REVIEW OF GENETIC PRINCIPLES

The genome contains the entire genetic content of a set of chromosomes present within a cell or an organism. Within the genome are genes that represent the smallest physical and functional units of inheritance that reside in specific sites (called loci, or locus for a single location). A gene can be defined as the entire DNA sequence necessary for the synthesis of a functional polypeptide molecule (production of a protein via a messenger RNA intermediate) or RNA molecule (transfer RNA and ribosomal RNA). Genotype generally refers to the set of genes that an individual carries and, in particular, usually refers to the specific pair of alleles (alternative forms of a particular gene) that a person has at a given location (locus) of their total collection of DNA, called their genome. In contrast, phenotype is the observable properties and physical characteristics of an individual, as determined by the individual’s genotype and the environment in which the individual develops over a period of time.

Remarkable advances in the biochemical techniques that are used to study cell molecular biology and DNA have taken researchers to the threshold of understanding the regulation of cell functions. To illustrate, not so long ago DNA analyses were performed on minute amounts (picograms) of DNA. This limitation was necessary because there was so little DNA available for study in samples. When the DNA polymerase enzyme was discovered that could replicate DNA through the polymerase chain reaction and make it by the gram, this sample problem disappeared. This advance facilitated completion of the human genome project, which resulted not only in definition of a single human genome sequence composed of overlapping parts from many humans, but also in an expanding catalogue of more than 1 million sites of variation in the human genome sequence. These variations (or polymorphisms) may be used as markers to perform genetic analysis (including analysis of genetic-environmental interaction) in human beings.² The genome varies from one individual to the next, most often in terms of single base changes of the DNA, called single nucleotide polymorphisms (SNPs, pronounced “snips”). The main use of this human SNP map will be to determine the contributions of genes to diseases (or nondisease phenotypes) that have complex, multifactorial bases.³

CELL DIFFERENTIATION AND DEVELOPMENTAL BIOLOGY

It is fascinating that a single fertilized ovum contains within itself the potential for development of the incredibly complicated human organism. Cellular differentiation is a critical component of this developmental process, and aside from the development of antibody diversity, typically occurs in the absence of genetic alteration or mutation. Different types of cells gain their specific identities by using a particular subset of the approximately 30,000 or more genes present within the genome. The types of polypeptides that a cell can synthesize include enzymes, which catalyze various activities of cellular metabolism and homeostasis; structural proteins, which form the intracellular and extracellular scaffolding or cellular matrix; and regulatory proteins, which convey signals from the outside of the cell to the nucleus and modulate or control specific gene expression. In a developing embryo, cells reside in a three-dimensional environment and are responsive to signals from themselves (autocrine), from nearby sources (paracrine), and from anatomically distant sources (endocrine). Many of these signals are mediated by soluble molecules (either peptide or nonpeptide in origin) that bind to specific receptors (proteins) that are present on the surface or on the inside of cells. In addition to signals from soluble factors, cells can respond to cell-to-cell or cell-to-extracellular matrix signals.⁴

The action of “turning on” or “turning off” specific genes, referred to as regulation of gene expression, is carefully orchestrated and remains a critical element in determining cell specificity and tissue morphogenesis. Transcription factors bind to DNA and either facilitate or suppress initiation of gene transcription, the most common control point of gene expression. In the development of the craniofacial complex there is increasing evidence for the role of homeobox-containing gene families that encode transcription factors. These then are critical for the control of complex interactions between genes that are subsequently expressed during development.⁵

In summary:

CHROMOSOMES

DNA is grouped into units called chromosomes. Humans have 46 chromosomes that contain an estimated 30,000 genes, including numerous duplicates. Of the 46 chromosomes, the sex chromosomes are the X and Y, with the remaining 44 chromosomes referred to as autosomes. Each autosome has a paired mate that is referred to as its homologue. Therefore, with the exception of some of the genes on the X and Y chromosomes in males, there are at least two copies of each gene unless a piece of DNA is deleted. Thus the human chromosome complement consists of 23 pairs of chromosomes (one pair of sex chromosomes and 22 pairs of autosomes).

One area of special interest to the clinician is cytogenetics, the study of chromosomes. This interest has been stimulated by the development of techniques in which cells are grown in culture and the chromosomes are examined under a microscope for changes in size, shape, and fine structure. This is called karyotyping. Fig. 6-1 shows the karyotypes of a normal human male and female. By applying this technique, Lejeune and colleagues demonstrated that the fundamental cause in Down syndrome is the presence of an extra specific chromosome (number 21) in the affected individual’s karyotype.⁶ When an entire extra chromosome is present, the condition is called a trisomy of the chromosome in question, for example, trisomy 21 for Down syndrome. Fig. 6-2 shows the karyotype of a male who has Down syndrome. The extra chromosome in the group of number 21 and number 22 chromosomes is readily apparent.

Figure 6-1 Banded karyotypes of a normal human male (left) and female (right). Group designations according to Denver nomenclature are also indicated.

(Courtesy Cytogenetic Laboratories, Indiana University School of Medicine.)

Figure 6-2 Banded karyotype of a male with trisomy of chromosome 21 (Down syndrome).

(Courtesy Cytogenetic Laboratories, Indiana University School of Medicine.)

Since this report in 1959, many disease states have been shown to be associated with an incorrect chromosome complement. By using this approach with considerable refinement, it was shown that alterations in the fine structure of chromosomes, as well as in their number, could be present. Monosomy of an autosome, or a missing autosomal chromosome, had not been believed to be compatible with life, but several monosomies in live-born children have now been reported. Monosomy of the sex chromosomes can be compatible with life and typically affects development of both internal and external sex organs of the individuals. The best known example of this is Turner syndrome, which occurs in approximately 1 in every 5000 live female births. These persons are phenotypic females who are usually missing one of the X chromosomes and are chromosomally designated as 45, X. Other aberrations of the X chromosome may also cause Turner syndrome. Affected individuals are typically short of stature, lack secondary sex characteristics, and are sterile. The Turner syndrome karyotype is shown in Fig. 6-3. Table 6-1 lists common chromosomal aberrations that produce clinical disease, including examples of translocations (the attachment of a broken piece from one chromosome to another, but not homologous, chromosome) and deletions (the absence of a piece of chromosome).

Figure 6-3 Banded karyotype of a female with missing X chromosome (Turner syndrome).

(Courtesy Cytogenetic Laboratories, Indiana University School of Medicine.)

Table 6-1 Common Chromosomal Aberrations

Chromosome abnormalities are an important cause of spontaneous abortion. About 15% of all recognized pregnancies end in spontaneous abortion, and the incidence of chromosome abnormalities in such abortions is greater than 50%. Only 0.3% to 0.5% of all live-born infants have a chromosome abnormality that is detectable with standard microscopic karyotyping. Microdeletions and microduplications of DNA, not visible by routine chromosome karyotype analysis, are a major cause of human malformation and mental retardation. A complementary analysis called comparative genomic hybridization (CGH) or array comparative genomic hybridization (arrayCGH or aCGH) can improve the diagnostic detection rate of these small chromosomal abnormalities. This technique attains such a high-resolution screening by hybridizing differentially labeled test and reference (“normal”) DNAs to arrays consisting of thousands of genomic clones. In this way, relatively small differences between the test and reference DNA sequences may be discovered and investigated further if indicated.^7,8

HEREDITARY TRAITS IN FAMILIES

Heritability is the proportion of the total phenotypic variance in a sample that is contributed by genetic variance.⁹ On an individual basis for a binary trait (i.e., a disease or trait that an individual either has or does not have), heritability is not the proportion of disease or the trait attributable to, or caused by, genetic factors. For a quantitative trait, heritability is not a measure of the proportion of an individual’s score attributable to genetic factors.¹⁰ A trait with a heritability of 1 is said to be expressed without any environmental influence, whereas a trait with a heritability of 0.5 has half its variability (from individual to individual) influenced by environmental factors and half by genotypic factors. Values greater than 1 may occur because the methodology provides an estimate of heritability under several simplifying assumptions that may be incorrect.

There is the common perception that knowing a trait’s heritability will somehow affect how a patient should be treated (e.g., for malocclusion) or that it will define the limits of tooth movement or the manipulation of jaw growth. This is not true. The ability of the patient to respond to changes in the environment (including treatment), which has nothing to do with heritability, defines these limits. Heritability estimates imply nothing about trait size or treatment limits based upon a presumed genetic “predetermination.”¹¹ Even so, the estimation of heritability can provide an indication of the relative importance of genetic factors on a trait in a group at that time. Confirming that there is a certain degree of genetic influence on a trait is a preliminary step to performing further specific genetic linkage studies (using DNA markers) to determine areas of the genome that appear to be associated with the characteristics of a given trait.¹²

When hereditary traits in families are to be studied, it is convenient to think of three classes of genetically influenced traits:.(1) monogenic, (2) polygenic, and (3) multifactorial. Recently the polygenic and multifactorial classes have often been combined into what are referred to as complex traits rather than Mendelian traits.¹³ Monogenic traits are produced and regulated by a single gene locus. Usually they are relatively rare in the general population (occurrence in fewer than 1 per 1000 individuals). However, if the appearance of an affected person is striking, there may be instant recognition of the disease, as with patients having albinism, achondroplasia, or neurofibromatosis. Monogenetic conditions often occur in families and show transmission characteristics of the Mendelian (dominant or recessive) traits.

Polygenic traits, too, are hereditary and typically exert influence over common characteristics such as height, skin, and intelligence. This influence takes place through many gene loci collectively asserting their regulation of the trait. Although each gene involved has a minimal effect by itself, the effect of all the genes involved is additive. The associated phenotype is rarely discrete and is most commonly continuous or quantitative. Because these traits show a quantitative distribution of their phenotypes in a population, they do not show Mendelian inheritance patterns. It is important to note that the very nature of their influence (multiple genes each with a small additive effect) dictates that their environment may readily influence them. Monogenic traits are not readily amenable on a large scale to environmental modification, although there can be variation, presumably secondary to other genetic and environmental factors. By contrast, one can easily think of a dozen environmental factors known to influence height and intelligence quotient.

Finally, multifactorial traits or conditions are influenced by multiple genes but differ significantly from polygenic traits in that the influence is achieved through an interaction of multiple genes and environmental factors, and occurs when a liability threshold is exceeded. Although typically the number of genes involved is many, occasionally a few genes, sometimes only two or three, influence the trait. The effect of these genes on the phenotype is therefore a net effect, not necessarily a simple additive one. Furthermore, phenotypic expression approaches that of a discrete Mendelian trait and therefore cannot be readily classed as a quantitative trait. Likewise, the effect of a gene influencing the phenotype may not be as great as that of a gene associated with a monogenic trait, but the gene may be referred to as having a major effect. Among the well-known hereditary types of conditions designated as multifactorial are many of the severe nonsyndromic congenital malformations such as cleft lip and palate (CLP), neural tube defects such as spina bifida-anencephaly, and hip dislocation. Multifactorial complex inheritance is discussed later.

The investigation of human heritable traits usually involves the observation of specific features in a family and the study of that family’s pedigree. The affected individual in a family who first brings that family to the attention of the geneticist is called the proband or propositus. This individual is the index case. Brothers and sisters of the proband are siblings or sibs. Thus a sibship consists of all the brothers and sisters in a nuclear family unit (parents and their offspring). The clinical appearance in an individual of a given trait, such as eye color or height, is that individual’s phenotype, whereas the specific genetic makeup that influences or is associated with the phenotype is the genotype.

In an earlier section, the point is made that the human chromosome complement has 22 homologous pairs of autosomes and one pair of sex chromosomes. Because of homologue pairing (excluding the X and Y chromosomes in the male), there are at least two copies of each gene, one located at the same position (locus) on each member of the homologous pair. Genes at the same locus on a pair of homologous chromosomes are alleles. When both members of a pair of alleles are identical, the individual is homozygous for that locus. When the two alleles at a specific locus are different, the individual is heterozygous for that locus.

A gene that results in the expression of a particular phenotype in single dose (i.e., heterozygous) is a dominant gene. If the gene must be present in double dose (homozygous) to express the phenotype, it is a recessive gene. It is actually the phenotype that is dominant or recessive and not the gene itself. The terms dominant gene and recessive gene, though, are commonly used to describe these types of inherited traits in families.

Construction of a pedigree, which is a shorthand method of classifying the family data, conveniently summarizes the family data for the study of inherited traits. The symbols used in constructing a pedigree are shown in Fig. 6-4. The observable inheritance patterns followed by such monogenic traits within families are determined by (1) whether the trait is dominant or recessive, (2) whether the gene is autosomal (on one of the autosomes) or X linked (on the X chromosome), and (3) the chance distribution in the offspring of those genes passed from parents in their gametes (sperm and ova). Pedigree construction is a valuable tool for the clinician who is concerned with the diagnosis of and counseling regarding hereditary traits. Every dentist should be able to construct and interpret a pedigree, because it is a certainty that patients will come to the dentist’s office with heritable oral diseases that need diagnosing before treatment is begun.

Figure 6-4 Pedigree symbols used in family studies.

The simple patterns of monogenic inheritance seen in families are described in the following discussion. Because all the Mendelian modes of inheritance are found in the amelogenesis imperfecta (AI) disorders, these are used to illustrate basic genetic principles.

DEVELOPMENTAL BIOLOGY OF ENAMEL

For a review of how molecular biologists are studying the genetic factors involved in dental development, there is a paper by Tucker and Sharpe.¹⁴ The two developmentally different cell layers involved in dentinogenesis, inner enamel epithelium (enamel) and neural crest (dentin), are separated by an extracellular matrix.¹⁵ Specific tooth development is then mutually dependent on reciprocal cell-to-cell signaling between these two developmentally different cell layers.¹⁶ The genes involved in the development of these tissues are candidates for DNA mutation analysis, especially if they are in a chromosome location that has been associated with or linked to an inherited defect of enamel or dentin. The most intriguing dental research today is (1) the attempt to localize the genes for these proteins to specific loci and (2) the biochemical identification of a specific defect in the protein that prevents it from functioning normally. The following is a discussion of genetic principles best exemplified by the heritable disorders of enamel. Further discussion of the molecular basis of the heritable disorders of dentin and enamel appears in Chapter 7.

Based on the clinical appearance, radiographic characteristics, and microscopic features, oral pathologists have recognized three major types of inherited enamel defects: hypoplasia, hypocalcification, and hypomaturation.¹⁷ These terms also provide the general description of the disease phenotypes. For example, in type 1, enamel hypoplasia, the enamel is hard and well calcified but defective in amount, so the teeth appear small. Two types of deficient enamel phenotypes are seen: generalized (all the enamel) and localized (pits and grooves in specific areas). Type 2, hypocalcification disorders, are those in which the enamel matrix is so drastically altered that normal calcification cannot occur, with the result that the clinical phenotype is a soft, mushy enamel that easily wears away. Type 3 defect, hypomaturation, involves the process of maturation of the enamel crystal. This occurs after an essentially normal enamel matrix has been established. The enamel is of normal thickness (not hypoplastic) and relatively normal hardness (slightly hypocalcified) with reduced radiographic density and discoloration.

From this collection of enamel diseases we can now draw out four examples of AI that illustrate the four major Mendelian modes of inheritance: autosomal dominant (AD), autosomal recessive (AR), X-linked dominant (XLD), and X-linked recessive (XLR).

One characteristic of inherited dental defects is that both dentitions (primary and permanent) are affected. Occasionally, the defect is expressed differently in the two dentitions, as in the case of dentin dysplasia type II.¹⁸ However, it is much more common to see the same clinical and radiographic picture in both dentitions. Both dentitions are affected in the AI disorders.

AUTOSOMAL DOMINANT INHERITANCE

From pedigrees such as that shown in Fig. 6-5, the following criteria for AD inheritance may be deduced:

Figure 6-5 Autosomal dominant inheritance in hypocalcified amelogenesis imperfecta.

The hypocalcification type of AI provides an excellent example of AD inheritance. For diagnosing this trait, several criteria are employed. First, enamel matrix is susceptible to abrasion. The clinical picture is typical-gross accumulation of plaque on teeth that are hypersensitive because of the exposed dentin. Second, radiographs show enamel of varying thickness interproximally but with a Swiss cheese appearance because of loss of mineral. Thus severe abrasion of this soft enamel is common.

AUTOSOMAL RECESSIVE INHERITANCE

Recessively inherited traits require that both genes of a given pair at a single locus code for defective proteins. Thus, of the two alleles at this genetic locus for AI, both must be mutants to show the trait. The following three gene pairs are recognized: AA—normal; Aa—heterozygote, showing an unaffected phenotype; aa—homozygous-affected. The most common genetic situation producing an affected child is that in which both parents are heterozygous at this genetic locus (Fig. 6-6).

Figure 6-6 Autosomal recessive inheritance in pigmented amelogenesis imperfecta.

The following significant points about recessive inheritance must be noted:

Of several AR types of AI, the one chosen for discussion here is the pigmented hypomaturation form. In this instance, the genetic defect probably lies in the protein needed in late tooth development to produce mature, hard, and dense enamel. The defective enamel present is softer than normal but not nearly as soft and easily abraded as in the hypocalcification defect. Remarkably, a brown pigment is found in these outer layers of enamel that are formed last, imparting a dark brown, unsightly appearance that necessitates restorative treatment. A pedigree illustrating AR inheritance of this hypomaturation defect is shown in Fig. 6-6.

X-LINKED OR SEX-LINKED INHERITANCE

Genes on the sex chromosomes are unequally distributed to males and females. This inequality is the result of the following facts: (1) males have one X and one Y chromosome, whereas females have two X chromosomes and (2) the genes active on the Y chromosome are essentially concerned with the development of the male reproductive system. For these reasons, then, males are hemizygous for X-linked genes, meaning that they have only half (or one each) of the X-linked genes. Because females have two X chromosomes, they may be either homozygous or heterozygous for X-linked genes, just as with autosomal genes.

Interesting genetic combinations are made possible by the male hemizygous condition. Because only one gene locus of each kind in the X chromosome is represented in the male, all recessive genes in single dose express themselves phenotypically and thereby behave as though they were dominant genes. On the other hand, X-linked recessive (XLR) genes must be present in double dose (homozygous) in females to fully express themselves. Consequently, full expression of rare XLR diseases in practice is restricted to males and is seen infrequently in females.

To this point we have considered heritable defects in two of the three major types of enamel disorders. The third type-AI, hypoplastic type-shows both autosomal and X-linked modes of inheritance, but only one X-linked type is described here.

X-LINKED DOMINANT

Fig. 6-7 is the pedigree of a family with an X-linked dominant (XLD) form of AI, hypoplastic type.¹⁹ The clinical features are diagnostic and in some females can be quite striking.

Figure 6-7 X-linked dominant inheritance in hypoplastic amelogenesis imperfecta.

Once again, both dentitions are affected similarly. The surface defect has been described as being granular, lobular, or even pitted. Conceivably, all these different forms of expression are the result of the action of a single gene (or at least its alleles). The enamel is hard but because of its thinness is more susceptible to fracture and abnormal wear. Under the appropriate conditions, this trait resembles a hypocalcification defect. However, radiographs quickly resolve this diagnostic problem and show enamel of normal density but with greatly reduced thickness.

X-LINKED RECESSIVE

A pedigree of a family with the X-linked recessive form of enamel hypomaturation is shown in Fig. 6-8. The genetic criteria for diagnosing an XLR trait are summarized as follows:

1. Because the gene cannot be passed from father to son, affected fathers almost never have affected sons. A son could be affected if the mother is a carrier of the XLR trait.

2. All daughters of an affected male receive his X-linked genes. Therefore affected males transmit the trait to their grandsons if they are affected through their daughters.

3. The incidence of the trait is much higher in males than in females. This is typified by the disease hemophilia, which is also caused by an XLR gene.

Figure 6-8 X-linked recessive inheritance in hypomaturation amelogenesis imperfecta.

The clinical features of XLR hypomaturation type AI are most striking. The enamel has a somewhat reduced hardness but is not soft. However, the crowns of the teeth look like mountains with snow on them. Hence the name given has been “snow-capped teeth.” Radiologically the enamel is hypomature; it shows a lack of contrast between enamel and dentin even though the enamel is of normal thickness.

It should be noted that heterozygous females occasionally show significant clinical expression of a single XLR gene. The reason for this apparent contradiction is the process of X-inactivation, termed lyonization after geneticist Mary Lyon. This occurs only in females. All normal female cells have two X chromosomes, but most of the genes on one of the two X chromosomes are inactivated approximately at the blastula stage of development. This has the effect of making the total number of active, X-linked genes about the same in both males and females. If the female is heterozygous for an X-linked trait, two populations of cells result. One cell population has genes on one X chromosome that are active, while the other cell population has genes on the other X chromosome that are active. When by chance the X chromosome with the deleterious gene is active in a significant proportion of the cells, its expression may be observed in that female. Chance dictates that this imbalance does not occur frequently, but because all females are, by definition of lyonization, mosaic with regard to X-linked traits, phenotypic expression of heterozygous genes may occur in them.

The previous statements concerning the distribution of XLR genes in males and females apply equally as well to XLD genes. The principal difference lies in the fact that when the gene is dominant more females than males will show the trait (see pedigree in Fig. 6-8). Because all XLR genes behave as dominant genes in males, no new criteria are made for their inheritance in males. The following criteria distinguish an XLD trait in families:

1. Affected males must transmit the trait to all of their daughters (as with XLR traits), but all of them are affected, because fathers give their X chromosome to their daughters and their Y chromosome to their sons.

2. Affected males cannot transmit the trait to their sons (just as with XLR traits).

3. Heterozygous females transmit the trait on the average to 50% of their children of both sexes, whereas homozygous-affected females will have only affected children. The latter situation is exceptionally rare for a dominant trait and is practically never observed. Thus all females affected with a dominantly inherited X-linked trait are considered to be heterozygotes until proven otherwise.

Two points are emphasized here. First, transmission of XLD genes by females follows a pattern indistinguishable from that of autosomal transmission. Thus these two types of dominant inheritance can be differentiated only by observation of the offspring of affected males. Second, it was noted that XLR disorders are much less common in females than in males. The reverse is true for XLD traits. An XLD trait should appear about twice as often in females as in males, because females have twice as many X chromosomes as males.

VARIATION IN GENE EXPRESSION

The patterns of inheritance shown in traits determined by genes at a single locus are usually easy to recognize. However, many factors may modify the expression of a gene in a family in such a way that a typical monogenic pattern of inheritance is not discernible. Two concepts related to modification of gene action are discussed here: penetrance and expressivity.