Cytogenetics

Chromosome Terminology and Classification

• Chromosome Number: Denoted as #, includes 1-22 and X & Y sex chromosomes.

• Arm Designation:

  • p-arm: Refers to the short arm of the chromosome, derived from the French word "petit".

  • q-arm: Refers to the long arm of the chromosome, derived from the word "queue".

• Region: Numbered regions from the centromere outward.

• Band Numbering: Numbered within the region in a hierarchical fashion, with the first band closest to the centromere being designated as 1, followed by subsequent bands as 2, 3, and so on, allowing for precise identification of chromosomal abnormalities.

  • Banding methods are utilized to identify specific regions of chromosomes.

Cytogenetic Banding Nomenclature

• Chromosome Identification Example:

  • For Chromosome 3:

  • p telomere: denoted as "ptel"

  • q telomere: denoted as "qtel"

  • Specific Regions:

    • Region 3p22 represents a specific location near the p telomere.

    • Regions 3p22.1 and 3p21 denote finer divisions within 3p22.

Types of Chromosomes (By Centromere Location)

• Metacentric Chromosomes: The centromere is near the center, resulting in arms of equal length.

• Submetacentric Chromosomes: The centromere is off-center, resulting in one arm being longer than the other.

• Acrocentric Chromosomes: The centromere is located significantly off-center, leading to a long and a very short arm.

Karyotype Analysis

• Definition: A laboratory test used to analyze chromosomes for abnormalities.

• Cells Used for Karyotype:

  • Any nucleated cells that can be induced to divide in culture.

  • Specimens used could be from blood, bone marrow, or amniotic fluid.

  • Routine Analysis: Commonly performed karyotypes, using leukocytes and fibroblasts.

  • Prenatal Analysis: Karyotypes performed for prenatal diagnostics, using amniotic cells and chorionic cells.

Karyotype Procedure

• Chromosome Harvesting:

  • Remove leukocytes from blood after centrifugation (RBCs fall to bottom, plasma remains above).

  • The middle layer consisting of leukocytes is called the "buffy coat" and is collected for further analysis.

• Culturing Cells: Add cells to nutrient broth with a mitogen to stimulate division, incubated at 37°C for approximately 70 hours to allow for cell division.

• Add colcemid to arrest mitosis in metaphase since it is when chromosomes are most condensed.

• Hypotonic Solution Addition: At 72 hours, cells are treated with a hypotonic solution, which allows them to swell without bursting.

  • Cells will burst upon hitting an ice-cold slide, resulting in the spread of chromosomes.

• Fixation and Staining: Chromosomes are fixed on the slide and stained for visualization.

Chromosome Banding Techniques

• Banding Patterns: Reproducible patterns of light and dark areas on chromosomes. Produced by chemical and or heat treatment of chromosomes on slides

  • G Bands: Stained using chemicals that digest proteins, developing a distinct light and dark pattern. Stain using Giemsa. Dark is the G bonds.

  • Q Bands: Produced similarly to G bands but using quinacrine mustard stain, fluorescing under UV light.

  • C Bands: Generated using chemicals that digest DNA, highlighting just the centromere. Used with G bands. Stain using Giemsa.

Chromosomal Abnormalities and Prenatal Diagnosis

• Prenatal Diagnosis Purpose: Diagnosing chromosomal abnormalities in the fetus, not specific gene defects.

  • Techniques involve obtaining fetal cells for chromosomal analysis.

• Indications for Prenatal Diagnosis:

  • Older maternal age.

  • Previous child diagnosed with a chromosomal abnormality.

  • Parents known to be carriers of chromosomal abnormalities.

Amniocentesis and CVS

• Amniocentesis:

  • Involves sampling amniotic fluid to obtain fetal cells, can be performed from 16 weeks of gestation.

  • Takes several weeks to grow cells

  • Results around 20 weeks total

  • <1% risk of complications

• Chorionic Villi Sampling (CVS):

  • Involves sampling placental tissue, which can be performed as early as 8 weeks of gestation.

  • Takes several days to get results

  • Similar risks as amniocentesis

Chromosomal Abnormalities

• Types of Chromosomal Abnormalities:

  • Polyploidy: Chromosomal number is a multiple of the haploid set (e.g., humans are normally diploid, with 46 chromosomes).

  • Triploidy: Simplest form of polyploidy, three haploid sets (69 chromosomes total).

  • Dispermy may cause triploidy with a high rate of spontaneous abortions. Two sperms fertilize same ova.

• Aneuploidy: Abnormality involving a non-polyploid number of chromosomes, typically pertaining to a single chromosome's addition or loss,

  • often resulting from non-separation of homologous pairs in Meiosis I, sister chromatids in Meiosis II and sister chromatids in Mitosis.

    • 50% of all conceptions may start as aneuploidy, with 70% of spontaneous abortions demonstrating this condition.

    • The incidence of live births with some form of aneuploidy is about 1 in 170.

    • Chimera (mosaic) - if it occurs in early embryo divisions some cells will be normal and some will be abnormal in the newborn.

  • older maternal age is a significant risk factor

    • 1. aging oocytes: primary oocytes stuck in prophase

    • 2. reduced implantation selection

Types of Aneuploidy in Humans

• Trisomy: One extra chromosome, leading to 47 chromosomes total.

• Monosomy: One chromosome missing, leading to 45 chromosomes total.

• Aneuploidy involving autosomal chromosomes typically results in miscarriage, with special exceptions for chromosomes 13, 18, and 21.

Specific Syndromes Associated with Trisomy

• Trisomy 13 (Patau Syndrome):

  • Occurs in 1 in 15,000 live births, with an average survival of 6 months. Characterized by severe congenital malformations. CNS and heart malformations.

• Trisomy 18 (Edward's Syndrome):

  • Occurs in 1 in 11,000 live births, with an average survival of 4 months, similar malformations to Trisomy 13. More females are affected.

• Trisomy 21 (Down syndrome):

  • Occurs in 1 in 900 live births, thought to account for 0.5% of all conceptions. Features include congenital defects, intellectual disability, heart defects, and characteristic physical features.

  • Reduced lifespan

Sex Chromosome Aneuploidy

• Aneuploidy of sex chromosomes is seen more frequently in live births compared to autosomal aneuploidy.

  • Turner Syndrome (45,XO):

  • Affects 1 in 10,000 female live births, characterized by short stature, neck folds, and infertility.

  • Klinefelter Syndrome (47,XXY):

  • Affects 1 in 1,000 male live births, features reduced intelligence, and tall stature, female secondary sexual characteristics develop at puberty.

  • Usually infertile

  • XYY Syndrome (47,XYY):

  • Affects 1 in 1,000 male births, often presents normal appearance with slightly elevated height and lower IQ.

  • Normally fertile

Chromosomal Structural Abnormalities

• These may disrupt gene function and contribute to clinical syndromes despite having a correct number of chromosomes.

• Types of Structural Abnormalities:

  • Duplication (dup+): A segment of a chromosome is repeated. Leads to gene dosage imbalances that can lead to clinical manifestations.

  • Deletion (del-): A segment of a chromosome is missing, e.g., Cri du Chat syndrome (5p15-). Can lead to gene disruptions at break points and deletions of genes in that region.

  • Inversion (inv): A segment of a chromosome is inverted. Can lead to gene disruption at break points and problems with gene regulation

    • Can be either

      • paracentric (not including the centromere) or

      • pericentric (including the centromere).

  • Translocation (t): A segment of a chromosome is transferred to a non-homologous chromosome.

  • Reciprocal Translocation: No gain/loss of genetic material; balanced exchange results in a normal appearance, but may yield unbalanced gametes.

  • Robertsonian Translocation: Results in the loss of genetic material, producing a very large chromosome and a smaller one that may be lost.

Gene-specific Syndromes

• Fragile X Syndrome: The most common form of intellectual disability in males, caused by a fragile site at the tip of the X chromosome (q arm) due to extensive CGG repeat regions.

• Prader-Willi Syndrome: Manifests as intellectual disability and uncontrollable appetite (maternal allele is present but is silenced via epigenetic imprinting)

  • 70% results from deletion of paternal chromosome 15q11 region and loss of SNRPN allele

  • 30% results from mutation of paternal allele

  • Uniparental disomy: in rare cases both 15 chromosomes were found to be maternal with no deletion or mutation… both maternal alleles were silenced = no gene activity

• Angelman Syndrome: Manifests as intellectual deficits, seizures, bouts of laughter (paternal allele is present in both cases but is silenced via epigenetic imprinting)

  • 70% due to a deletion of maternal chromosome 15Q11 region and loss of UBE3A allele

  • 30% due to mutation of maternal allele

  • Uniparental disomy = in rare cases both 15 chromosomes were found to be paternal with no deletion or mutation… both paternal alleles were silenced = no gene activity

Population Genetics Concepts

• Hardy-Weinberg Equilibrium (HWE): A principle stating that allele frequencies within a population remain constant over generations in the absence of evolutionary influences.

  • Conditions for HWE:

  1. Population must be large (>100).

  2. Random mating without selection.

  3. Rare migration into/out of the population.

  4. Mutation is rare

  5. Simple dominant and recessive inheritance

• Calculating Allele Frequencies: If in equilibrium, the equation $p^2 + 2pq + q^2 = 1$ can be used to represent genotype frequencies.

Example Calculation of Genotype Proportions

• Given a population of 100 individuals with allele frequency $p = 0.6$:

  • Calculate $q = 1 - p = 0.4$.

  • Genotypes:

  • Homozygous Dominant: $p^2 = (0.6)^2 = 0.36$.

  • Heterozygous: $2pq = 2(0.6)(0.4) = 0.48$.

  • Homozygous Recessive: $q^2 = (0.4)^2 = 0.16$.