Basic Concepts Of Chromosomal Disease

Deoxyribonucleic acid (DNA) is the fundamental unit that directs the orchestration of cellular function and transmits traits from one generation to the next. It is a self-reproducing macromolecule that determines the composition of proteins within the cell. The gene is a sequence of DNA that encodes for a single, specific protein or regulates the expression of a gene; just as the DNA is arranged as beads on a necklace, the genes (specific segments of DNA) are similarly aligned. The information contained in the DNA is transcribed to ribonu-cleic acid (RNA), an intermediary template, which is, in turn, translated to the specific protein. The DNA of functional genes is arranged in exons and introns; exonic sequences are transcribed into RNA, and the intronic sequences are removed. Sequence variation is considerably greater for the introns. The nuclear genes and other untranslated DNA are organized as chromosomes, discrete organelles within the nucleus. The majority of the DNA of a cell is arranged as chromosomes within the nucleus; a small percentage of DNA is within the mitochondria in the cytoplasm of the cell and primarily orchestrates oxidative metabolism. Different species of animals and plants have different numbers of chromosomes; for example, a mouse has 40 and a tomato 24. Humans have 23 pairs, of which 2, an X and a Y, determine gender. Each nucleated cell of the organism has the same DNA as every other cell unless a mutation or chromosomal anomaly has occurred after conception.

Humans are diploid organisms with two sets of each chromosome and, therefore, two sets of each gene, one member of each pair being inherited from a parent; the X and Y chromosomes are the exception as they are structurally different but paired. An individual is homozygous for a gene pair if the DNA sequence of each is identical and is heterozygous (or a het-ererozygote) if the sequences differ. Alternative forms of a gene are called alleles; a null allele is one in which a protein is not evident. Many enzymatic deficiencies are inherited as autosomal recessive diseases; the heterozygote shows no evidence of the disease because one chromosome is normal and 50% activity of the enzyme is functionally adequate. Many autosomal dominant disorders are caused by mutations of structural genes. Autosomal dominant disorders are manifest with only one chromosome abnormal (heterozgote) because all structural building blocks must be normal to build a normal structure. If a portion of chromosome is missing, the deleted genes are not transcribed or, if adjacent to the deletion, a gene may be transcribed abnormally. Conversely, if a portion of a chromosome is duplicated, the genes within the duplicated region are transcribed, resulting in an abundance of protein or, as occurs with deletions, the adjacent genes may be transcribed abnormally.

DNA consists of 2 strands of nucleotides, each with a base, a pentose (five-carbon) sugar (deoxyribose in the case of DNA), and a phosphoric acid; RNA has a similar structure with the exception of the base uracil instead of cytosine. The 2 strands of nucleotides are bound together at each base forming a complementary sequence of base pairs. Hydrogen bonding between the bases adenine (A, a purine) from one strand and thymine (T, a pyrimidine) from the other or guanine (G, a purine) and cytosine (C, pyrimidine) maintain the alignment of the two strands. The tertiary structure of the double strand makes the classic double helix. Because the pairing of A with T and G with C is essential to the secondary and tertiary structure, the two strands are complementary.

The precise order of the base pairs within the exons of a gene determines the amino acid sequence of the protein it encodes. For each amino acid, a triplet of DNA bases (a codon) provides the information for the amino acid of the protein, and some amino acids are represented by more than one codon. For example, the amino acid phenylalanine is encoded by either of the triplets uracil-uracil-uracil or uracil-uracil-cytosine. Protein sequence can be determined on the basis of the DNA sequence.

Some of the chromosomal DNA may be repetitive, and such sequences are transcribed; the evolutionary significance is unknown.

"Genetic" defects occur at many different molecular levels. Single gene defects may be produced by a single gene mutation, an alteration of the DNA sequence or a deletion or duplication of DNA within a single gene, which causes a change of one or more amino acids of the protein product. Point mutations of functional significance to the organism usually occur within an exon or exon-intron boundary or in a regulatory sequence. Mutations of single genes are the basis of Mendelian inheritance patterns including autosomal recessive, autosomal dominant, and X-linked. Chromosomal abnormalities encompass deletions (loss) or duplications (additional) of larger regions of DNA and alter the function of more than one gene; such abnormalities, if sufficiently large, may be identified under the microscope. The term cytoplasmic or mitochondrial inheritance was coined to describe diseases such as Leber optic atrophy, which are passed from a mother to offspring but not from the father to his progeny because sperm have few mitochondria. Such diseases are caused by abnormalities of the mitochondrial DNA. Last, multifactorial inheritance is poorly understood and difficult to prove scientifically; it is defined as a genetic predisposition to a disease with the manifestations or expression of the disease being influenced by environmental factors in either the intrauterine or postnatal period.

During the process of cell replication (mitosis), nuclear DNA is duplicated and each daughter cell receives the same information as the parent unless a mutation or chromosomal anomaly occurs. The process of gamete (spermatozoa or ova) formation (meiosis) involves a halving of the number of chromosomes. During meiosis, a single cell forms four gametes (ova or sperm), each of which has half the number of chromosomes (haploid), and therefore genes, of the parent cell. Crossing-over (exchange of DNA) between homologous chromosomes (a pair with the same gene loci in the same order) probably occurs during the replication process, and genetic material is exchanged. This process reorganizes the alleles on a chromosome and increases genetic variability. Errors may occur in the duplicative process of mitosis or meiosis and result in somatic and germinal defects, respectively. During fertilization, two haploid cells fuse to form a diploid cell with the normal number of chromosomes. A germi nal defect in a gene or chromosome is present in all cells of the body and is caused by an error in one or both of the gametes. Alternatively, a chromosomal abnormality may occur relatively early in gestation, resulting in a portion of cells with the defect; termed mosaicism, the condition is usually defined by the percentage of abnormal cells and/or the cell types associated with the abnormality. Rarely, the testicles or ovaries, may exhibit mosaicism with some cell lines containing a mutation of a gene or chromosomal aberration.

A phenotype is the physical, biochemical, and physiological features of an individual as determined by genotype (genetic constitution). A similar phenotype may occur as a result of different gene defects or chromosomal abnormalities and is termed genetic heterogeneity. Examples of genetic heterogeneity are common in ophthalmology. Retinitis pigmentosa is an important ophthalmic disease exhibiting genetic heterogeneity, as this disease may be inherited by different gene defects; as an autosomal dominant, an autosomal recessive, or an X-linked recessive disorder. Within the category of autosomal dominant retinitis pigmentosa, mutations of rhodopsin and peripherin proteins have been identified as the basis of the disease. Similarly, different chromosomal anomalies may result in overlap of pheno-typic features. Thus, classification of diseases is most reliably made on the basis of specific chromosomal abnormalities.

Chromosomes may be studied under the microscope by arresting the progression of the mitotic (duplicative) process. Using this technique (Karyotyping) the chromosomes can be visualized under the microscope and the specific chromosomal identification can be made on the basis of length and by using stains such as trypsin-Giemsa (G banding) (Fig. 3-1),320 quinacrine mustard (Q banding),45 "reverse" (following controlled denaturation by heat, followed by Giemsa staining), silver (stains nucleolar organizing regions), and C banding (the centromere and regions with heterochromatin stain). All methods identify bands or specific regions and are useful for studying the specific structure of a chromosome. The number of chromosomes can be counted and the bands of each studied for deletions, duplications, and other anomalies. Chromosomes were initially classified by size and given numbers when banding permitted identification. Relatively new techniques for arresting the progression of mitosis earlier in the cell cycle have been developed. In the late prophase or early metaphase stage of

FIGURE 3-1A-C. Chromosome 1. (A) Diagrammatic representation of G (Geimsa) bands. (B) Metaphase banding pattern. (C) Early metaphase (high-resolution) banding pattern. (Courtesy of Dr. R.S. Sparkes.)

mitosis, the chromosomes are longer and less condensed, permitting identification of more bands; therefore, smaller deletions or duplications can be detected by analyzing the chromosomes in these stages. This "extended" analysis, called high-resolution banding, is more time consuming but is particularly useful if a specific chromosomal anomaly is suspected (Fig. 3-1). Techniques have been developed to identify duplicated or missing portions of chromosomes using DNA probes of known chromosomal location with fluorescent in situ hybridization (FISH). The probes may be specific for a chromosomal region (single-copy cosmid) (Fig. 3-2) or represent sequences on an entire chromosome (Fig. 3-3).

In 1956, Tjio and Levan identified the correct number of human chromosomes as 46351; previously, the total had been

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