Cytogenetics – Aneuploidy, Meiosis & Chromosome Abnormalities

Cytogenetics: Core Concepts

• Definition
  • “Cytogenetics” = laboratory study of chromosomes in tissue, blood, bone-marrow or cultured cells.
  • Detects broken, missing, duplicated, rearranged or extra chromosomes.
  • Two analytic families:
  • Microscopy-based: Karyotype & FISH.
  • DNA-based: Array (CGH/SNP) & PCR-derived (e.g. STR).
• Clinical applications
  • Prenatal diagnosis, confirmation of genetic syndromes, cancer cytogenetics, pre-implantation genetic diagnosis (PGD).

Chromosome Number & Morphology

• Species variability
  • Jumping-jack ant: $1$ pair (♀); ♂ haploid.
  • Plains viscacha rat: $56$ pairs.
  • Adder’s-tongue fern: $631$ pairs.
• Human karyogram conventions
  • $46,XX$ or $46,XY$ = normal diploid complement.
  • Numbering by size except chromosomes $21,22$.
  • Short arm = p, long arm = q; bands numbered from centromere outward.
  • Centromere positions define metacentric, sub-metacentric, acrocentric.
  • Acrocentric satellites (chromosomes $13,14,15,21,22$) house rDNA.

Microscopy-Based Methods

Conventional Karyotyping

• Cells stimulated, arrested in metaphase, swollen, fixed & dropped onto slide.
• Giemsa (G-banding) → highly reproducible, chromosome-specific band pattern (~$5\,\text{Mb}$ resolution).
• Pros: global view, rapid, cheap, detects unknown large anomalies.
• Cons: cannot see <$5\,\text{Mb}$ changes; misses balanced/cryptic lesions.

Fluorescent In-Situ Hybridisation (FISH)

• Denatured metaphase/interphase DNA hybridised with fluorescently labelled probe.
• Detects presence, absence, location, or copy number of specific loci.
• Resolution kb-Mb depending on probe size.
• Multiplex forms: spectral karyotyping, subtelomeric, break-apart, fusion probes.

DNA-Based Methods

• Array-CGH
  • Co-hybridise patient & reference DNAs to oligo array → log$_2$ ratios.
  • Resolution ≈ $1.6\,\text{Mb}$ (clinical) to <50\,\text{kb} (research).
  • Detects gains/losses; misses balanced events.
• SNP arrays
  • Patient DNA amplified, fragmented, hybridised to SNP probes.
  • Measures both copy number (signal intensity) & genotype (allelic ratios).
  • Useful for uniparental disomy, consanguinity, LOH.
• STR (microsatellite) PCR
  • Used for rapid trisomy $21$ screen: three alleles or two alleles with 2:1 peak ratio.

Comparative Strengths / Weaknesses

• Array-CGH: +high sensitivity | −misses balanced.
• SNP array: +detects CNV & UPD | −limited to queried SNPs.
• STR: +fast/cheap | −preferential amplification biases.
• Karyotype: +balanced/large events | −low resolution.
• FISH: +gene/locus-specific | −one locus per assay, labour-intensive.

Structural Chromosome Abnormalities

• Translocations (reciprocal / Robertsonian)
  • Often balanced; phenotypically silent unless breakpoint disrupts gene or during meiosis → unbalanced gametes.
  • Commonly associated with leukaemias (e.g., $t(9;22)$).
• Deletions
  • Terminal: must acquire telomere.
  • Interstitial: internal loss.
• Duplications, Inversions (paracentric/pericentric), Ring chromosomes – each alters gene dosage or meiotic pairing.
• Chromothripsis
  • One-off chromosome “shattering” with haphazard re-assembly; $\approx2\%$ of cancers; rare curative event in WHIM (loss of mutant CXCR4).

Meiosis & Recombination Basics

• Two divisions:
  • Meiosis I (reductional): homologues separate.
  • Meiosis II (equational): sister chromatids separate.
• Crossing-over at chiasmata during prophase I assures proper segregation & increases genetic diversity.
• Closer loci (A & B) → lower recombination frequency (linkage).
• Random assortment of $23$ chromosome pairs yields $2^{23}$ (~$8.4\times10^{6}$) combinations before crossing-over.

Sex-Specific Features

• Male: continuous post-pubertal meiosis; $\sim65$ days.
• Female:
  1. Meiosis initiates $11{-}12$ wk fetal life.
  2. Arrest in dictyate (post-recombination prophase I) for years.
  3. Resume at ovulation → metaphase II arrest.
  4. Completion only upon fertilisation.
• Prolonged arrest + declining cohesin integrity ⇒ maternal-age aneuploidy.

Aneuploidy

• Definition: chromosome number not exact multiple of haploid set; gain/loss of one/few chromosomes.
• Viable human autosomal trisomies: $13$ (Patau), $18$ (Edwards), $21$ (Down).
• Sex chromosome aneuploidies:
  • Turner $45,X$: short stature, streak ovaries, normal IQ.
  • Klinefelter $47,XXY$: tall, infertility, gynecoid fat.
  • Triple-X $47,XXX$: often asymptomatic.
  • Double-Y $47,XYY$: tall; variable traits.

Maternal-Age Effect for Trisomy 21

• Risk table (approx.)
  • $20\,\text{yrs}: 1/1500$
  • $30\,\text{yrs}: 1/900$
  • $34\,\text{yrs}: 1/500$
  • $36\,\text{yrs}: 1/300$
  • $38\,\text{yrs}: 1/200$
  • $40\,\text{yrs}: 1/100$
  • $42\,\text{yrs}: 1/60$
  • $45\,\text{yrs}: 1/30$.
• $\approx70\%$ Down cases = nondisjunction in maternal meiosis I.

Down-Syndrome Phenotype

• Universal: cognitive impairment, characteristic facies.
• Common: congenital heart defect, acute megakaryocytic leukaemia.
• Neuro-degeneration: $10\%$ Alzheimer at $40{-}49$ yrs, $\sim100\%$ by $70$ yrs (triplicated APP).

Diagnostic Modalities

• Karyotype (identifies free vs translocation trisomy).
• Interphase FISH on uncultured cells.
• Array-CGH/SNP array.
• STR dosage analysis.

Microdeletion (Contiguous-Gene) Syndromes

• Definition: heterozygous deletion $1{-}5\,\text{Mb}$; invisible in karyotype; haploinsufficiency.
• Mechanism: unequal meiotic crossing-over via low-copy repeats.
• Examples & key genes:
  • 22q11.2 Deletion – VCFS / DiGeorge (TBX1); congenital heart, palatal, immune defects.
  • Williams-Beuren (7q11.23, $1.4\,\text{Mb}$) – loss of ELN (elastin) → SVAS; hypercalcaemia, elfin facies, sociable behaviour.
  • Angelman / Prader-Willi (15q11-13; imprinting, addressed elsewhere).

VCFS Case (Gillian)

• Presentation: VSD, global developmental delay.
• Karyotype normal; suspicion → FISH with 22q11 probe → single signal ⇒ deletion confirmed.

WBS Case

• Family with SVAS only had t(6;7)(p21.1;q11.23) disrupting ELN.
• Showed elastin haploinsufficiency causes SVAS; additional deletion of neighbouring $\approx26$ genes explains full WBS phenotype.

Chromosome Rearrangement Case Study (Ellen & Elizabeth)

• Mother Ellen: balanced $\text{t}(1;22)(q25;q13)$ (phenotypically normal).
• Daughter Elizabeth: inherited normal chr 1 + der(22) → partial trisomy 1q & monosomy 22q; results in undiagnosed syndrome.
• CGH plot: gains above baseline, losses below.
• Genetic counselling issues: quantify risk, offer prenatal/FISH/array-CGH, consider PGD.

Summary of Diagnostic Assays

• Microscopy
  • Karyotype: detects >5\,\text{Mb} aneuploidies/rearrangements.
  • FISH: locus specific; interphase-compatible.
• Molecular
  • Array-CGH: genome-wide CNV $\geq1.6\,\text{Mb}$.
  • SNP array: CNV + genotype + UPD.
  • STR: fast trisomy test; allele height ratios.
• Balanced anomalies only visible by karyotype/FISH, not arrays.

Learning Outcomes (Re-visited)

• Describe & compare chromosome-analysis techniques.
• Recognise numerical & structural abnormalities, including microdeletions.
• Explain meiotic mechanisms leading to diversity & aneuploidy.

Example MCQ (Lecture Slide 45)

• Haploid gamete = $n$ chromosomes, DNA $C$.
• Diploid G1: $2n$ chromosomes, $2C$ DNA.
• Diploid G2 (post-S): chromosomes still $2n$ (paired chromatids count as one chromosome each in cytogenetics), DNA $4C$.
• Correct choice: d) $2n$ & $2C$ in G1; $2n$ & $4C$ in G2.

Further Reading

• New Clinical Genetics, Ch. 2.
• Nagaoka SI, Hassold TJ, Hunt PA (2012) Nat Rev Genet $13$:$493{-}504$.
• Antonarakis SE et al. (2004) Nat Rev Genet $5$:$725{-}738$.
• Cereda A, Carey JC (2012) Orphanet J Rare Dis $7$:$81$.