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Investigation of Genetic Factors Affecting Complex Traits Using External Apical Root Resorption as a Model
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 environmental 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 determinants 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 complex 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 common clinical complication of orthodontic 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 maxillary incisors. Seven to 13% of individuals who have not had orthodontic treatment show 1 to 3 mm of EARR on radiographs.1 Severe EARR,
E
Assistant Professor of Dental Science, Faculty of Dentistry, University 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 orthodontic 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 problem for orthodontists and patients who undergo orthodontic treatment. There is a significant variability for EARR susceptibility among individuals. This variability can be due to an innate or systemic predisposition to resorption in permanent and primary teeth in these individuals.4-9 It has been pro-
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posed that when extreme susceptibility exists, EARR can occur in absence of any specific causative factor.7,10 A racial dichotomy has also been reported with Asian patients having less EARR than white or Hispanic patients.9 Familial clustering of EARR has also been reported, although no Mendelian pattern of inheritance was identified.6 Although racial differences and familial clustering may suggest a genetic component, it may also reflect different and common environmental factors respectively. Although many factors 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 environmental 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 genetic factors associated with a complex trait such as EARR.
Genetic Basis of Disease The genetic background (genome) and environmental (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 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 is the simple or Mendelian traits. These traits follow the two genetic laws of heredity: the law of segregation and the law of independent assortment identified by Gregor Mendel in 1985. These traits have a “simple” pattern of inheritance (autosomal dominant, autosomal recessive, or Xlinked) 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 information is available on human genetics, including a glossary, at the National Library of Medicine National Institutes of Health “Genetics Home Reference” http://ghr.nlm.nih.gov Web site. Pinpointing a genotype-phenotype relationship is relatively easy to establish in Mendelian traits or diseases because a single gene mutation usually results in a recognizable phenotype. Environmental factors and other genes may modify the clinical expression of the disease,11 but are not of crucial importance for disease development (Fig 1). Such single gene traits or diseases are relatively rare except in certain isolated communities. 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.
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Examples of simple traits include amelogenesis imperfecta and cleidocranial dysplasia. For a list of genetic traits that follow Mendelian inheritance, 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 mutation responsible for a particular Mendelian trait allows the screening of individuals carrying the mutation, and also allows for predicting the disease transmission from parents to offspring (genetic 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 determinants 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 nonaffected. The interplay of these genetic polymorphisms at different loci with environmental factors leads to the manifestation of such complex traits. Unlike Mendelian traits, environmental factors and multiple genes are critical to the development of such complex traits (Fig 2). These
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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. Examples of such physical traits include height and weight (which are measurable, not dichomatous). Disease 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 genetic aspect of a trait is studying families, especially 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 environmental conditions, which should reduce the noise of environmental factors. It should be understood that this is variably not the case (see the discussion of heritability estimates in “Interpreting 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.
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(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 concordant. If the trait of interest has a higher concordance in MZ twins compared with DZ twins, this implies a genetic component. A recent retrospective twin study on EARR that included 16 MZ and 10 DZ twins showed that the concordance scores for MZ twins were approximately 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 environmental 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 investigated for the trait of interest, since they share similar (50% of their genes in common on average) 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), family 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 relative risk value the greater the familial aggregation of the disease due to common familial genetic and environmental factors. For quantitative traits (complex traits) this family resemblance is expressed 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 genetic to total (additive genetic plus environmental) variation does not take into account geneto-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 expressed 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 environment analyzed at that point in time. Although it may seem strange, heritability estimated 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 estimate for the trait being analyzed will change (decrease in this example). Also heritability estimates are population estimates and not applicable to an individual (Fig 3). Using a sib-pair model, Harris and coworkers have shown that the h2 estimates for EARR averaged 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 implies that siblings experience similar levels of EARR in response to orthodontic treatment compared with unrelated individuals. The study conducted 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 narrow sense, estimating the effect of an indefinitely large number of genes that all contribute equally to the phenotype. It cannot take into account alleleallele interactions at a gene locus (termed dominance) and gene-gene interactions involving two or more loci (termed epistasis).
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Strategies of Identifying EARR Genes
Linkage Analysis
Tools to Identify Disease Genes
Linkage analysis tests for the cosegregation of a marker and a trait (disease) locus in families with affected individuals. During meiosis the genetic material crosses over from one parental derived chromosome to the other through chromosomal recombination. Chromosome segments and their associated genes that are far apart are more likely to recombine during meiosis. Chromosome segments and their associated 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 chromosomes, the recombination fraction is 0.5, which means that the two markers are inherited independently following Mendel’s law of independent assortment. For genes that tend to be inherited together (linked) in a part of a chromosome, the recombination fraction will be less than 0.5. The results of linkage studies are reported 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 (recombination 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 information see “Genetic Factors and Orofacial Clefting” by Lidral and coworkers in this volume). 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.
Besides mitochondrial DNA, human genetic information 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 chromosomes 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 unknown functions. These noncoding regions of the DNA provide an important tool for the study of the genome. The DNA sequence varies at a particular chromosomal location in both the coding and noncoding 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 genetic analysis of diseases (for further information 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 susceptibility. 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 families. These repeated nucleotide sequences are usually found in noncoding regions and are thus typically nonfunctional polymorphisms. Another form of polymorphism widely found in the genome is single nucleotide polymorphisms (SNPs). SNPs can be in both the coding and the noncoding DNA regions. Although most of the SNPs are found within noncoding regions, SNPs found within coding regions are of particular importance, since depending on their codon position and genetic code they may alter the amino acid sequence and potentially the biological function of the protein.
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EARR, however, is a complex disease, with multiple genetic and environmental factors contributing 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 provide a way to perform linkage analysis without knowing the model of inheritance. In allele-sharing 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 multigenerational families, it does require a large numbers of sib pairs to gain enough power for the localization 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 manifestation 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 (monogenic) or major gene disorders, since in complex traits several genes contribute to the disease process. Linkage analysis and allele-sharing studies are performed by studying a panel of polymorphisms on the whole genome (genome-wide approach). In this way chromosome areas of interest 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 approach). Candidate genes are usually selected because of previous knowledge of the function of the gene and its possible affect on the development of the disease if mutated. In an effort to identify genetic factors in EARR Al-Qawasmi and coworkers used the candidate gene approach to look for evidence of linkage 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 disequilibrium (LD), which is the consistency of association of alleles at two linked loci. It is assumed that the disease-associated polymorphism occurs more frequently with a set of markers. LD analysis tests the hypothesis that a marker and the disease allele occur more frequently in a sample of affected individuals compared with unaffected 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 controls to compare the allele’s frequencies at a marker in a candidate gene. In such populationbased association studies (case-control), LD results can be complicated by inherited differences 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 stratification. One such method is the affected familybased control (AFBAC) method, in which the frequency distribution of marker alleles transmitted from parents to an affected child is compared with those not transmitted. The transmitted and nontransmitted alleles come from the same parent. Another method is the transmission disequilibrium test (TDT), in which the frequency of the transmission of a particular allele from heterozygous parents to an affected child is compared with the 50% Mendelian ratio. Because in such a triad (parents and affected child) the
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transmitted allele acts as a case and the untransmitted 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 disequilibrium for an IL-1B polymorphism with EARR was reported.16 The analysis of 35 families indicated that the IL-1B polymorphism accounts for 15% of the total variation seen for EARR seen in the maxillary central incisor in the sample studied. When locating susceptibility genes, both linkage and association analysis may be useful in the same families. Linkage analysis usually identifies regions of the genome that contain the susceptibility 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 disequilibrium to identify the trait-associated polymorphism at that region. Another approach is to identify certain candidate genes and then analyze 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 Genetics in Practice?” in this volume, the rapid growth in genome-wide association studies presents both the greatest challenge and the likelihood 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 (diseases) have a complex genetic etiology. Multiple genetic loci and the environment interact to modulate susceptibility to such complex diseases. 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 environmental factors. This is facilitated by the
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mouse and human genome showing a high degree 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 environmental factors and ease of conducting crosses among the different strains makes them a perfect model in genetic studies. Mice carrying a specific gene deletion (knockout) 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 physiology. 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 repeated 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 further generations, inbred mice become homozygous in all loci providing a colony of genetically identical mice. With exception to the sex chromosomes, 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 phenotypic 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 (differences in background genes between the inbred 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
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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 generation that has a unique combination of progenitor genes. The genetic diversity of F2 animals 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) analysis. QTL analysis has proved to be the method of choice in identifying genes responsible for complex traits, particularly in absence of evidence 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 markers allows the identification of both major and minor effect loci. By only analyzing the F2 generation members who have different extremes of the phenotype, one can identify major loci affecting the trait. Software programs are now
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 using recombinant inbred (RI) strains. RI strains are developed by mating pairs of sister-brother from the F2 generation to produce an F3 generation. This process of sib mating is repeated for 20 generations within each RI line. The result 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 commercial availability for many inbred strains, minimizing breeding time. The repetitive backcrossing of F1 (donor) animals 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 otherwise 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
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.
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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 congenic inbred strains are generated by two backcrosses followed by intercrosses for 20 generations, leading to the fixation of the desired strain as homozygous (Fig 4). To investigate genes that affect the susceptibility 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 extensively 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 (IL1) 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 markedly 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 addition 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 variation 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
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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 further 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 (monogenic). Abass and coworkers crossed A/J (resistant 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 implied 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 maxillary central incisors during orthodontic treatment is associated with genetic variation.10,14 Apparent 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 genetic factors, and their interaction with environmental factors, that influence variation in EARR. A better understanding of the genetic factors that may predispose an individual to EARR, particularly if combined with orthodontic treatment, 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 extractions may be avoided, and or a more frequent schedule of radiographic monitoring of the case may be pursued. Ultimately, recognition of and
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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 variation that may alter or even cover up the apparent effect of other factors on the trait will be ignored.
References 1. Harris EF, Robinson QC, Woods MA: An analysis of causes of apical root resorption in patients not treated orthodontically. Quintessence Int 24:417-428, 1993 2. Taithongchai R, Sookkorn K, Killiany DM: Facial and dentoalveolar structure and the prediction of apical root shortening. Am J Orthod Dentofacial Orthop 110:296302, 1996 3. Killiany DM: Root resorption caused by orthodontic treatment: an evidence-based review of literature. Semin Orthod 5:128-133, 1999 4. Becks H: Root resorptions and their relation to pathological bone formation, part 1: statistical data and roentgenographic aspects. Int J Orthod Oral Surg 22:445-482, 1936 5. Reitan K: Some factors determining the evaluation of forces in orthodontics. Am J Orthod 43:32-45, 1957 6. Newman WG: Possible etiologic factors in external root resorption. Am J Orthod 67:522-539, 1975 7. Massler M, Perreault JG: Root resorption in the permanent teeth of young adults. J Dent Child 21:158-164, 1954 8. Massler M, Malone AJ: Root resorption in human permanent teeth: a roentgenographic study. Am J Orthod 40:619-633, 1954 9. Sameshima GT, Sinclair PM: Predicting and preventing root resorption: part II. Treatment factors. Am J Orthod Dentofacial Orthop 119:511-515, 2001 10. Harris EF, Kineret SE, Tolley EA: A heritable component for external apical root resorption in patients treated orthodontically. Am J Orthod Dentofacial Orthop 111: 301-309, 1997 11. Chanock S, Wacholder S: One gene and one outcome? No way. Trends Mol Med 8:266-269, 2002 12. Martin N, Boomsma D, Machin G: A twin-pronged attack on complex traits. Nat Genet 17:387-392, 1997
13. Ngan DC, Kharbanda OP, Byloff FK, et al: The genetic contribution to orthodontic root resorption: a retrospective twin study. Aust Orthod J 20:1-9, 2004 14. Hartsfield JK Jr, Everett ET, Al-Qawasmi RA: Genetic factors in external apical root resorption and orthodontic treatment. Crit Rev Oral Biol Med 15:115-122, 2004 15. Nakagawa N, Kinosaki M, Yamaguchi K, et al: RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem Biophys Res Commun 253:395-400, 1998 16. Al-Qawasmi RA, Hartsfield JK Jr, Everett ET, et al: Genetic predisposition to external apical root resorption in orthodontic patients: linkage of chromosome-18 marker. J Dent Res 82:356-360, 2003 17. Meisler MH: The role of the laboratory mouse in the human genome project. Am J Hum Genet 59:764-771, 1996 18. Ehrlich J, Sankoff D, Nadeau JH: Synteny conservation and chromosome rearrangements during mammalian evolution. Genetics 147:289-296, 1997 19. Nadeau JH, Dunn PJ: Genomic strategies for defining and dissecting developmental and physiological pathways [review]. Curr Opin Genet Dev 8:311-315, 1998 20. Silver L: Mouse Genetics. New York, Oxford University Press, 1995 21. Grisel JE: Quantitative trait locus analysis. Alcohol Res Health 24:169-174, 2000 22. Brudvik P, Rygh P: The initial phase of orthodontic root resorption incident to local compression of the periodontal ligament. Eur J Orthod 15:249-263, 1993 23. Al-Qawasmi RA, Hartsfield JK Jr, Everett ET, et al: Root resorption associated with orthodontic force in IL-1beta knockout mouse. J Musculoskelet Neuronal Interact 4:383-385, 2004 24. Roberts WE, Hartsfield JK Jr, Al-Qawasmi R, et al: Physiologic interaction of root resorption, remodeling, tooth movement and ankylosis, in Davidovitch Z, Mah J, Suthanarak S (eds): Biological Mechanisms of Tooth Eruption, Resorption and Movement. Boston, MA, Harvard Society for the Advancement of Orthodontics, 2006, pp 191-198 25. Al-Qawasmi RA, Hartsfield JK Jr, Everett ET, et al: Root resorption associated with orthodontic force in inbred mice: genetic contributions. Eur J Orthod 28: 13-19, 2006 26. Abass S, Hartsfield JK Jr, Al-Qawasmi R, Everett ET, Foroud T, Roberts WE: Inheritance of susceptibility to root resorption associated with orthodontic force in mice. Am J Orthod Dentofacial Orthop. In press
xternal apical root resorption (EARR) is a common clinical complication of orthodontic 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 maxillary incisors. Seven to 13% of individuals who have not had orthodontic treatment show 1 to 3 mm of EARR on radiographs.1 Severe EARR,
E
Assistant Professor of Dental Science, Faculty of Dentistry, University 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 orthodontic 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 problem for orthodontists and patients who undergo orthodontic treatment. There is a significant variability for EARR susceptibility among individuals. This variability can be due to an innate or systemic predisposition to resorption in permanent and primary teeth in these individuals.4-9 It has been pro-
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posed that when extreme susceptibility exists, EARR can occur in absence of any specific causative factor.7,10 A racial dichotomy has also been reported with Asian patients having less EARR than white or Hispanic patients.9 Familial clustering of EARR has also been reported, although no Mendelian pattern of inheritance was identified.6 Although racial differences and familial clustering may suggest a genetic component, it may also reflect different and common environmental factors respectively. Although many factors 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 environmental 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 genetic factors associated with a complex trait such as EARR.
Genetic Basis of Disease The genetic background (genome) and environmental (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 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 is the simple or Mendelian traits. These traits follow the two genetic laws of heredity: the law of segregation and the law of independent assortment identified by Gregor Mendel in 1985. These traits have a “simple” pattern of inheritance (autosomal dominant, autosomal recessive, or Xlinked) 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 information is available on human genetics, including a glossary, at the National Library of Medicine National Institutes of Health “Genetics Home Reference” http://ghr.nlm.nih.gov Web site. Pinpointing a genotype-phenotype relationship is relatively easy to establish in Mendelian traits or diseases because a single gene mutation usually results in a recognizable phenotype. Environmental factors and other genes may modify the clinical expression of the disease,11 but are not of crucial importance for disease development (Fig 1). Such single gene traits or diseases are relatively rare except in certain isolated communities. 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.
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Examples of simple traits include amelogenesis imperfecta and cleidocranial dysplasia. For a list of genetic traits that follow Mendelian inheritance, 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 mutation responsible for a particular Mendelian trait allows the screening of individuals carrying the mutation, and also allows for predicting the disease transmission from parents to offspring (genetic 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 determinants 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 nonaffected. The interplay of these genetic polymorphisms at different loci with environmental factors leads to the manifestation of such complex traits. Unlike Mendelian traits, environmental factors and multiple genes are critical to the development of such complex traits (Fig 2). These
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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. Examples of such physical traits include height and weight (which are measurable, not dichomatous). Disease 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 genetic aspect of a trait is studying families, especially 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 environmental conditions, which should reduce the noise of environmental factors. It should be understood that this is variably not the case (see the discussion of heritability estimates in “Interpreting 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.
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(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 concordant. If the trait of interest has a higher concordance in MZ twins compared with DZ twins, this implies a genetic component. A recent retrospective twin study on EARR that included 16 MZ and 10 DZ twins showed that the concordance scores for MZ twins were approximately 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 environmental 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 investigated for the trait of interest, since they share similar (50% of their genes in common on average) 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), family 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 relative risk value the greater the familial aggregation of the disease due to common familial genetic and environmental factors. For quantitative traits (complex traits) this family resemblance is expressed 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 genetic to total (additive genetic plus environmental) variation does not take into account geneto-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 expressed 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 environment analyzed at that point in time. Although it may seem strange, heritability estimated 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 estimate for the trait being analyzed will change (decrease in this example). Also heritability estimates are population estimates and not applicable to an individual (Fig 3). Using a sib-pair model, Harris and coworkers have shown that the h2 estimates for EARR averaged 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 implies that siblings experience similar levels of EARR in response to orthodontic treatment compared with unrelated individuals. The study conducted 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 narrow sense, estimating the effect of an indefinitely large number of genes that all contribute equally to the phenotype. It cannot take into account alleleallele interactions at a gene locus (termed dominance) and gene-gene interactions involving two or more loci (termed epistasis).
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Strategies of Identifying EARR Genes
Linkage Analysis
Tools to Identify Disease Genes
Linkage analysis tests for the cosegregation of a marker and a trait (disease) locus in families with affected individuals. During meiosis the genetic material crosses over from one parental derived chromosome to the other through chromosomal recombination. Chromosome segments and their associated genes that are far apart are more likely to recombine during meiosis. Chromosome segments and their associated 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 chromosomes, the recombination fraction is 0.5, which means that the two markers are inherited independently following Mendel’s law of independent assortment. For genes that tend to be inherited together (linked) in a part of a chromosome, the recombination fraction will be less than 0.5. The results of linkage studies are reported 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 (recombination 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 information see “Genetic Factors and Orofacial Clefting” by Lidral and coworkers in this volume). 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.
Besides mitochondrial DNA, human genetic information 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 chromosomes 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 unknown functions. These noncoding regions of the DNA provide an important tool for the study of the genome. The DNA sequence varies at a particular chromosomal location in both the coding and noncoding 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 genetic analysis of diseases (for further information 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 susceptibility. 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 families. These repeated nucleotide sequences are usually found in noncoding regions and are thus typically nonfunctional polymorphisms. Another form of polymorphism widely found in the genome is single nucleotide polymorphisms (SNPs). SNPs can be in both the coding and the noncoding DNA regions. Although most of the SNPs are found within noncoding regions, SNPs found within coding regions are of particular importance, since depending on their codon position and genetic code they may alter the amino acid sequence and potentially the biological function of the protein.
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EARR, however, is a complex disease, with multiple genetic and environmental factors contributing 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 provide a way to perform linkage analysis without knowing the model of inheritance. In allele-sharing 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 multigenerational families, it does require a large numbers of sib pairs to gain enough power for the localization 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 manifestation 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 (monogenic) or major gene disorders, since in complex traits several genes contribute to the disease process. Linkage analysis and allele-sharing studies are performed by studying a panel of polymorphisms on the whole genome (genome-wide approach). In this way chromosome areas of interest 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 approach). Candidate genes are usually selected because of previous knowledge of the function of the gene and its possible affect on the development of the disease if mutated. In an effort to identify genetic factors in EARR Al-Qawasmi and coworkers used the candidate gene approach to look for evidence of linkage 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 disequilibrium (LD), which is the consistency of association of alleles at two linked loci. It is assumed that the disease-associated polymorphism occurs more frequently with a set of markers. LD analysis tests the hypothesis that a marker and the disease allele occur more frequently in a sample of affected individuals compared with unaffected 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 controls to compare the allele’s frequencies at a marker in a candidate gene. In such populationbased association studies (case-control), LD results can be complicated by inherited differences 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 stratification. One such method is the affected familybased control (AFBAC) method, in which the frequency distribution of marker alleles transmitted from parents to an affected child is compared with those not transmitted. The transmitted and nontransmitted alleles come from the same parent. Another method is the transmission disequilibrium test (TDT), in which the frequency of the transmission of a particular allele from heterozygous parents to an affected child is compared with the 50% Mendelian ratio. Because in such a triad (parents and affected child) the
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transmitted allele acts as a case and the untransmitted 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 disequilibrium for an IL-1B polymorphism with EARR was reported.16 The analysis of 35 families indicated that the IL-1B polymorphism accounts for 15% of the total variation seen for EARR seen in the maxillary central incisor in the sample studied. When locating susceptibility genes, both linkage and association analysis may be useful in the same families. Linkage analysis usually identifies regions of the genome that contain the susceptibility 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 disequilibrium to identify the trait-associated polymorphism at that region. Another approach is to identify certain candidate genes and then analyze 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 Genetics in Practice?” in this volume, the rapid growth in genome-wide association studies presents both the greatest challenge and the likelihood 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 (diseases) have a complex genetic etiology. Multiple genetic loci and the environment interact to modulate susceptibility to such complex diseases. 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 environmental factors. This is facilitated by the
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mouse and human genome showing a high degree 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 environmental factors and ease of conducting crosses among the different strains makes them a perfect model in genetic studies. Mice carrying a specific gene deletion (knockout) 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 physiology. 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 repeated 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 further generations, inbred mice become homozygous in all loci providing a colony of genetically identical mice. With exception to the sex chromosomes, 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 phenotypic 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 (differences in background genes between the inbred 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
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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 generation that has a unique combination of progenitor genes. The genetic diversity of F2 animals 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) analysis. QTL analysis has proved to be the method of choice in identifying genes responsible for complex traits, particularly in absence of evidence 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 markers allows the identification of both major and minor effect loci. By only analyzing the F2 generation members who have different extremes of the phenotype, one can identify major loci affecting the trait. Software programs are now
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 using recombinant inbred (RI) strains. RI strains are developed by mating pairs of sister-brother from the F2 generation to produce an F3 generation. This process of sib mating is repeated for 20 generations within each RI line. The result 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 commercial availability for many inbred strains, minimizing breeding time. The repetitive backcrossing of F1 (donor) animals 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 otherwise 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
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.
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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 congenic inbred strains are generated by two backcrosses followed by intercrosses for 20 generations, leading to the fixation of the desired strain as homozygous (Fig 4). To investigate genes that affect the susceptibility 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 extensively 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 (IL1) 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 markedly 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 addition 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 variation 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
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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 further 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 (monogenic). Abass and coworkers crossed A/J (resistant 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 implied 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 maxillary central incisors during orthodontic treatment is associated with genetic variation.10,14 Apparent 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 genetic factors, and their interaction with environmental factors, that influence variation in EARR. A better understanding of the genetic factors that may predispose an individual to EARR, particularly if combined with orthodontic treatment, 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 extractions may be avoided, and or a more frequent schedule of radiographic monitoring of the case may be pursued. Ultimately, recognition of and
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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 variation that may alter or even cover up the apparent effect of other factors on the trait will be ignored.
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