Investigation of Genetic Factors Affecting

Complex Traits Using External Apical Root

Resorption as a Model
Shaza K. Abass and James K. Hartsfield, Jr.

Genetic (genomic) and environmental (non-genetic) factors are the two

main aspects that determine phenotype. Although there has long been a
tendency to separate them (nature versus nurture), in essentially all traits
(diseases) besides those secondary to trauma, both the genetic and envi-
ronmental factors interact to develop the phenotype (nature and nurture).
Traits (diseases) can be divided into two broad categories based on their
genetic components pattern of transmission. The first category include so
called simple (Mendelian) traits. The second category includes genetically
complex traits. These are more common than Mendelian traits. They do not
follow a clear pattern of inheritance, but they tend to run in families.
Relatives of an affected individual or one who has the trait have an increased
risk of developing the disease and or having the trait. The genetic determi-
nants of such traits are difficult to identify since the trait or disease results
from a set of genetic polymorphisms that may be common within the
population, both affected and non-affected. The interplay of these genetic
polymorphisms at different loci with environmental factors leads to the
manifestation of such complex traits. Some approaches to analyzing com-
plex traits using external apical root resorption as an example are reviewed.
(Semin Orthod 2008;14:115-124.) © 2008 Elsevier Inc. All rights reserved.

xternal apical root resorption (EARR) is a
E common clinical complication of orth-
odontic treatment. It is a permanent shortening
of the end of the root that can be seen on routine
dental radiographs. Although EARR may occur in
any or all teeth, it most often involves the maxil-
lary incisors. Seven to 13% of individuals who
have not had orthodontic treatment show 1 to 3
mm of EARR on radiographs.1 Severe EARR,
Assistant Professor of Dental Science, Faculty of Dentistry, Uni-
versity of Khartoum, Khartoum, Sudan; Professor and Director of
Oral Facial Genetics, Professor of Orthodontics, Indiana University
School of Dentistry, Professor of Medical and Molecular Genetics,
Indiana University School of Medicine, Indianapolis, IN.
Address correspondence to James K. Hartsfield, Jr., DMD, PhD,
Department of Orthodontics and Oral Facial Genetics, Indiana
University School of Dentistry, 1121 W. Michigan St., Indianapolis,
IN 46202-5186. Phone: 317-278-1148; E-mail: jhartsfi@iupui.
edu.
© 2008 Elsevier Inc. All rights reserved.
1073-8746/08/1402-0$30.00/0
doi:10.1053/j.sodo.2008.02.008
which is root loss of more than 5 mm, has been
reported to occur in 2% to 5% of patients
treated with orthodontics.2,3
The American Association of Orthodontists
(AAO; St. Louis, MO) estimated that in the year
2000 there were 4.5 million people being treated
by its members. Extrapolation estimates that
90,000 to 225,000 patients undergoing orth-
odontic treatment during 2000 may develop
EARR of more than 5 mm. This number of
possible affected individuals does not include
those treated by orthodontists that are not AAO
members, and nonorthodontists. This estimate
of EARR incidence hallmarks a potential prob-
lem for orthodontists and patients who undergo
orthodontic treatment.
There is a significant variability for EARR sus-
ceptibility among individuals. This variability
can be due to an innate or systemic predisposi-
tion to resorption in permanent and primary
teeth in these individuals.4-9 It has been pro-

Seminars in Orthodontics, Vol 14, No 2 (June), 2008: pp 115-124 115

116 S.K. Abass and J.K. Hartsfield, Jr.

posed that when extreme susceptibility exists,
EARR can occur in absence of any specific caus-
ative factor.7,10 A racial dichotomy has also been
reported with Asian patients having less EARR
than white or Hispanic patients.9 Familial clus-
tering of EARR has also been reported, although
no Mendelian pattern of inheritance was identi-
fied.6 Although racial differences and familial
clustering may suggest a genetic component, it
may also reflect different and common environ-
mental factors respectively. Although many fac-
tors have been suggested to affect EARR, none
have by themselves been sufficient to explain the
variance in individual incidence or susceptibility
to EARR.
Since mechanical forces and other environ-
mental factors do not adequately explain the
variation in EARR seen among individuals, an
increased interest has focused on the role of
genetic factors influencing the susceptibility to
EARR. In this study we will describe strategies
that have been used to estimate and identify
genetic influences on EARR. We will start by
identifying basic analyses used in identifying ge-
netic factors associated with a complex trait such
as EARR.
Genetic Basis of Disease
The genetic background (genome) and environ-
mental (nongenetic) factors are the two main
aspects that determine phenotype. Although
there has long been a tendency to separate them
(nature versus nurture), in essentially all traits
(diseases) besides those secondary to trauma,
both the genetic and environmental factors in-
teract to develop the phenotype (nature and
nurture).
Traits (diseases) can be divided into two broad
categories based on their genetic components’
pattern of transmission. The first category is the
simple or Mendelian traits. These traits follow
the two genetic laws of heredity: the law of seg-
regation and the law of independent assortment
identified by Gregor Mendel in 1985. These
traits have a “simple” pattern of inheritance (au-
tosomal dominant, autosomal recessive, or X-
linked) and in most of the cases they result from
a mutation of a single locus (see the article titled
“Genetic Factors and Orofacial Clefting” by
Lidral and coworkers in this volume). More in-
formation is available on human genetics, includ-
ing a glossary, at the National Library of Medicine
National Institutes of Health “Genetics Home Ref-
erence” http://ghr.nlm.nih.gov Web site.
Pinpointing a genotype-phenotype relation-
ship is relatively easy to establish in Mendelian
traits or diseases because a single gene mutation
usually results in a recognizable phenotype. En-
vironmental factors and other genes may modify
the clinical expression of the disease,11 but are
not of crucial importance for disease develop-
ment (Fig 1). Such single gene traits or diseases
are relatively rare except in certain isolated com-
munities. They are also referred to as discrete or
qualitative traits since they are dichotomous,
that is, they are either present or not (although
some aspects of them may still be measured).

Figure 1. Mendelian (monogenic) traits or diseases result because a single gene polymorphism or mutation
usually results in a recognizable phenotype. Environmental factors and other genes may modify the clinical
expression of the disease or other type of trait, but are not of crucial importance for its development.
 Genetic Factors Affecting Complex Traits 117

Examples of simple traits include amelogenesis
imperfecta and cleidocranial dysplasia. For a list
of genetic traits that follow Mendelian inheri-
tance, one can search the Online Mendelian
Inheritance in Man (OMIM) at the http://www.
ncbi.nlm.nih.gov/sites/entrez?db ⫽ OMIM
Web site. The identification of the gene muta-
tion responsible for a particular Mendelian trait
allows the screening of individuals carrying the
mutation, and also allows for predicting the dis-
ease transmission from parents to offspring (ge-
netic counseling).
The second category includes genetically
complex disease. These traits are more common
in the population than Mendelian traits. They
do not follow a clear pattern of inheritance, but
they tend to run in families. In other words,
relatives of an affected individual or one who has
the trait have an increased risk of developing the
disease or having the trait. The genetic determi-
nants of such traits are very difficult to identify
since the trait or disease results from a set of
genetic polymorphisms that may be common
within the population, both affected and nonaf-
fected. The interplay of these genetic polymor-
phisms at different loci with environmental fac-
tors leads to the manifestation of such complex
traits.
Unlike Mendelian traits, environmental fac-
tors and multiple genes are critical to the devel-
opment of such complex traits (Fig 2). These
type of physical traits are continuous rather than
discrete (although diseases of this type can still
be present or not). Such traits are referred to as
quantitative traits or multifactorial, since they
are caused by some number of genes in combi-
nation with environmental factors. Examples of
such physical traits include height and weight
(which are measurable, not dichomatous). Dis-
ease traits of this type include cardiovascular
disease, periodontal disease, and nonsyndromic
cleft lip and palate.
Evidence of a Genetic component to
EARR
One of the classic methods to estimate the ge-
netic aspect of a trait is studying families, espe-
cially twins.12 Twin studies provide a powerful
method to recognize genetic and environmental
effects on the manifestation of a trait. This is
usually done by comparing monozygotic (MZ)
twins and dizygotic (DZ) twins. It is assumed in
such studies that the twins share the same envi-
ronmental conditions, which should reduce the
noise of environmental factors. It should be un-
derstood that this is variably not the case (see the
discussion of heritability estimates in “Interpret-
ing Heritability Estimates in the Orthodontic
Literature” by Harris in this volume).
MZ twins share 100% of their genes whereas
DZ twins share 50% of their genes. If the trait

Figure 2. Unlike Mendelian traits, environmental factors and multiple genes are critical to the development of
complex (polygenic) traits. These type of physical traits are continuous rather than discrete (although diseases
of this type can still be present or not). Such traits are referred to as quantitative traits or multifactorial, since
they are caused by some number of genes in combination with environmental factors.
118 S.K. Abass and J.K. Hartsfield, Jr.
(disease) of interest has a genetic component it
is expected to occur more in MZ twins than DZ
twins regardless of it being a simple trait or a
complex trait. When the two related individuals
express the same trait they are said to be con-
cordant. If the trait of interest has a higher
concordance in MZ twins compared with DZ
twins, this implies a genetic component. A re-
cent retrospective twin study on EARR that in-
cluded 16 MZ and 10 DZ twins showed that the
concordance scores for MZ twins were approxi-
mately twice those of DZ twins, indicating a
strong genetic influence on EARR.13 However,
the concordance of the MZ twins was less than
100%, indicating that there was an environmen-
tal influence on EARR as well.
In addition to twins, genetic influence on a
trait (disease) can also be studied by looking at
the resemblance between other family members.
Parent-offspring and sib pairs can be investi-
gated for the trait of interest, since they share
similar (50% of their genes in common on aver-
age) genetic backgrounds. In case of a strong
genetic influence the trait of interest is usually
more common in certain families compared
with the general population. Care must be taken
again to take common environmental factors
into account as well.
For qualitative traits (present or absent), fam-
ily resemblance is expressed in terms of relative
risk (␭), which is the prevalence of the disease
among relatives as compared with its prevalence
in the general population. The greater the rela-
tive risk value the greater the familial aggrega-
tion of the disease due to common familial genetic
and environmental factors. For quantitative traits
(complex traits) this family resemblance is ex-
pressed in the terms of heritability (h2), which is
the ratio of additive genetic variance to the total
variance of the trait. This ratio of additive ge-
netic to total (additive genetic plus environmen-
tal) variation does not take into account gene-
to-gene interaction or the gene-environment
interaction.
The value of heritability varies between 0 and
1. A heritability of 0 means that genetic variation
does not contribute at all to variation in the
phenotype. A heritability of 0.5 means that both
genetic and environmental variation contribute
equally to the phenotypic variation. A trait with
a heritability estimate of 1 is theoretically ex-
pressed with all of its variation related with ge-
netic variation, and no variation related with
environmental variation.
When estimating heritability it is important to
realize that it reflects the population and envi-
ronment analyzed at that point in time. Al-
though it may seem strange, heritability esti-
mated in this manner may change in the future,
and is not necessarily predictive of the future.
Since environmental factors may change that
may affect the phenotype more (although the
genes have not changed), the heritability esti-
mate for the trait being analyzed will change
(decrease in this example). Also heritability es-
timates are population estimates and not appli-
cable to an individual (Fig 3).
Using a sib-pair model, Harris and coworkers
have shown that the h2 estimates for EARR aver-
aged about 0.7 for the maxillary incisors and
mandibular first molar roots.10 They concluded
that such a moderately high heritability accounts
for half of the total phenotypic variation seen
among the individuals in their sample. This im-
plies that siblings experience similar levels of
EARR in response to orthodontic treatment com-
pared with unrelated individuals. The study con-
ducted by Harris and coworkers was the first to
quantitate a transmissible component of EARR,
and was confirmed by Hartsfield and coworkers14
Figure 3. Aspects of (additive) heritability in the nar-
row sense, estimating the effect of an indefinitely
large number of genes that all contribute equally to
the phenotype. It cannot take into account allele-
allele interactions at a gene locus (termed domi-
nance) and gene-gene interactions involving two or
more loci (termed epistasis).
 Genetic Factors Affecting Complex Traits
Strategies of Identifying EARR Genes
Tools to Identify Disease Genes
Besides mitochondrial DNA, human genetic in-
formation in each cell is contained in 23 pairs
of chromosomes (22 autosomes and one pair
of X chromosomes in the female, or an X and
Y chromosome in the male). Each of the chro-
mosomes is formed from a DNA molecule that
contains many genes. The nucleotide sequence
that makes up the DNA encodes the genetic
information. In addition to genes, each of the
chromosomes carries regulatory sequences as
well as other nucleotide sequences with still un-
known functions. These noncoding regions of
the DNA provide an important tool for the study
of the genome.
The DNA sequence varies at a particular chro-
mosomal location in both the coding and non-
coding regions. This genetic variation is called
genetic polymorphism and the different DNA
variants arising from such polymorphism are
called alleles. The study of alleles allows for ge-
netic analysis of diseases (for further informa-
tion see “Genetic Factors and Orofacial Clefting”
by Lidral and coworkers, and “Genetic Factors
and Tooth Movement” by Iwasaki and coworkers
in this volume). Even though 99.9% of the genes
are similar among individuals, the remaining
0.1% variation accounts for the differences seen
among individuals, including disease susceptibil-
ity.
Several types of polymorphisms are commonly
used for genetic analyses, including microsatellite
markers that are DNA sequences in which a short
sequence (2-4 nucleotides) sequence is repeated
several times. The number of repeats varies
among individuals, but is inherited within fami-
lies. These repeated nucleotide sequences are usu-
ally found in noncoding regions and are thus typ-
ically nonfunctional polymorphisms. Another
form of polymorphism widely found in the ge-
nome is single nucleotide polymorphisms (SNPs).
SNPs can be in both the coding and the non-
coding DNA regions. Although most of the SNPs
are found within noncoding regions, SNPs found
within coding regions are of particular impor-
tance, since depending on their codon position
and genetic code they may alter the amino acid
sequence and potentially the biological function of
the protein.
119
Linkage Analysis
Linkage analysis tests for the cosegregation of a
marker and a trait (disease) locus in families
with affected individuals. During meiosis the ge-
netic material crosses over from one parental
derived chromosome to the other through chro-
mosomal recombination. Chromosome seg-
ments and their associated genes that are far
apart are more likely to recombine during mei-
osis. Chromosome segments and their associ-
ated genes that are close together have a lesser
chance of recombination and are more likely to
cosegregate and be inherited together and thus
be linked. The recombination fraction is the
probability that two DNA markers will appear in
a new combination on a chromosome not seen
in the parental generation.
When two genes are far apart (unlinked) on
the same chromosome, or on two different chro-
mosomes, the recombination fraction is 0.5,
which means that the two markers are inherited
independently following Mendel’s law of inde-
pendent assortment. For genes that tend to be
inherited together (linked) in a part of a chro-
mosome, the recombination fraction will be less
than 0.5. The results of linkage studies are re-
ported by calculating the LOD score, which is
the logarithm of odds that the disease locus and
the marker are linked (recombination fraction
less than 0.5) rather than unlinked (recombina-
tion fraction of 0.5). A LOD score below –2.0
excludes any linkage, whereas a LOD score
above 1.9 is suggestive of linkage. A LOD above
3.0 suggests a significant linkage.
By using markers that are evenly spaced
across all chromosomes, one can analyze the
entire genome using this approach and identify
genes that are linked to the trait (disease) of
interest. To perform parametric linkage analysis,
one must specify an inheritance model of the
disease of interest as well as the allele frequency
distribution. This is relatively difficult to achieve
for a trait in which more than one genetic locus
plays a Mendelian or major role (for more in-
formation see “Genetic Factors and Orofacial
Clefting” by Lidral and coworkers in this vol-
ume). Classical linkage is usually performed in
large extended families with multiple affected
individuals, which makes it difficult with rare
diseases and or those that markedly decrease
procreation.
120 S.K. Abass and J.K. Hartsfield, Jr.
EARR, however, is a complex disease, with
multiple genetic and environmental factors con-
tributing to its occurrence and severity. In such
complex diseases with no Mendelian pattern of
inheritance, nonparametric linkage analyses are
the best tools to identify contributing genes. These
nonparametric linkage analyses are mainly based
on a concept called identity by descent (IBD)
marker allele sharing. Allele-sharing studies pro-
vide a way to perform linkage analysis without
knowing the model of inheritance. In allele-shar-
ing studies, sib pairs who are both affected by
the disorder are genotyped to determine if they
inherit the same alleles at a certain region of the
genome more than would be expected by
chance. Although allele-sharing analysis does
not require the collection of large multigenera-
tional families, it does require a large numbers
of sib pairs to gain enough power for the local-
ization of genes that at least moderately affect
the disease susceptibility.
In general, the goal of linkage analysis is to
identify the region(s) of the chromosome that
contains the gene(s) responsible for the mani-
festation of the disease of interest. The marker
allele segregating with the disease gene varies
from family to family. This kind of analysis tends
to be more powerful with single gene (mono-
genic) or major gene disorders, since in com-
plex traits several genes contribute to the disease
process.
Linkage analysis and allele-sharing studies are
performed by studying a panel of polymor-
phisms on the whole genome (genome-wide ap-
proach). In this way chromosome areas of inter-
est may be further investigated, including those
with no previous indication of importance to the
trait (disease) of interest. Linkage analysis and
allele-sharing studies can also be performed by
studying polymorphic markers within suitable
candidate genes or loci (candidate gene ap-
proach). Candidate genes are usually selected
because of previous knowledge of the function
of the gene and its possible affect on the devel-
opment of the disease if mutated.
In an effort to identify genetic factors in
EARR Al-Qawasmi and coworkers used the can-
didate gene approach to look for evidence of link-
age in 38 pedigrees. In their study they found
suggestive evidence of linkage between EARR of
maxillary central incisors and a polymorphic
marker D18S64 (LOD score 2.51). This polymor-
phism marker lies close to the TNFRSF11A gene
suggesting that this locus or a closely linked one
contributes to the susceptibility to EARR. The
TNFRSF11A gene codes for RANK, an essential
signaling molecule in osteoclasts differentiation
and function.15
Association Analysis
Association analysis is a method to determine if
a particular marker allele is more frequent in a
group of subjects with the disease compared
with a control group. Association studies can be
used in both quantitative and qualitative traits.
They rely on the concept of linkage disequilib-
rium (LD), which is the consistency of associa-
tion of alleles at two linked loci. It is assumed
that the disease-associated polymorphism occurs
more frequently with a set of markers. LD anal-
ysis tests the hypothesis that a marker and the
disease allele occur more frequently in a sample
of affected individuals compared with unaf-
fected controls. The disease mutation might be
homogenous in all individuals reflecting a
founder effect.
To evaluate evidence of association, samples are
collected from affected subjects and matched con-
trols to compare the allele’s frequencies at a
marker in a candidate gene. In such population-
based association studies (case-control), LD re-
sults can be complicated by inherited differ-
ences in the genetic background between the
cases and controls rather than actual association
with the disease. That is why it is essential to
closely match cases and controls to control for
false positives that lead to spurious association
results.
Family-based association methods have been
developed to control for population stratifica-
tion. One such method is the affected family-
based control (AFBAC) method, in which the
frequency distribution of marker alleles trans-
mitted from parents to an affected child is com-
pared with those not transmitted. The transmit-
ted and nontransmitted alleles come from the
same parent.
Another method is the transmission disequi-
librium test (TDT), in which the frequency of
the transmission of a particular allele from het-
erozygous parents to an affected child is com-
pared with the 50% Mendelian ratio. Because in
such a triad (parents and affected child) the
 Genetic Factors Affecting Complex Traits
transmitted allele acts as a case and the untrans-
mitted alleles act as a control, TDT results in a
well-matched genetic background and controls
for population stratification. Limiting factors for
the TDT is the unavailability of parents due to
death, refusal of genetic analysis, or in late-onset
diseases. Significant evidence of linkage disequi-
librium for an IL-1B polymorphism with EARR
was reported.16 The analysis of 35 families indi-
cated that the IL-1B polymorphism accounts for
15% of the total variation seen for EARR seen in
the maxillary central incisor in the sample stud-
ied.
When locating susceptibility genes, both link-
age and association analysis may be useful in the
same families. Linkage analysis usually identifies
regions of the genome that contain the suscep-
tibility genes, although the marker allele used to
identify this region varies from one family to the
other. When linkage is found, the linked region
can be further investigated for linkage disequi-
librium to identify the trait-associated polymor-
phism at that region. Another approach is to
identify certain candidate genes and then ana-
lyze markers in and around that gene for linkage
and association. Linkage and association are
complementary methods for identification of
genes responsible for complex traits.
As Hartsfield discusses in the article titled
“Personalized Orthodontics, The Future of Ge-
netics in Practice?” in this volume, the rapid
growth in genome-wide association studies pre-
sents both the greatest challenge and the likeli-
hood of truly understanding the interaction of
genetic and environmental (treatment) factors
affecting root resorption in the clinic.
Animal Studies
As mentioned earlier most common traits (dis-
eases) have a complex genetic etiology. Multiple
genetic loci and the environment interact to
modulate susceptibility to such complex dis-
eases. In such cases the identification of genes
that predict a trait are challenging in humans.
Using the mouse model to study genetic effects
on complex traits is becoming popular.
The use of mice to study genetic influences
on traits is very appealing, since it allows for
controlling two major factors that complicate
human studies: genetic heterogeneity and envi-
ronmental factors. This is facilitated by the
121
mouse and human genome showing a high de-
gree of homology (80%).17-19 There is a marked
genomic conservation of gene order (synteny)
between the two species. The availability of a
dense and detailed genetic map makes gene
mapping in mice practical and efficient. Mice
are easy to breed with a short gestational period
and relatively large litter sizes. They also have a
short life span and can be raised easily and
economically in small facilities. Control of envi-
ronmental factors and ease of conducting crosses
among the different strains makes them a perfect
model in genetic studies.
Mice carrying a specific gene deletion (knock-
out) or gene addition (transgenic) have been
very useful in examining how the manipulated
gene affects the trait or the disease. Both the
knockout and transgenic mice will examine the
effect of a single gene on the disease process.
They often result in a major change in physiol-
ogy. In complex diseases, this may not be the
case, since more than one gene is involved in the
development of the trait.
The use of inbred strains of mice to uncover
genetic determinants of various diseases with
either a single or polygenic basis is increasing in
popularity. Inbred mice are produced by re-
peated brother and sister mating for at least 20
consecutive generations.20 This results in about
100% homozygosity in all alleles across the
mouse genome. By even-crossing them for fur-
ther generations, inbred mice become homozy-
gous in all loci providing a colony of genetically
identical mice. With exception to the sex chro-
mosomes, each inbred mouse is essentially an
identical twin to the other, each carrying the
same genome. Because of the allelic variation
between different strains, each of the strains
differs from the others with its own set of phe-
notypic characteristics. Finding two inbred
mouse strains that differ in the expression of the
trait of interest while controlling for all other
factors indicates that genetic components (dif-
ferences in background genes between the in-
bred strains) strongly affect the trait of interest.
Crossing two inbred mice that differ in the
trait of interest results in hybrid mice called F1s
that carry a single set of alleles from each parent.
These F1 mice are genetically identical mice and
heterozygous in all loci in the alleles arising
from the two parental strains. The F1 animals
can provide significant information about the
122 S.K. Abass and J.K. Hartsfield, Jr.
monogenic or polygenic status of the trait of
interest. Furthermore these F1 animals can be
further mated to each other (intercross) or
mated to one of the parental strains (backcross)
to further elucidate contributing genetic factors
(Fig 4).
Intercrossing F1 animals results in an F2 gen-
eration that has a unique combination of pro-
genitor genes. The genetic diversity of F2 ani-
mals results from recombination and random
assortment of DNA from the progenitor mice.
The use of F2 mice is very popular in analyzing
the loci that influence a complex trait through
the process of quantitative trait loci (QTL) anal-
ysis. QTL analysis has proved to be the method
of choice in identifying genes responsible for
complex traits, particularly in absence of evi-
dence implicating a candidate gene.21 Areas of
the genome from each parent can be identified
by using readily available polymorphic markers.
These polymorphic markers vary from one strain
to another and thus allow the identification of
loci that affect the trait. The analysis of the F2
phenotypes and relating them to genomic mark-
ers allows the identification of both major and
minor effect loci. By only analyzing the F2 gen-
eration members who have different extremes of
the phenotype, one can identify major loci af-
fecting the trait. Software programs are now
Figure 4. Different types of crosses (breeding) of inbred animals of different strains are useful for different
aspects of genetic studies as described in the text.
available to test the relationship of any of the
markers with the expression of the phenotype
and thus find linkage.
Another method for mapping QTLs is by us-
ing recombinant inbred (RI) strains. RI strains
are developed by mating pairs of sister-brother
from the F2 generation to produce an F3 gen-
eration. This process of sib mating is repeated
for 20 generations within each RI line. The re-
sult is a new inbred strain that is homozygous in
every locus but has different combination of
genes from the original parental inbred strains.
One advantage of using RI mice is their com-
mercial availability for many inbred strains, min-
imizing breeding time.
The repetitive backcrossing of F1 (donor) an-
imals to one of the parental strains (recipient)
for 20 generations results in a congenic strain
that carries a segment of the chromosome of the
donor strain. At each generation produced, only
those offspring who have received the desired
donor allele and display the desirable phenotype
are selected for the next round of backcrossing.
This results in the transfer of the chromosomal
region of interest from the donor to the other-
wise inbred recipient mouse strain. Congenic
strains are generated to test the effect of a single
or multiple linked loci from a donor strain
placed on the genetic background of a recipient
 Genetic Factors Affecting Complex Traits
strain.20 The use of congenic mice provides a
valuable tool to confirm QTLs and their fine
mapping, and allows for studying the biology
regulated by the donor loci. Recombinant con-
genic inbred strains are generated by two back-
crosses followed by intercrosses for 20 genera-
tions, leading to the fixation of the desired strain
as homozygous (Fig 4).
To investigate genes that affect the suscepti-
bility to histological root resorption associated
with orthodontic force, inbred strains of mice
are being used to investigate their fitness as a
model. In general rodents have been used ex-
tensively to study tissue reaction to orthodontic
tooth movement. Rats are generally used more
than mice to study histological root resorption
associated with orthodontic force (RRAOF), at
least in part because their larger size makes
their manipulation easier. Brudvik and Rygh
were the first to verify mice as a model to
investigate tissue response to orthodontic force,
including RRAOF.22 One challenge is the small
size of mice, which makes the insertion and
calibration of an orthodontic force difficult.
Following the previously cited clinical study,
the potential for effect of the interleukin-1␤ (IL-
1␤) protein on root resorption was confirmed in
an IL-1␤ knockout (KO) mouse model, with the
wild-type (normal) C57BL/6J and the KO mice
having the same baseline RRAOF without any
orthodontic force. When orthodontic force was
applied, in the KO mice RRAOF increased mark-
edly compared with that seen in the wild-type
mice.23 This not only confirms some role of
IL-1␤ in root resorption as at least one possible
factor, but also suggests that in this case the
mechanism is not an increase in inflammation
because of IL-1␤, since the KO mice have been
demonstrated to have no IL-1␤ activity. In addi-
tion to being a cytokine, IL-1␤ also has an affect
on bone resorption, a decrease of which may
increase stress and strain on the root, which may
lead to an increase in root resorption.24 This
could also have an impact on tooth movement,
as discussed in the article titled “Genetic Factors
and Tooth Movement” by Iwasaki and coworkers
in this volume.
Al-Qawasmi and coworkers examined the vari-
ation in severity of RRAOF among eight inbred
strains of mice. In their study they used a coil
spring to tip the maxillary first molar mesially
while controlling for all other factors such as
123
animal age, food, housing, and duration of the
force among the different strains. Results of that
study showed that inbred strains of mice differed
in their response to the same controlled force
with all other environmental factors controlled
for. This implied that RRAOF is a trait in mice
that is influenced by genetic factors. The mice
were grouped into resistant (A/J, C57BL/6J,
and SJL/J), intermediate (C3H/HeJ and AKR/
J), and susceptible (BALB/cJ, DBA/2J, and
129P3/J) strains.25 These strains identified at
the opposite ends of the spectrum can be fur-
ther used to perform QTL analysis.
Inbred strains may also be used to analyze
whether the genetic factors that influence a trait
are complex (polygenic) or Mendelian (mono-
genic). Abass and coworkers crossed A/J (resis-
tant to RRAOF) mice with either DBA/2J or
BALB/cJ (susceptible mice) to identify the
mode of inheritance of RRAOF. F1 animals from
the A/J ⫻ DBA/2J cross showed RRAOF scores
intermediate between the parental strains (A/J
and DBA/2J), whereas F1 animals from the
A/J ⫻ BALB/cJ cross had RRAOF scores that
were closer to the resistant A/J strain. This im-
plied that the trait is polygenic with a major gene
influence.26
Implication to Clinical Practice
Human studies have shown that more than half
of the variation seen clinically in EARR of max-
illary central incisors during orthodontic treat-
ment is associated with genetic variation.10,14 Ap-
parent interaction with environmental factors,
including presumably occlusal and orthodontic
forces when present, makes EARR appear to be
a complex trait in most individuals. Clinical and
animal studies can further investigate the ge-
netic factors, and their interaction with environ-
mental factors, that influence variation in EARR.
A better understanding of the genetic factors
that may predispose an individual to EARR, par-
ticularly if combined with orthodontic treat-
ment, may help to identify the individual who is
more susceptible before treatment.
Depending on the possible likelihood of
EARR with treatment, treatment itself may be
contraindicated, or in a “borderline” case extrac-
tions may be avoided, and or a more frequent
schedule of radiographic monitoring of the case
may be pursued. Ultimately, recognition of and
124 S.K. Abass and J.K. Hartsfield, Jr.
accounting for the genetic factors that vary with
a part of the total variation seen in any clinical
trait is also important to help understand the
effect that environmental factors, including
treatment, have on the clinical trait. If this is not
done, then a significant (both in a statistical and
a real sense) unexplained source of clinical vari-
ation that may alter or even cover up the appar-
ent effect of other factors on the trait will be
ignored.
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