In humans, the normal diploid number of chromosomes is 46, consisting of 22 pairs of autosomal chromosomes (numbered 1–22 in decreasing size) and one pair of sex chromosomes (XX in females and XY in males). The genome is estimated to contain approximately 25,000 genes. Even the smallest autosome contains between 200 and 300 genes. Not surprisingly, duplications or deletions of chromosomes, or even small chromosome segments, have profound consequences on normal gene expression, leading to severe developmental and physiologic abnormalities.
Deviations in number or structure of the 46 human chromosomes are astonishingly common, despite severe deleterious consequences. Chromosomal disorders occur in an estimated 10–25% of all pregnancies. They are the leading cause of fetal loss and, among pregnancies surviving to term, the leading known cause of birth defects and mental retardation.
In recent years, the practice of cytogenetics has shifted from conventional cytogenetic methodology to a union of cytogenetic and molecular techniques. Formerly the province of research laboratories, fluorescence in situ hybridization (FISH), genomic array analysis, and related molecular cytogenetic technologies have been incorporated into everyday practice in clinical laboratories. As a result, there is an increased appreciation of the importance of "subtle" constitutional cytogenetic abnormalities such as micro-deletions and imprinting disorders, as well as previously recognized translocations and disorders of chromosome number.
Conventional Cytogenetic Analysis
In theory, chromosome preparations can be obtained from any actively dividing tissue by causing the cells to arrest in metaphase, the stage of the cell cycle when chromosomes are maximally condensed. In practice, only a small number of tissues are used for routine chromosome analysis: amniocytes or chorionic villi for prenatal testing and blood, bone marrow, or skin fibroblasts for postnatal studies. Samples of blood, bone marrow, and chorionic villi can be processed using short-term culture techniques that yield results in 1–3 days. Analysis of other tissue types typically involves long-term cell culture, requiring 1–3 weeks of processing before cytogenetic analysis is possible.
Cells are isolated at metaphase or prometaphase and treated chemically or enzymatically to reveal chromosome "bands" (Fig. 62-1). Analysis of the number of chromosomes in the cell and the distribution of bands on individual chromosomes allow the identification of numerical or structural abnormalities. This strategy is useful for characterizing the normal chromosome complement and determining the incidence and types of major chromosome abnormalities.
A. An idealized human chromosome, showing the centromere (cen), long (q) and short (p) arms, and telomeres (tel). B. A G-banded human karyotype from a normal (46,XX) female.
Each human chromosome contains two specialized structures: a centromere and two telomeres. The centromere, or primary constriction, divides the chromosome into short (p) and long (q) arms and is responsible for the segregation of chromosomes during cell division. The telomeres, or chromosome ends, "cap" the p and q arms and are important for allowing DNA replication at the ends of the chromosomes. Prior to DNA replication, each chromosome consists of a single chromatid copy of the DNA double helix. After DNA replication and continuing until the time of cell division (including metaphase, when chromosomes are typically visualized), each chromosome consists of two identical sister chromatids (Fig. 62-1).
The introduction of FISH methodologies in the late 1980s revolutionized the field of cytogenetics. In principle, FISH is similar to other DNA–DNA hybridization methodologies. In most instances, the probe is labeled directly with a fluorochrome to allow detection. After the hybridization step, the specimen is counterstained and the preparations are visualized with a fluorescence microscope.
A variety of probes are available for use with FISH, including chromosome-specific paints (chromosome libraries), repetitive probes, and single-copy probes (Fig. 62-2). Chromosome libraries hybridize to sequences that span the entirety of the chromosome from which they are derived and, as a result, they can be used to "paint" individual chromosomes.
Repetitive probes recognize amplified DNA sequences present in chromosomes. The most common are α-satellite DNA probes that are complementary to DNA sequences ...