Chromosome Structure and Nomenclature

The human genome is composed of deoxyribonucleic acid (DNA), the genetic code for all organisms. Chromosomes are located in the cell nucleus and are composed of DNA packaged with proteins that exist in 23 pairs in every human diploid cell—22 pairs of autosomes (numbered 1-22) and one pair of sex chromosomes (X and Y). Each pair of chromosomes is connected at a region called the centromere, creating two arms for each pair of chromosomes (Figure 116-1). In most chromosomes, the centromere is located closer to one end than the other, forming a short p arm and a longer q arm. Chromosomes can be visualized under a microscope when they are captured during the metaphase stage of the cell cycle. Using a special technique known as Giemsa staining, metaphase chromosomes can be used to determine a karyotype for an individual (see Figure 116-1). A karyotype result indicates the total number of chromosomes identified in the cell, typically 46, followed by the sex chromosomes observed, usually XX or XY. Therefore, a normal female karyotype is 46,XX, and a normal male karyotype is 46,XY.

Figure 116-1 Chromosome structure and karyotype.

Giemsa staining also allows chromosomal structural differences, and aneuploidy, or imbalance in genetic material, to be identified. The most easily identified forms of aneuploidy include monosomy, the presence of only one of two normal copies of a pair of chromosomes, and trisomy, three copies of a chromosome. Karyotypes are performed on 20 or more cells to determine whether aneuploidy is present in all cells from a patient. To denote aneuploidy, a “+” or “–” is used to indicate an extra or missing chromosome or portion of a chromosome. For example, a female with trisomy 21 would be denoted 47,XX,+21. Another monosomy involving the short arm of chromosome 4, causing Wolf-Hirschhorn syndrome, would be designated in an affected male as 46,XY,4p-. Smaller chromosomal alterations that result from duplicated or deleted region of a chromosome are designated as dup or del followed by the chromosome segment that is missing or extra [e.g., 46,XY del(1)(p36)].

Methods Of Detecting Aneuploidy

With advances in molecular biology, more specific techniques for evaluating aneuploidy have been developed. In the late 1980s, fluorescent in situ hybridization (FISH) was developed as a method to detect specific chromosomal deletions (Figure 116-2). A fluorescent-labeled DNA sequence that is complementary to a known region of a chromosome is hybridized to a set of chromosomes in a cell. If two copies of the chromosome sequence are present, two fluorescent probes are visualized; if only one probe is seen, it is inferred that one copy of the chromosomal segment is deleted. FISH is used to confirm karyotype findings or to detect deletions too small to be visualized with a standard karyotype. FISH also permits analysis of uncultured cells, permitting diagnoses to be made more rapidly.

Figure 116-2 Fluorescent in situ hybridization (FISH) detection of aneuploidy.

Duplicated or deleted regions of the genome must be more than 3 million base pairs in size to be visualized via karyotyping and more than 100 kilobases to be visualized by FISH. Recently, more sophisticated methods of identifying chromosomal alterations of 10 to 100 kilobases to be detected have been introduced. These array-based methods use bacterial artificial chromosome (BAC) probe comparative genomic hybridization (CGH) or synthetic oligonucleotide hybridization probes or single nucleotide polymorphism (SNP) probes. All approaches analyze DNA from a test subject and compare the amount of signal at 5000 with 2 million positions across the genome with that of a normal reference patient’s sample. Often, both pathologic and normal variants are noted and collectively are called copy number variants (CNVs). Those that are believed to cause disease have been termed copy number alterations (CNAs).

Mitosis, Meiosis, and Gamete Formation

Diploid cells are maintained through the process of mitosis in dividing somatic cell types. Typical cells have four stages to their growth and division cycle (Figure 116-3). The first stage, G1 (for growth 1 or gap 1), is largely composed of growth and has no active cell division. During the next stage (S), DNA is replicated or synthesized to form a four-copied, or tetraploid, genome. After another growth phase (G2) during which the replicated genome is surveyed to correct any aberrations, a division phase (D), during which mitosis occurs, results in two new daughter cells. During mitosis, the replicated tetraploid genome is divided so that each daughter cell receives a diploid complement of chromosomes after cell division. Mitosis is divided into four stages—prophase, metaphase, anaphase, and telophase. At the beginning of prophase, the duplicated chromosomes are elongated and appear as a single chromosome, although two sister chromatids have already been formed by this stage. At the end of prophase, the sister chromatids have become more condensed, and the centrioles are seen at opposite poles of the nucleus. During metaphase, the nuclear membrane disintegrates, and the formation of the mitotic spindle made up of fibers that connect each chromatid of a sister chromatid pair to opposite centrioles, causing them to align in the middle of the nucleus. The centromere of each chromosome also divides in preparation for migration of sister chromatids to opposite centriole spindles. It is during metaphase that chromosomes are typically viewed under the microscope because this is when the chromosomes are most condensed and the mitotic spindle can be arrested with the chemical colchicine. During anaphase, sister chromatics are segregated and migrate toward opposite poles along the spindle. Telophase is marked by the arrival of chromosomes at opposite poles, the disintegration of the spindle, and reformation of a nuclear membrane.

Figure 116-3 Cell division and meiosis.

Haploid cells are generated via a special type of germ cell division called meiosis, so that each egg or sperm contributes half of the genetic material to an embryo (see Figure 116-3). A haploid complement of chromosomes consists of one of every autosome (a non-sex chromosome) and an X or Y. In this way, each parent contributes 23 chromosomes to produce a full complement of 46 in an embryo. Meiosis also permits some exchange of genetic material, thus creating genetic variability between gametes. Recombination events, usually at least one per chromosome, result in no two haploid gametes being identical. Recombination events are thought to occur twice as frequently in females as in males. Meiosis is divided into two stages, meiosis I and II. In meiosis I, similar to mitosis, a cell replicates its genome and then divides to create two cells with a full complement of 23 chromosome pairs. In meiosis II, daughter chromatids are divided among daughter cells, such that each contains a single copy of each of the 22 autosomes and an X or Y sex chromosome. In males, all four resulting haploid cells differentiate into sperm. In females, only one of these four haploid set of chromosomes ends up as an ova; the other products become polar bodies. A common cause of aneuploidy is thought to arise from nondisjunction, or failure of the chromosomes to divide equally between daughter cells during meiosis.

Gene Structure

DNA is a double-stranded macromolecule composed of complementary pairs of the purine bases, adenosine and guanine, and the pyrimidine bases, thymine and cytosine (Figure 116-4). The entire haploid genome is composed of some 3 billion base pairs, with each chromosome composed of 50 to 150 million base pairs. Of the entire genome, only about 2% is coding sequence (i.e., encodes genes that are transcribed into proteins). The remainder of the genome is noncoding sequence presumably involved in structural integrity of chromosomes or control of gene expression. A typical gene is transcribed into RNA (RNA), which in turn is translated into a functional protein composed of amino acids. Elements of the genomic DNA surrounding a gene, known as promoters and enhancers, serve to regulate the location, timing. and level of transcription. Before translation, transcribed RNA is processed to form messenger RNA (mRNA) by removing or splicing out intervening sequences known as introns to result in a mature mRNA containing only exons. Mature mRNA moves from the nucleus to the cytoplasm of a cell, where it can be translated into protein in a structure called the ribosome. The ribosome scans the mRNA codons, triplets of nucleotides that encode a specific amino acid, or a start or stop signal. Using the mRNA as a template, ribosomal RNA (rRNA), ribosomal proteins, and transfer RNA (tRNA), the ribosome delivers the appropriate amino acid into the chain of amino acids in the formation of a translated protein. At any stage in the process of transcription or translation, several types of errors can occur, resulting in alterations of protein structure, function, or both (see Figure 116-4).

Figure 116-4 Gene structure and expression.

Modes of Inheritance

An individual inherits genetic material from the mother and father when an egg and sperm fuse during fertilization. Thus, both a maternal and paternal copy of every gene, or alleles, are present in the genome. There are exceptions to this rule, as is evident in males, because the Y chromosome does not carry a matching allele for every gene on the X chromosome. The combination of two specific alleles determines the genotype. The nature of the interaction between two alleles of a given gene determines the mode of inheritance of a disorder caused by mutations in that gene.

Genetic diseases can also be partially inherited as illustrated by the principles of penetrance and expressivity. Penetrance is the likelihood that an individual who carries a disease-causing gene will manifest signs and symptoms of the disorder. A highly penetrant disorder will manifest in nearly all individuals who carry the gene. Expressivity describes the variation in the phenotype, or clinical presentation, that results from a gene alteration, or genotype. Expressivity can be variable, meaning that individuals with the same genotype may have significantly different manifestations in their phenotype or in the severity of their disease.