Basic Principles of Cytogenetics

Introduction to Cytogenetics

Cytogenetics is the branch of genetics focused on the study of the structure, function, and behavior of chromosomes. Human cytogenetics applies these studies to the diagnosis, prognosis, and evolution of diseases with a genetic basis. Progress in this field has been significantly favored by the use of cell cultures, allowing for deeper insights into cellular development and chromosomal characteristics.

The study of chromosomes requires an understanding of their morphology, the different existing types, and their changes throughout the cell cycle. This foundational knowledge is essential for human cytogenetic procedures and methodologies used in chromosomal analysis.

Structure of the Chromosome

A chromosome in a eukaryotic cell is an organized structure formed by the association of a linear DNA molecule, which carries part of the individual's genetic information, and a set of proteins. The cytogenetic study is primarily conducted during the metaphase period of the cell cycle, as this is when the chromosome reaches its maximum degree of compaction, making it perfectly visible and identifiable under an optical microscope. This is referred to as the metaphasic chromosome.

In a metaphasic chromosome, several distinct structures can be identified:

Cromátidas (Chromatids): These are two linear and parallel filaments, exactly identical to each other, held together by protein complexes called cohesins. Each chromatid represents one of the two identical DNA molecules resulting from DNA replication prior to cell division, at its maximum state of supercoiling. During mitosis, the two chromatids separate, and each moves to one of the daughter cells, ensuring the daughter cells possess the same genetic endowment as the parental cell.

Centrómero (Centromere) or Primary Constriction: This is a narrowing that divides the chromosome into two arms. Its position is characteristic for each chromosome and is used for morphological classification. It is a region rich in repetitive DNA, specifically satellite DNA. At the centromere, each chromatid presents a protein structure called the cinetocoro (kinetochore), which serves as the docking point for the microtubules of the mitotic spindle.

Constricciones secundarias (Secondary Constrictions): These are narrowings of the chromatids that are less deep than the centromere. Some are related to the formation of the nucleolus and are therefore called organizadores nucleolares (nucleolar organizers).

Telómeros (Telomeres): These are the ends of the chromatids. They consist of thousands of repetitions of the same sequence and perform vital functions in chromosome replication, interaction with the nuclear membrane, protection against nucleases, and structural stability. They are also linked to cellular aging and certain types of cancer.

Morphological Classification of Chromosomes

Under a microscope, metaphase chromosomes appear as elongated structures divided into two arms by the centromeric constriction. The length of each arm is determined by the position of the centromere, resulting in a short arm, designated as brazo p, and a long arm, designated as brazo q.

The centromeric index ($Ic$) is the ratio between the length of the short arm and the total length of the chromosome. It is calculated using the following mathematical expression:

$Ic = \frac{p \times 100}{p + q}$

Based on this parameter, chromosomes are classified into four morphological types:

Metacéntricos (Metacentric): The centromere is located in the central region, and both arms are approximately equal. The $Ic$ is usually close to 50 (approximate range: 45–50).

Submetacéntricos (Submetacentric): The centromere is displaced from the center, resulting in unequal arms. The $Ic$ typically ranges between 25 and 45.

Acrocéntricos (Acrocentric): The centromere is close to one end, making the short arm very small. The $Ic$ is less than 25. Some acrocentric chromosomes feature a secondary constriction on the short arm called a tallo (stalk), which leaves a small amount of DNA at the tip known as a satélite (satellite).

Telocéntricos (Telocentric): The centromere is at the very end, so only a long arm exists ($Ic = 0$).

Chromosome Count and Ploidy

Higher organisms originate from the union of two gametes, each contributing a complete set of chromosomes, denoted by the number $n$, which is characteristic of the species. Consequently, each individual has two complete sets ($2n$), one from each parent, forming pairs of homologous chromosomes. In humans, the chromosome count is $n = 23$ (23 pairs), totaling 46 chromosomes.

Chromosomes responsible for sex determination are called sexual chromosomes or heterocromosomas (in humans, X and Y). The remaining chromosomes are called autosomas (autosomes). Species or cells with two chromosomal sets are called diploides (diploid), while those with a single set (like gametes) are called haploides (haploid).

The Cell Cycle: Phases and Characteristics

The cell cycle is the set of activities and changes experienced by a cell from its appearance (after parental division) until it transforms into two daughter cells. The term cycle applies only to dividing cells. Differentiated cells that will not divide again are considered to be in a state of repose or quiescence, known as the Phase $G_0$. Some quiescent cells can re-enter the cycle under specific stimuli.

The cell cycle is divided into Interphase and Mitosis (Phase M).

Interphase

Interphase is the period between two consecutive mitoses. During this time, the cell grows and prepares for division. The DNA-protein association exists as chromatin fibers in varying states of condensation, which are indistinguishable under an optical microscope. Interphase is divided into three periods:

Period $G_1$: A period of cell growth starting after mitosis and lasting until the start of DNA replication. Its duration varies by cell type and determines the length of the interphase.

Period $S$: The phase where all cellular DNA is replicated. Each chromosome then consists of two identical DNA molecules joined by cohesins. In mammal cells, this lasts between 5 and 10 hours.

Period $G_2$: The phase where genes encoding proteins for cell division are expressed. It is the shortest interphase period (roughly 3 hours in mammals) and concludes with the duplication of the centrosome.

Mitosis (Phase M)

Mitosis is the process of generating two daughter cells with identical genetic endowments. In mammals, it lasts about 2 hours and consists of four phases:

Profase (Prophase): Chromatin fibers condense and chromosomes become visible. Kinetochores form at the centromeres. Centrosomes move to opposite poles, and the mitotic spindle fibers polymerize. The nucleolus disappears, and the nuclear membrane dissolves, dispersing chromosomes into the cytoplasm.

Metafase (Metaphase): Chromosomes anchor to spindle fibers at the kinetochores and migrate to the equatorial region. At the end of this phase, they reach maximum compaction and align at the placa metafásica (metaphase plate). Chromatids are oriented toward opposite poles.

Anafase (Anaphase): Microtubules shorten, pulling sister chromatids apart starting at the centromere. Metacentric chromatids form a "V" shape, while submetacentric and acrocentric ones form an "L" shape. The phase ends when chromatids reach cellular poles.

Telofase (Telophase): Processes involve the disappearance of spindle fibers, formation of new nuclear membranes around each set of chromosomes, and decondensation of chromosomes back into chromatin. Nucleoli reappear, resulting in a binucleated cell prior to separation.

Citocinesis (Cytokinesis): This is the distribution of cytoplasm and organelles. In animal cells, it occurs via a contractile ring of proteins that constricts the equatorial region until the two daughter cells are separated.

Visualization and Banding Techniques

Chromosomes are best studied between late prophase and metaphase due to high condensation. There are two primary categories of optical study:

Conventional Staining Methods

Methods using Giemsa or orceína (orcein) stain chromosomes uniformly and intensely. These allow for counting, size sorting, and centromere positioning ($Ic$ calculation), but often fail to identify individual chromosomes with similar morphologies or detect structural anomalies.

Chromosomal Banding Techniques

These techniques stain chromosomes discontinuously, creating transverse bands and specific patterns for unequivocal identification. Major techniques include:

Bandeo G (G-banding): Known as the GTG technique (G-bands by Trypsin and Giemsa). Aged metaphase preparations are treated with trypsin and stained with Giemsa. It produces dark bands ($G+$) rich in AT and clear bands ($G-$) rich in GC. It is the most common technique.

Bandeo R (R-banding): The reverse of G-banding. Preparations are treated with heat (Sorensen buffer at $85\,^{\circ}\text{C}$) and then stained with Giemsa.

Bandeo Q (Q-banding): A fluorescent technique using quinacrine. Patterns of varying intensity are observed under a fluorescence microscope; bright bands coincide with $G+$ bands.

Bandeo C (C-banding): Stains heterochromatin-rich regions. In humans, it stains centromeres, pericentromeric regions of the q-arm of chromosomes 1, 9, and 16, and the distal q-arm of the Y chromosome. It involves treatment with hydrochloric acid, an alkali (barium hydroxide), heat (SSC buffer at $60-65\,^{\circ}\text{C}$), and Giemsa staining.

Idiograms and ISCN Nomenclature

An idiograma (idiogram) is a simplified graphical representation of a banded chromosome. It maps the centromere position, arm ratios ($p$ and $q$), stalks, satellites, and specific banding patterns. The ISCN (International System for Human Cytogenetic Nomenclature) provides a standardized code to identify specific areas.

Chromosomes are represented with the short arm ($p$) on top. Arms are divided into regions, and regions into bands, both numbered consecutively from the centromere toward the telomere. Region 1 is always closest to the centromere. The code format is: Chromosome – Arm – Region – Band. For example, $2q24$ is the fourth band of the second region of the long arm of chromosome 2.

Metaphase G-banding has a resolution of roughly 400 bands. Prophase banding allows for higher resolutions (550 to 850 bands), revealing sub-bands and sub-sub-bands. These are denoted with a dot followed by the number (e.g., $14q32.13$ represents Chromosome 14, arm q, region 3, band 2, sub-band 1, sub-sub-band 3).

Mutations: Genic and Chromosomal

Mutations are spontaneous, sudden changes in DNA sequence or structure. If they occur in somatic cells, they affect the organism's clones (mosaicismo) and can cause neoplasms. If they occur in the germline, they are passed to offspring and drive evolution.

Genic Mutations

These involve one or few nucleotides and are detected via molecular techniques like PCR or sequencing.

Sustituciones (Substitutions): Puntual mutations (single nucleotide change). In coding regions, these can be Silent (synonymous codon, no protein change), Missense (different amino acid, e.g., sickle cell anemia where Glu changes to Val at position 6), or Nonsense (premature stop codon, truncated protein).

Inserciones y deleciones (Insertions and Deletions): Gain or loss of nucleotides. Within exons, they usually cause a frameshift (frameshift mutation), altering all subsequent codons. If multiples of 3 are added/lost, the reading frame is preserved but the protein gains/loses amino acids. Mutations can also affect Splicing (mRNA maturation) or Regulatory regions (expression levels).

Chromosomal Mutations

These affect chromosome number or large-scale structure, visible via optical microscopy.

Numerical Alterations

Individuals with complete sets are euploides (euploid). Variations include:

Poliploidía (Polyploidy): Multiple complete sets (3n = triploidy, 4n = tetraploidy).

Aneuploidía (Aneuploidy): Excess or lack of specific chromosomes. Monosomía (missing one homolog), Trisomía (three homologs), Tetrasomía (four homologs).

Structural Alterations

Deleción (Deletion): Loss of a fragment; can be terminal (includes telomere) or intersticial (internal).

Duplicación (Duplication): Repetition of a fragment. Can be in tándem (adjacent) or desplazada (separated). Can also be direct or inverted.

Inversión (Inversion): A segment flips orientation. Pericéntricas include the centromere; Paracéntricas do not.

Translocación (Translocation): Exchange between non-homologous chromosomes. Can be non-reciprocal (insertion into another), reciprocal (mutual exchange), or Robertsonian (fusion of two acrocentric chromosomes at the centromere, often losing the short arms).

Cromosoma en anillo (Ring Chromosome): Double terminal deletion followed by circularization.

Isocromosoma: An aberrant chromosome with two identical arms in mirror orientation (e.g., two p arms or two q arms).