 5 Clusters of Class II Malocclusion

46
Table 9 PC1 Cephalometric Variables
Cephalometric Measures Definition

Angle formed at intersection of Sella-Nasion with Gonion-
SNGoGNDeg (intermaxillary)
Gnathion Line
Angle formed at intersection of Frankfort Horizontal (Porion-
FHMPDeg (intermaxillary)
Orbitale) with Mandibular Plane (Gonion-Menton)
Angle formed by Nasion-Sella and Sella-Gnathion lines at
NSGnDeg (Y-axis)
Sella
mm distance from Pg to N Horizontal, which is a vertical line
PgNhorizmm (mandibular)
from Nasion constructed perpendicular to Horizontal Plane.
Horizontal Plane is 7 degrees superior to Sella-Nasion (S-N).
47
Figure 10 PC1 Extremes: referring to vertical dimension in regards to the angulation of
the mandibular plane.
Figure 11 PC1 Quartiles
Diagram shows cephalometric profiles of individuals within the minimum,

25%, 50%, 75% and maximum component scores of PC1.
48

Angle formed at the intersection of Sella-Nasion
U1SNDeg (dental measurement)
with line connecting U1 tip and U1 root
Angle formed at the intersection of a line
U1L1Deg (dental measurement)
connecting U1 tip-U1 root with line connecting L1
tip-L1 root
Angle formed at the intersection of Frankfort
U1FHDeg (dental measurement)
Horizontal (Porion-Orbitale) with line connecting
U1 tip and U1 root
mm distance from Lower Lip to Soft Tissue N
LLipSTNPerpmm (soft tissue
Perpendicular, which is a vertical line from ST
measurement) Nasion constructed perpendicular to Frankfort
Horizontal (Po-Or)
49
Figure 12 PC2 Extremes: referring to maxillary incisor angulation.

50

mm distance from Condylion to Gnathion
CoGnmm (mandibular unit length)
mm distance from Condylion to Gonion
CoGomm (posterior facial height)
mm distance from Nasion to Menton
NMemm (anterior facial height)
mm distance from U6 cusp tip to palatal plane
U6PPmm (dental measurement)
(ANS-PNS)
51
Figure 14 PC3 Extremes: referring to mandibular horizontal and vertical lengths
(anterior and posterior facial heights).

52

Angle formed at the intersection of Nasion-A with line
U1NaDeg (dental measurement)
connecting U1 tip and U1 root
mm distance from Nasion-A line to U1 tip
U1Namm (dental measurement)
Angle formed by Sella-Nasion and Nasion-A lines at
SNADeg (maxilla to cranial base)
Nasion
mm distance from A to N Horizontal, which is a vertical
ANHorizmm (maxilla)
line from Nasion constructed perpendicular to
Horizontal Plane. Horizontal Plane is 7 degrees superior
to Sella-Nasion (S-N).
53
Figure 16 PC4 Extremes: referring to the position of the maxilla, especially in regards to
the maxillary incisor angulation.

54

Angle formed at the intersection of Mandibular Plane
L1MPDeg (dental measurement)
(Gonion-Menton) with line connecting L1 tip and L1
root
Angle formed by Gonion-Gnathion and Gnathion-
NGnGoDeg (facial taper)
Nasion lines at Gnathion
Angle formed at the intersection of Mandibular Plane
IdPgMPDeg (chin angle)
(Gonion-Menton) with a line connecting Pogonion to
the L1 Labial Gingival Border (Pg-Id)
mm distance from Sella-Gonion divided by mm
SGoNMePerc (P-A Face height)
distance from Nasion-Menton
55
Figure 18 PC5 Extremes: referring to the position of the mandibular incisor to the
mandibular plane and the degree of facial taper.

56
FHSNDeg (intermaxillary) Angle formed at intersection of Sella-Nasion with Frankfort
Horizontal (Porion-Orbitale)
ANPerpmm (maxilla) mm distance from A to N Perpendicular, which is a vertical
line from Nasion constructed perpendicular to Frankfort
Horizontal (Po-Or)
ULipSTNPerpmm (soft tissue) mm distance from Upper Lip to Soft Tissue N
Perpendicular, which is a vertical line from ST Nasion
constructed perpendicular to Frankfort Horizontal (Po-Or)
LLipSTNPerpmm (soft tissue) mm distance from Lower Lip to Soft Tissue N
Perpendicular, which is a vertical line from ST Nasion
constructed perpendicular to Frankfort Horizontal (Po-Or)

57
Figure 20 PC6 Extremes: referring to the cranial base angulation and position of the
maxilla.
Figure 21 PC6 Quartiles.

58
AOBOmm (Wits Appraisal) mm distance between two points located on the occlusal
plane, projected from A and B perpendicular to the occlusal
plane. Occlusal plane is defined as a line constructed
between the midpoint of the U6 and L6 cusp tips and
bisection of U1 and L1 tips
OJmm (dental measurement) horizontal mm distance: L1 tip to U1 tip measured along
occlusal plane, which is defined as a line constructed
between the midpoint of the U6 and L6 cusp tips and
bisection of U1 and L1 tips

Angle formed at the intersection of Facial Plane
ABNPgDeg (intermaxillary)
(Nasion-Pogonion) with A-B line
ANBDeg (intermaxillary) Angle formed by A-Nasion and Nasion-B lines at Nasion

59
Figure 22 PC7 Extremes: referring to the WITS analysis and amount of overjet
Figure 23 PC7 Quartiles.

60
Figure 24 Cluster Centroids with Descriptions

61
Figure 25 Cluster 1 Centroid and Core with Description

62

63

64

65

66
DISCUSSION
There have been no studies that have delved into the phenotypic makeup of Class
II malocclusion like this study. Using similar statistical procedures and methods as Bui
et al. (2006) had used in the study of phenotypic characterization of Class III
malocclusion, this study has produced specific components describing most of the Class
II variation and identified 5 distinct clusters depicting the most common phenotypic
characteristics found in the Class II malocclusion. Unlike the study by Bui, we have
included only Caucasian adult patients presenting with Class II malocclusion who most
likely had full expression of growth completed and all subjects were standardized in
regards to age and gender.
This study also differed from the older study conducted by Moyers et al., which
did not have set inclusion/exclusion criteria or as well defined statistical methods. In
Moyers‟ study, all subjects were Caucasian children, however, the age ranges were not
specified. Moyers‟ study divided Class II malocclusion into separate horizontal and
vertical subtypes. While separating the horizontal and vertical components may be
convenient for description purposes and a good tool for classifying patients, it was not
fully representative of the Class II malocclusion subphenotypes, like our findings. The
current study produced clusters representing the most common combinations of
horizontal and vertical components. It is beneficial to illustrate the interactions between
the two dimensions, as viewing them as separate entities may not reveal the true
expression of the malocclusion. Additionally, in Moyers‟ study, 115 out of the 610
horizontally-classified patients were not classified into a vertical type. Therefore, no
direct comparison can be made between the clusters in this study and the different
horizontal and vertical subtypes in Moyers‟ study. The findings of this study showed all
of the phenotypic variation of Class II malocclusion seen in the studies using the single
67
variable analysis including McNamara (1981), Pancherz et al. (1997), Sayin and
Turkkahraman (2005), as well as the multivariate analysis of Moyers (1980).
The present study has conducted more in-depth statistical analyses with more
stringent methodological criteria and detailed cephalometric landmark identification.
With this, we have elicited the principal components or specific features that can explain
most of the variation of Class II malocclusion, something that has not been produced
before. The statistical methods in Moyers‟ study were not described in detail, however, a
cluster analysis was used. By using a principal component analysis prior to a cluster
analysis, as in our study, we have elicited the most important independent variables and
used that information to form the specific clusters.
The PCA reduced the 63 variables and identified seven principal components that
explain 81% of the variation of our Class II sample. Each component represents a
specific phenotype or trait of the malocclusion. The most variation was explained by the
first component (PC1), and subsequent components explain progressively less variation.
The first component describes the vertical dimension in regards to the angulation of the
mandibular plane to the cranial base. Verticality of patients plays a significant role in
determining orthodontic correction for Class II patients; for example, the treatment and
treatment response may differ in patients with a flat plane versus a steeper plane.
PC2 referred to maxillary incisor inclination and the subjects representing each
quartile demonstrated progressively increasing incisor proclination. Incisor angulation is
a significant feature distinguishing Class II subdivisions. As reviewed from the literature,
those divisions present with very different skeletal and dental characteristics (E. A. Al-
Khateeb & Al-Khateeb, 2009).
PC3 differentiates the range of mandibular lengths present in Class II patients. A
common finding of Class II malocclusion, as described previously, is smaller mandibles,

however, some patients who present with this malocclusion have normal size mandibles
68
and are Class II skeletally based on a malpositioned maxilla. For each patient the jaw
size and position will affect the treatment.
The fourth component refers to the position of the maxilla, especially in regards to
the maxillary incisor angulation. As the incisors become more upright, the position of the
maxilla, represented by A point, moves forward. This can create the appearance of a
more protrusive maxilla due to the positioning of the maxillary incisors.
Principal components 5, 6, and 7 explain less of the variation and it should be
noted that as progressively less variation is explained, the clinical differences may be
more difficult to observe. Viewing the quartiles in PC5, it is apparent that the mandibular
incisors become more proclined and there are differences in degree of facial taper. The
position of the maxilla is referred to in the PC6 and is seen by the distance from A-point
to nasion perpendicular, where the low extreme has A-point set back from N-
perpendicular and the high extreme has A-point much more forward of that line,
depicting a more protrusive maxilla. Inclination of the anterior cranial base can create
variation in the measurements based on these landmarks. Smaller than normal
measurements of the SNA and SNB angles can be due to actual maxillo-mandibular
retrusion, or can be an outcome of a low sella or high nasion point creating a steeper
cranial base. In PC7, it is obvious between the low and high extreme that the overjet
increases significantly. This component describes the differences in the underlying
skeletal discrepancy and it is apparent that the patient on the high end is a more severe
Class II, while the low end has normal overjet and may have only mild Class II skeletal
tendencies. Obviously, treatment options for the two extremes will differ, where the
patient with minimal overjet may benefit from orthodontics alone and the patient with
more significant amount of overjet may need orthodontic and surgical correction for the
underlying skeletal discrepancy.

69
The specific components produced illustrate the most important variables
explaining the variation of Class II malocclusion. Having more descriptive clinical
information will aid in a more accurate diagnoses and better treatment decisions. Patients
with similar growth patterns should have comparable treatment modalities. If the amount
of horizontal and vertical components in Class II patients can be assessed earlier on, more
specific and appropriate treatment can be rendered. Other Class II patients can be
described through this model and their specific phenotypes can be evaluated and assigned
to an appropriate cluster. Each cluster will require different treatment and the most
befitting treatment for each cluster will be determined. Future patients assigned to a
particular cluster can be assumed to have a similar growth patterns and course of
treatment. Thus, these specific phenotypic groups will elicit more valuable information
on the timing and treatment of orthodontics and help elucidate the etiology of
malocclusion.
Limitations of the Study
There are several inherent limitations to this study. First, our sample represents
those people seeking orthodontic treatment and may not be representative of the general
population, as possibly those with more severe malocclusion may present to the
orthodontic clinic. Also, there is some subjectivity involved in determining the number
of clusters to describe Class II malocclusion. Although there are statistical computations
and numerical values in the process, identifying distinct clusters both spatially and
clinically is left open to some interpretation. Lastly, our study used and analyzed 2
dimensional data, which can create difficulty analyzing 3-dimensional craniofacial
features. The ability to landmark 3-D images could provide more information when
standardized cephalometric analyses are available. As technology improves, replicating

this study with 3-D images will prove beneficial.
70
Future Projects
Future projects could include dividing Class II subjects into division 1 and
division 2 subgroups and analyzing the principle components and clusters amongst the
divisions to note differences. However, Class II division 2 patients are not as prevalent in
the population and an adequate sample size for this group may be difficult to attain. As
noted before, the division 2 group can be viewed as a distinct entity and has significant
skeletal and dental differences from those patients with Class I or Class 2 division 1
malocclusion.
More importantly, phenotypic characterization of Class II malocclusion will be
the data infrastructure to future large-scale genetic studies that will allow an in-depth
analysis of the etiology of malocclusion. Hopefully, with specific clusters of Class II
patients, identifying a causative gene will be more attainable. Having the ability to
distinguish between those patients with more vertical versus more horizontal growth
components and identifying genetic variants that account for these differences will
facilitate the design of preventative measures and the most beneficial treatment for
specific phenotypes. Although it is clear that there is a genetic role in Class II

malocclusion, there are limited studies on the specific genes involved. Having
characterized Class II patients into commonly occurring phenotypic subgroups will
hopefully allow a more accurate depiction of causative genes creating various
components of this malocclusion.

71
SUMMARY AND CONCLUSIONS
Malocclusion is a complex trait, with both environmental and genetic influences
interacting to create disharmony of the jaws and teeth. This can create a distorted facial
appearance and have significant effects on the quality of life, both psychosocially and
functionally. The etiology of malocclusion is not fully understood and has not been
studied in great detail due to the lack of well-defined phenotypes. The purpose of this
study was to reduce the heterogeneity of Class II malocclusion and produce distinct
phenotypic groups that will allow future studies to determine the etiology of this
malocclusion.
This study has produced seven components that help to explain the most
important features of Class II malocclusion. Furthermore, five distinct clusters have been
identified that divide this malocclusion into homogenous phenotypic groups. This study
will lay the foundation for future studies to identify a causative gene in this multifactorial
problem. The identification of genetic influences in Class II malocclusion can aid in the
prevention and improve treatment modalities of maxillo-mandibular discrepancies.

72
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University of Iowa

Masthead Logo Iowa Research Online

Phenotypic characterization of Class II

Copyright 2012 Sara Howe

This thesis is available at Iowa Research Online: https://ir.uiowa.edu/etd/2896

Follow this and additional works at: https://ir.uiowa.edu/etd

Part of the Orthodontics and Orthodontology Commons

A thesis submitted in partial fulfillment

Thesis Supervisor: Assistant Professor Lina M. Moreno Uribe

This is to certify that the Master's thesis of

Sara Christine Howe

has been approved by the Examining Committee

University of Iowa, Department of Orthodontics, for giving me the opportunity to further

my education and pursue a career in orthodontics.

they have given me.

LIST OF TABLES .............................................................................................................. v

LIST OF FIGURES ........................................................................................................... vi

Purpose of this Study ...................................................................................................... 2

LITERATURE REVIEW ................................................................................................... 3

Etiology of Class II Malocclusion .................................................................................. 3

MATERIALS AND METHODS:..................................................................................... 23

Limitations of the Study................................................................................................ 69

SUMMARY AND CONCLUSIONS ............................................................................... 71

Table 1 Eligibility Criteria ..............................................................................................24

Table 2 Cephalometric Variables ....................................................................................26

Table 3 Cephalometric Landmarks .................................................................................27

Table 4 CII Intra Rater Ceph Variables ..........................................................................34

Table 5 CIII Intra Rater Ceph Variables .........................................................................35

Table 7 Eigenvalues of the Correlation Matrix ...............................................................42

Table 9 PC1 Cephalometric Variables ............................................................................46

Table 10 PC2 Cephalometric Variables ...........................................................................48

Table 11 PC3 Cephalometric Variables ...........................................................................50

Table 12 PC4 Cephalometric Variables ...........................................................................52

Table 13 PC5 Cephalometric Variables ...........................................................................54

Table 14 PC6 Cephalometric Variables ...........................................................................56

Table 15 PC7 Cephalometric Variables ...........................................................................58

Figure 1 CII division 1 Malocclusion.............................................................................5

Figure 2 CII division 2 Malocclusion.............................................................................5

Figure 3 Six Horizontal Subgroups from Moyers 1980 ...............................................14

Figure 4 Five Vertical Types from Moyers 1980 .........................................................15

Figure 5 Scree Plot .......................................................................................................41

Figure 6 Component Scores 95% Prediction Ellipse ...................................................42

Figure 7 Pseudo F and CCC Values by Cluster Number .............................................43

Figure 9 3D Plot of 5 Clusters of Class II Malocclusion .............................................45

Figure 10 PC1 Extremes.................................................................................................47

Figure 11 PC1 Quartiles .................................................................................................47

Figure 12 PC2 Extremes.................................................................................................49

Figure 13 PC2 Quartiles .................................................................................................49

Figure 14 PC3 Extremes.................................................................................................51

Figure 15 PC3 Quartiles .................................................................................................51

Figure 16 PC4 Extremes.................................................................................................53

Figure 17 PC4 Quartiles .................................................................................................53

Figure 18 PC5 Extremes.................................................................................................55

Figure 20 PC6 Extremes.................................................................................................57

Figure 21 PC6 Quartiles. ................................................................................................57

Figure 22 PC7 Extremes.................................................................................................59

Figure 23 PC7 Quartiles. ................................................................................................59

Figure 24 Cluster Centroids with Descriptions ..............................................................60

Figure 26 Cluster 2 Centroid and Core with Description ...............................................62

Figure 28 Cluster 4 Centroid and Core with Description ...............................................64

Figure 29 Cluster 5 Centroid and Core with Description ...............................................65

Malocclusion, which may involve misalignment of the teeth, mal-positioning of

of malocclusion based on molar positioning. Class I malocclusion occurs when molars