The Genome

The term genome refers to the collection of all of the genes that an organism possesses. It has been estimated that there are between 40,000 and 140,000 genes in the human genome. The genes are assembled into lengthy strands of deoxyribonucleic acid (DNA), which are organized linearly into chromosomes. The chromosomes are made up of the DNA that forms the genes and the intervening DNA as well as chromatin, which is a protein that assists in the maintenance of the structure and regulation of chromosomal expression. The nuclei of most human cells contain 46 chromosomes that are organized as 23 pairs. Except for the mitochondria, all genes that are contained within the human genome lie on one or the other of these chromosomes. The linear order of the genes on the chromosomes allows one to create maps of the human gene order; these maps are generally invariant throughout any given species. It is these maps that allow us to associate specific genes with specific traits through gene mapping.^7,8 Genes are transmitted in groups that correspond with the chromosomes. Aside from cases of the exception known as “crossing over,” all of the genes of one chromosome (e.g., the paternal one) are transmitted together to the exclusion of the other one. Crossing over allows one chromosome to become a mosaic of both paternal and maternal genes; the frequency with which crossing over occurs has been studied and forms the basis for one of the two ways of measuring distances between genes. The physical distance between two genes is the number of bases between genes and is measured in megabases or kilobases. The genetic distance between two genes, however, is based on the frequency of observed recombination between them and can only be estimated by the study of informative matings and their offspring. Genetic distance is the result of a biologic phenomenon and is imperfectly correlated with physical distance. The order of genes on a chromosome is constant.

The amount of information stored in the genome is tremendous. In the human genome there are approximately 3 × 10⁹ base pairs of DNA that make up the haploid genome. The largest chromosome, 1, contains about 10% of the total, whereas the smallest autosome, 21, contains about 2.5%. Given that the estimated number of genes is between 20,000 and 30,000, the expected number of genes per megabase is between 20 and 30. The average high-resolution chromosome band is about three megabases in size and would be expected to contain between 60 and 90 genes.

Another way of appreciating the size of the genome is to compare the amount of information in it with that in the typical encyclopedia. The encyclopedia would need to have 200 volumes of 1000 pages each to contain the information found in the human genome. In this analogy, gene size would vary from about a third of a page up to several pages. In actuality, the human genetic encyclopedia is packaged into 23 volumes, and the genes are not as easily demarcated as are chapters in a real encyclopedia. However, the analogy serves well when trying to understand the importance of deciphering the genome. This biologic encyclopedia is in truth a manual for the construction and maintenance of a human being. By understanding the information contained within the genes, we will come to understand the basics of our own biology.

Unfortunately, the information in the genome is simply not organized into rational groupings. One purpose of the genome project had been to develop an index of the genome that would allow researchers who are trying to connect specific genes with specific disorders to do so efficiently.^9,10 From the perspective of the otolaryngologist, this means first that gene-specific diagnoses are now available for many ENT-related disorders and that ultimately better therapies will emerge as more is learned about the basic nature of hereditary disorders that affect hearing, speech, and the structures of the head and neck.

The genome revolution has placed mankind at the brink of the development of new and exciting therapies for both rare and common genetic disorders.¹¹ It will thus become increasingly important for the ENT practitioner to recognize those disorders that have a strong genetic component.

DNA Structure and the Genetic Code

Humans store genetic information in DNA, which is a linear polymer made up of four different nucleotides: adenine (A), guanine (G), thymidine (T), and cytosine (C). Nucleotides (also called bases) are linked together by phosphodiester bonds into a single strand. Nucleotides also have the capability of pairing with each other (A with T and G with C) through hydrogen bonds. Two strands of DNA can pair with each other in a complementary fashion (again, A with T and G with C) to form a double helix. The two strands are perfectly complementary; for example, if one strand has an order of ATGGGCCATA, its complement would be TACCCGGTAT. During replication, the two strands separate, and the base sequence of each strand would dictate the construction of a new, complementary strand. In this way, the sequence of the double strands is preserved in the two new identical double strands that are produced.

A single strand has an orientation that reflects the direction of the phosphodiester bond, which is usually referred to as going from the 5′ to the 3′ end; genes are transcribed in this direction. Because there is a double helix, the actual transcription occurs from only one strand, called the template strand. The antiparallel strand is referred to as the coding strand, because its base sequence corresponds with the sequence of the message; however, uracil is substituted for thymidine in the message.

The sequence of bases is what determines all parts of the gene, and it specifically determines the sequence of amino acids in the protein that results from the process of translation. The nucleotides within the coding region are arranged in groups of three, called codons, which determine the precise amino acid sequence. Because there are four bases, there are 64 possible combinations of nucleotides, but there are only 20 amino acids. Thus the code is said to be degenerate, because most amino acids are specified by more than one codon. For example, the code for valine can be GTT, GTC, GTA, or GTG. The third nucleotide can vary for most amino acids and is often called the wobble nucleotide. A specific codon, ATG, codes for methionine and also indicates the beginning of a coding sequence. There are three stop codons: TAA, TAG, and TGA.

Gene Structure and Expression

The definition of a gene has gone through several stages and is now neither simple nor straightforward. The gene is the basic unit of biologic information that can be transmitted from parents to offspring, and it typically provides information about structural or functional components of the cell. The information transfer occurs not only between parent and daughter cells but also between the nucleus and the cytoplasmic machinery. The transfer of this information is called inheritance when it occurs between parent and child. The transfer of information from genome within the nucleus to the cell proper would be referred to as gene expression. Gene structure has presumably evolved in a way that facilitates the transfer of genetic information, but the true molecular boundaries that define any specific gene are often poorly recognized.

The basic eukaryotic gene is made up of exons and introns. Exons make up the coding part of the gene; the intron is DNA that is interspersed between the exons. During the process of creating a message, introns are spliced out of the message, thus leaving only the exons to be translated into protein. The gene is thus an interrupted sequence of code that must be further processed into a usable message. Figure 1-1 illustrates how the structure of the gene is related to the process of transcription. During transcription, the whole gene is copied—exons and introns together—into a premessage. The premessage is then processed by excising the introns and joining the exons together to make a series of bases that code for a protein. The sites at which the excisions and rejoining take place are called splice sites, and specific sequences of bases are used to signal the cellular machinery to recognize these places. There are specific start and end points for the transcription of the gene as well; within the genes, there are specific signals in the form of three-base sequences (the start and stop codons) that indicate where translation into protein is to begin and end. The gene is thus made up of a coding sequence and punctuation.

Figure 1-1. Gene structure and transcription. The ultimate primary function of most genes is to produce a protein in a cell at a time at which it is needed. Genes have structures that are adapted to this function. Not only does a gene have syntax that establishes the amino acid sequence of the protein, but it also has punctuation and regulatory sequences that control its expression. The exons make up the coding or information content of the gene; these are interspersed with introns, which are DNA sequences with a function that is only poorly understood. The first step of expression is transcription. The gene is transcribed from the 5′ cap to the end of the polyadenylation signal. This transcript contains both introns and exons, and so the next step is for the introns to be excised. Recognizable splice-site junctions are needed for this to occur normally, and so are other sites that assist in the recognition of the appropriate splice sites. The final mRNA product contains the code that is necessary for the final step of translation, after which a protein is made.

In addition to the basic structure of a gene, there are elements that are both 5′ and 3′ of the gene that regulate its expression. These are called cis-acting elements, because they are on the same strand of DNA as the gene they regulate. Some of the regulatory elements may be inside one or more introns. In fact, the first few introns of a gene frequently contain such regulatory elements. Although some of the cis-acting elements are close to the actual start of the gene, others may be as much as 50 kilobases in front of (upstream) or in back of (downstream) the genes. Regulatory elements act predominantly by controlling the rate of transcription, and they respond to signals in the nucleoplasm to control the cellular specificity of gene action.

A basic understanding of gene structure is critical to an understanding of how mutations can disrupt gene function. Mutations can change the code, the punctuation, or the elements that regulate the expression of the gene; their detection and analysis are discussed in more detail in subsequent paragraphs.

The Molecular Basis of Patterns of Inheritance

There are three broad categories of genetic disease: chromosomal, monogenic, and complex. Chromosomal disorders are those in which relatively large segments of one or more chromosomes occur in only one copy or in more than the expected two copies. Examples would be Down syndrome (trisomy 21) and Turner’s syndrome (missing an X or a Y, depending on your perspective). The term single-gene disorder refers to the large group of disorders that are due to mutations in only one gene. Many hearing loss disorders are known to be the result of mutations in specific genes; almost all single-gene disorders show a specific pattern of inheritance (e.g., dominant, recessive, X-linked). Complex traits are those in which the genetics are not clear. The commonly accepted idea is that complex disorders—like cleft lip and/or palate, reading disability, cancer, and hypertension, to name a few—are the result of the interaction between several genes, the environment, and random factors. The otolaryngologist will encounter each category of genetic disease. Chromosomal disorders are generally severe but will frequently present with hearing and head and neck problems. Single-gene disorders are also severe, but many are not associated with multiple anomalies and/or mental retardation. The bulk of an ENT physician’s practice involves individuals with complex disorders, because these are typically more frequent.

Chromosomal Disorders

With some exceptions, chromosomal disorders are generally not heritable. Physical abnormalities associated with chromosomal imbalance are the result of rather extensive duplications or deletions of genetic material and involve multiple genes. The most common chromosomal disorder that involves the autosomes is trisomy 21.¹² Chromosomal disorders can be classified into one of four groups:

A chromosomal abnormality can occur in all or just some of the cells; the latter instance is called mosaicism. For example, most malignant cell lines show extensive chromosomal mosaicism, with multiple cell lines present in the tumor. Many females with only one X chromosome (designated as 45,X) are mosaic with a minor cell line that has a normal female constitution, 46,XX. The degree of mosaicism and the distribution in different tissues is believed to determine the severity of some cytogenetic disorders.

Aneuploidies

A trisomy is demonstrated when three copies of a whole chromosome occur in an offspring. This happens because of nondisjunction, which is the movement of a pair of chromosomes to the same pole during cell division; this results in one daughter cell lacking that chromosome and the other daughter cell possessing an extra copy of that chromosome.¹³ The three major autosomal trisomies are 21, 18, and 13.

Trisomies 13 and 18 are not compatible with long life, and the average life span of individuals with either of these conditions is less than 1 year. The majority of infants with trisomy 13 are profoundly deaf and have a cleft lip and palate in addition to multiple other congenital anomalies.^14,15 Hearing loss is frequent in trisomy 18 as well.¹⁶ However, hearing and head and neck anomalies are unlikely to be a serious concern because of the limited survival.

Patients with trisomy 21 have ears that are smaller than normal. About 75% have hearing loss, which can be sensorineural, conductive, or mixed.¹⁷ The prognosis of a child with trisomy 21 is generally good, and correction to normal hearing is important for helping such a child achieve maximal abilities.¹⁸

The common aneuploidies that involve the sex chromosomes include 45,X (Turner’s syndrome, phenotypic female) and 47,XXY (Klinefelter’s syndrome, phenotypic male). Although profound hearing loss is infrequent, mild to moderate hearing loss is common in 45,X individuals.^19–21 Females with Turner’s syndrome are highly susceptible to otitis media, but whether this changes with hormone replacement therapy remains to be investigated.^20,22,23 About 25% of children with Klinefelter’s syndrome have a mild sensorineural hearing loss.^24,25 Hearing losses in both Turner’s and Klinefelter’s syndromes often remain undetected.

Chromosomal Rearrangements

Usually aneuploidies are not heritable; however, rearrangements, translocations, and inversions can be. There is a heritable form of trisomy 21 that involves a translocation between chromosome 21 and another chromosome, usually chromosome 14. This is a so-called centric fusion translocation that results in the loss of both short arms and the fusion of the two long arms of chromosomes 21 and 14. A balanced chromosome complement would be 45 chromosomes. Because of the abnormal way in which the chromosomes need to be paired, this translocation sets the stage for an abnormal separation of chromosomes during meiosis. The result can be a balanced carrier, like the parent (a normal), or a carrier who has three copies of chromosome 21 material. Both translocations and inversions can be heritable and could result in a family that has multiple instances of children with multiple anomalies. The heritable forms can be distinguished by a straightforward cytogenetic evaluation.

Single-Gene Disorders

The terms dominant and recessive usually refer to the pattern of inheritance of a particular disorder, but, more importantly, they communicate the way in which combinations of two alleles produce a specific (usually abnormal) phenotype. With a dominant inheritance pattern, individuals who carry one copy of the mutant allele (heterozygote) or two copies of the mutant allele (homozygote) are equally affected. With recessive inheritance, a person must be homozygous for the mutant alleles; individuals who are heterozygous are normal. When using these terms to describe a disease, if the disease is called dominant, then normal is recessive, and vice versa. True dominance is probably uncommon. Branchio-oto-renal syndrome (BOR) is described as dominant,²⁶ but because it is uncommon a true mutant homozygous individual has probably never been born. Most geneticists would expect that patients with the homozygous mutant form of BOR would have a more severe phenotype, one that is possibly even lethal. Similarly, one might expect that patients with many of the recessive nonsyndromic deafness disorders could have a mild manifestation in the heterozygote, possibly contributing to the liability of the development of age-related hearing loss. An example of true dominance occurs with Huntington’s disease, in which a homozygous patient who is affected has the same phenotype as heterozygous individuals.^27,28

Dominant Disorders

Figure 1-2 shows a typical family pedigree of an autosomal dominant disorder. Under full penetrance, each affected individual has an affected parent. Because they are heterozygous, each affected individual has a 50% chance of transmitting the abnormal gene to offspring, each of whom would be similarly affected. The only reasonable instance in which a person could be homozygous and affected would be if both parents were affected. Dominant mutations are recognized through their pattern of inheritance, which typically shows vertical transmission and the involvement of several generations and several sibships.

Figure 1-2. Pedigree typical of a dominantly inherited trait; in this case, autosomal dominant congenital deafness. The arrow points to the proband of the family (a proband is the person though whom the family came to your attention). Note that the pattern of transmission in the family covers multiple generations and that both sexes are affected equally. A slash though a circle or square indicates that the person is deceased.

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