Lecture 4: Alterations in Chromosomes and Genes

How common are cytogenetic disorders?

• Cytogenetic disorders: diseases caused by abnormal chromosomes (number or structure).

• Nearly 1% of live births have chromosomal abnormalities.

• 2% of pregnancies in women >35 (older eggs have been in meiosis longer → more errors).

• 10% of stillbirths.

• 50% of spontaneous abortions (miscarriages).

• Many cancers involve chromosomal changes.

How are cytogenetic studies performed?

• Take a blood sample (use white blood cells).

• Culture cells and stimulate them to divide.

• Arrest at metaphase with colcemid (chromosomes are easiest to see).

  • Colcemid a drug that halts cells in metaphase so chromosomes can be seen.

• Use a hypotonic solution to swell cells and spread chromosomes.

  • Hypotonic: Lower solute concentrations outside; higher solute concentrations inside.

• Fix and stain chromosomes on a slide for viewing.

• Resolution: detects large changes (~1-10 Mb).

  • Big chromosomal abnormalities.

• Can also use fetal cells from amniotic fluid, chorionic villis, or bone marrow.

What is G banding?

• Treat cells with trypsin and Giemsa stain.

• Creates pattern of light and dark bands on chromosomes.

• Pattern correlates with:

  • % GC vs. AT content.

  • Distribution of repetitive elements.

• Each chromosome has unique pattern → can identify all 24 human chromosomes.

What is a karyotype?

• Output of g-banding.

• Standard chromosomal set of an individual.

• Chromosomes arranged by size: 1-22 (autosomes) + gender chromosomes (XX or XY).

• Used to identify chromosomal abnormalities.

• Karyotype vs.kkaryogram:

  • Karyotype: the full set of chromosomes in a cell; includes number, size, shape; also refers to the analysis.

  • Karyogram: the arranged picture of chromosomes after analysis.

What is FISH (Fluorescence In Situ Hybridization)?

• Uses fluorescent probes to detect specific DNA sequences on chromosomes.

• Process:

  • Fix chromosomes, denature DNA.

    • The chromosomes are in interphase or metaphase.

      • Interphase = gene location or copy number.

      • Metaphase = structure.

  • Labeled probe hybridizes to target sequence.

  • View under fluorescence microscope.

• Can work on dividing (metaphase) or non-dividing (interphase) cells.

• Resolution: several megabases.

What is spectral karyotyping (SKY)?

• "Chromosome paint" - colors each chromosome differently.

  • Uses combinatorial labeling (different ratios of fluorophores).'

  • Different ratios are assigned pseudocolors.

    • Pseudocolors = artificial colors added to visualize different DNA probes.

• Makes it easy to see translocations and large rearrangements.

• Useful for studying cancer (complex chromosomal changes).

• To view:

  • Epifluorescence microscopy: view fluorescent probes.

  • CCD camera: captures bright, clear images.

  • Fourier spectroscopy: sees all emission colors at once.

What is array CGH (comparative genome hybridization)?

• Detects DNA copy number changes (gains/losses) across genome.

• Method:

  • Label patient DNA and control DNA with different colors.

    • Green = control DNA; red = sample DNA.

  • Hybridize to chip with 100,000+ oligonucleotides.

    • Oligonucleotide: synthetic, short DNA/RNA sequences used to target specific genetic sequences.

  • Measure fluorescence ratio.

    • Red = Green (ratio ≈ 1): Normal / balanced signal. Nothing unusual.

    • Red ≠ Green (ratio ≠ 1): Indicates a difference.

• Shows duplications and deletions.

• Cannot detect translocations, inversions, small mutations, or rearrangements.

What are the chromosome classifications by centromere position?

• Metacentric: centromere in middle (equal arms).

• Acrocentric: centromere near end (one very short arm).

• Telocentric: centromere at end (does NOT occur in humans).

What is triploidy and tetraploidy?

• Triploidy: 3 complete chromosome sets (69 chromosomes).

  • Caused by fertilization by 2 sperm OR failure in meiotic division.

  • Almost always lethal.

• Tetraploidy: 4 complete chromosome sets (92 chromosomes).

  • Usually arises from mitotic error in the zygote.

  • Always lethal.

What is aneuploidy?

• Abnormal number of chromosomes (not a multiple of 23).

• About 5% of pregnancies.

• Types:

  • Monosomy: missing one chromosome (45 total).

  • Trisomy: extra chromosome (47 total).

• Caused by nondisjunction (chromosomes fail to separate properly in meiosis; before fertilization).

  • Affects all of the cells in the body.

What is mosaicism?

• Two or more cell populations with different chromosome numbers in same person.

  • Example: 47,XXX/46,XX (some cells have extra X, some normal).

• Caused by nondisjunction during early embryonic development (not in egg/sperm).

  • Occurs in mitosis; after fertilization.

  • Only affects some cells.

• Usually causes milder symptoms than if all cells affected.

What are the main structural chromosomal abnormalities?

• Deletion.

• Duplication.

• Inversion.

• Translocation.

• Ring chromosome.

• Isochromosome.

Deletion

Piece of the chromosome missing.

Duplication

Piece present in extra copies.

Inversion

Segment flipped 180°.

Translocation

• Segment moves to another chromosome.

  • Reciprocal:

    • Two non-homologous chromosomes exchange segments.

    • Can be balanced (no net gain/loss of material) or unbalanced.

    • Balanced carriers usually healthy but at risk for unbalanced offspring.

  • Robertsonian: two acrocentric chromosomes join.

    • Acrocentric: centromere near the end, tiny p arm, long q arm.

Ring chromosome

Ends join together forming a circle.

Isochromosome

Chromosome with two identical arms.

Frameshift

The second image highlights how a single base shift causes a "domino effect," altering the entire protein downstream from the mutation.

Point mutations (missense and nonsense)

• Missense: One nucleotide changes → different amino acid in the protein (e.g., His → Pro). Can alter protein function.

• Nonsense mutation: Nucleotide change creates a premature STOP codon → protein is truncated and usually non-functional.

Large scale & structural mutations

• Duplication Mutation: A section of DNA is copied twice. Can cause an “overdose” of protein instructions → may affect traits like lip shape or size.

• Repeat Expansion: A short DNA sequence (e.g., “CAG”) repeats too many times. Can cause neurological or developmental disorders.

What are other factors affecting inheritance?

• Reduced penetrance: not everyone with mutation shows symptoms.

• Variable expressivity: same mutation causes different severity in different people.

• Modifying loci: other genes affect how mutation is expressed.

• Anticipation: disease gets worse/earlier in successive generations.

• Genomic imprinting: parent of origin matters for gene expression.

• Uniparental disomy: both copies of chromosome from one parent.

What are the three types of mutations by origin?

• Inherited (germline): passed from parents to offspring.

• De novo: occur in egg, sperm, or just after fertilization (new mutation, not in parents).

• Acquired (somatic): occur during person's life in body cells.

  • NOT passed to offspring (unless in germline cells).

What are loss of function mutations?

• Reduce or eliminate normal protein function.

• Mechanisms:

  • Nucleotide substitutions, deletions, rearrangements.

  • Premature stop codons.

  • Impaired protein folding.

• Examples:

  • Turner syndrome (missing X chromosome).

  • Thalassemias (reduced hemoglobin).

What are gain of function mutations?

• Enhance or alter normal protein function.

• Types:

  • Increase amount of protein.

  • Increase protein's normal function.

  • Novel property in new protein.

  • Wrong timing/location of production.

• Illustrates importance of gene regulation.

What mutations can affect RNA?

• Splice junction mutations: affect where introns are removed.

• Intron mutations: affect splicing signals.

• Coding sequence mutations: can affect splicing even in exons.

• Defects in capping and tailing: affect mRNA stability and translation.

What are housekeeping proteins vs. specialty proteins?

• Housekeeping proteins:

  • Present in every cell.

  • Maintain basic cell structure and function.

  • 90% of expressed genes.

• Specialty proteins:

  • Tissue-specific.

  • Produced in limited cell types.

  • Have unique functions.

  • 10% of expressed genes.

What is genetic heterogeneity?

• Allelic heterogeneity: different mutations in SAME gene cause similar disease.

  • Example: many different CFTR mutations all cause cystic fibrosis.

• Locus heterogeneity: mutations in DIFFERENT genes cause similar disease.

  • Example: hearing loss can be caused by mutations in many different genes.

• Modifier genes: genes unrelated to main defect can alter how disease manifests.

Autosomal disorder

• Definition: Mutation on chromosomes 1–22.

• Note: Can be dominant or recessive; affects males & females equally.

X-linked disorder

• Definition: Mutation on the X chromosome.

• Note: Males more affected; females may be carriers if recessive.

Codominant disorder

• Definition: Both alleles are fully expressed.

• Example: AB blood type, sickle cell trait.

Mitochondrial disorder

• Definition: Mutation in mtDNA.

• Note: Maternal inheritance only, often affects energy-demanding tissues.