Mednotes

L06 Genetics Whole genome analysis 1

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tagsgenetics, lecture
aliasesL06 Genetics Whole genome analysis 1

21 min read

Overview

This lecture covers the analysis of chromosomal disorders in the clinic: how chromosomes are prepared, banded and named, why chromosomal balance is the essence of chromosomal disease, and how gene dosage links imbalance to phenotype. It then works through the clinical indications for chromosomal analysis in turn: a disorder of growth and development (Turner syndrome, with X-inactivation and Klinefelter syndrome alongside it), intellectual disability (the aneuploidies survivable to term, with Down syndrome as the worked example and its prenatal screening), and recurrent miscarriage (balanced reciprocal and Robertsonian rearrangements and the unbalanced gametes they generate). It closes with the place of chromosomal microarray and the karyotype in current practice.

Chromosome structure, banding and the karyogram

  • Chromosomes are visualised at high magnification as a replicated X shape: two sister chromatids joined at a central constriction, alongside smaller, rounder unreplicated forms.
  • Preparation and staining: cells are grown and Giemsa stained (G-banding).
  • Band meaning: dark bands are inactive (heterochromatin); light bands are active (euchromatin).
  • Ordering: chromosomes are ordered by size and by centromere position: acrocentric (centromere near the end) and metacentric (centromere central).
  • The short arms of acrocentric chromosomes house repetitive, duplicated DNA.
  • Arm names: long arm is q (queue), short arm is p (petite).
  • Band numbering: bands are numbered outwards from the centromere (cen) to the telomere (tel). The worked ideogram runs p and q arm bands out from the centromere in the series 13, 12, 11.2, 11.1, 11, 12.1, 12.2, 12.3, 13, 14.1, 14.2, 14.3, 21.1, 21.2, 21.3, 22, 31, 32, 33, 34.
  • Replication anatomy: a pair of homologous chromosomes, each with its centromere, undergoes DNA replication so that each homolog becomes two sister chromatids joined at the centromere.
  • The karyogram: a full human karyogram displays all 24 chromosome types (1 to 22, X and Y), arranged by size, each drawn with its characteristic G-banding pattern and centromere position and labelled underneath with its number or letter.

Karyotype nomenclature

Symbols used in a karyotype string:

  • 1 to 22: autosome numbers; X, Y: sex chromosomes
  • /: mosaicism, e.g. 46/47
  • p: short arm; q: long arm; ter: terminal (pter, qter)
  • del: deletion; dup: duplication; ins: insertion; inv: inversion
  • der: derivative (rearranged) chromosome; t: translocation
  • i: isochromosome; r: ring chromosome
  • + or - before a chromosome number indicates gain or loss of a whole chromosome (+21); placed after, it implies gain or loss of a part (5q-)

Worked examples given on the slide:

  • 46,XY,t(5;10)(p13;q25): 46 chromosomes, male, with a translocation between chromosomes 5 and 10, breakpoints at 5p13 and 10q25.
  • 47,XY,+21: 47 chromosomes, male, with gain of a whole extra chromosome 21.
  • 46,XX,+13,der(13;21)(q10;q10): 46 chromosomes, female, with gain of a whole chromosome 13 that is carried on a derivative chromosome formed from chromosomes 13 and 21 joined at q10 and q10.
  • 46,XX,dup(2)(p13p22): 46 chromosomes, female, with a duplication of chromosome 2 between bands p13 and p22.

Warning

On the nomenclature slide the two accompanying ideograms carry tick marks at the breakpoints, but they are not themselves labelled “5” and “10”; which ideogram is which is inferred from the adjacent karyotype text rather than stated on the slide.

Chromosomal balance and gene dosage

  • The essence of the chromosomal basis of disease states is balance. Chromosomal variants are common, but not all cause disease.
  • Frequency of abnormality: 50% of spontaneously aborted fetuses, 10% of stillborn babies, 0.7% of liveborn babies.
  • Incidence among newborns: all chromosome abnormalities ~0.65% (1/154). Aneuploidy total 1/263, of which X and Y chromosomes 1/475 and autosomes 1/700. Structural abnormalities total 1/375, of which balanced 1/500 and unbalanced 1/1600.
  • Gene dosage: there are normally two copies of each autosomal gene, so the appropriate autosomal gene dosage is two.
  • For the sex chromosomes the appropriate dosage is (mostly) one: males have one copy of X genes plus one copy of Y genes; females have two copies of X, but one X has 80% of its content inactivated.
  • Chromosomal abnormalities can give rise to loss or gain of genes, both of which may cause disease, often resulting from cell death and/or abnormal growth.
  • Chromosomal microarray at high resolution reveals we are all “unbalanced”: a genome-wide plot of 3400 healthy children shows deletions (red) and duplications (blue and green) scattered across all 22 autosomes. The challenge is distinguishing pathogenic copy number variants from the “normal” background variation seen in the general population.

Indications for chromosomal analysis

Four clinical indications for assessing chromosomal structure:

  1. Abnormality of growth or development (a “syndrome”)
  2. Intellectual disability
  3. Recurrent miscarriage
  4. Investigation of cancers (e.g. leukaemia)

Turner syndrome (indication I: a disorder of growth and development)

  • Presentation: neonatal oedema/webbed neck, coarctation of the aorta, linear growth delay, primary amenorrhoea.
  • Incidence: 1:3000.
  • A growth chart plotting height against age compares girls without Turner syndrome with untreated girls with Turner syndrome: the untreated Turner curve runs below the normal female curve.
  • Causes: 75% have a 45,X0 karyotype (one third of these are clearly mosaic); 25% have a structural abnormality of one X chromosome, such as a deletion or a ring chromosome. Deletions are illustrated as terminal or interstitial; a ring forms when a terminal fragment breaks off and the chromosome ends join into a circle.
  • How it occurs: the classical view is meiotic non-disjunction; the emerging view is post-zygotic loss of an X, producing chromosomal mosaicism. The recurrence risk is therefore negligible.
  • What families ask:
    • Is the clinical phenotype predictable? Often not, with two factors at play: mosaicism (often present) and variable strength of expression of alleles on the remaining X.
    • Will it recur in a subsequent sibling? For whole chromosomal monosomy X, no. The mechanism of chromosome loss is thought to be post-zygotic, so the recurrence risk equals the population risk.

X-inactivation

The slide poses the question: why, if one X chromosome is normally inactivated in girls, do females with Turner syndrome have problems?

  • The process: from a fertilised ovum carrying a paternal X and a maternal X, mitosis proceeds and one X is inactivated at random. This branches into the possible inactivation patterns across daughter cell lineages (maternal X inactivated with paternal X expressed, and the reciprocal), which are then propagated through further mitosis.
  • The consequence is mosaicism of X-inactivation across tissues, illustrated by body silhouettes shaded in a mosaic green and pink pattern.
  • The gene figure: an X chromosome ideogram (Xp and Xq arms) annotates genes at specific bands, labelling some as inactivated and some as escaping inactivation. Named genes include PGPL, SHOX, CSF2RA, IL3RA, ANT3, ASMT, MIC2 and XG in the PAR1 pseudoautosomal region; GSIX, STS, KAL1, OTC, CYBB, UBE1, TIMP1, SB1.8, UTX, SMCX/XE169X, ZXDA, ZXDB, AR, RPS4X, PGK2, XIST, GLA and HPRT along the arms; G6PD; and SYBL1/IL9R in the PAR2 region.
  • The slide shows only that some X-linked genes are inactivated and some escape inactivation, and names the genes on the figure. [slide does not state which of the named genes escape inactivation]

Klinefelter syndrome

  • Karyotype: 47,XXY; the second X undergoes X-inactivation.
  • Features: reduced testicular function, tall stature, IQ in the normal range but a bit left-shifted. The IQ distribution curve for 47,XXY individuals sits shifted towards lower scores relative to controls, over a range of roughly 50 to 130.
  • Incidence: 1:500 to 1:1000.
  • Management: testosterone replacement; fertility treatment options.
  • Cause: meiotic non-disjunction.

Warning

The metaphase karyotype image on this slide is small and the pair circled in red is not clearly legible. Given the slide topic it likely marks the XXY sex-chromosome complement, but that is not certain from the image alone.

Intellectual disability and the aneuploidies (indication II)

  • Chromosomal analysis is applied across the paediatric age group.
  • Some syndromes are clinically identifiable.
  • Some whole aneuploidies are survivable to term: the ones named are trisomy 13, trisomy 18 and trisomy 21 (Down syndrome).
  • Clinical patterns of presentation are prenatal and postnatal.
  • Prenatal ultrasound features of trisomy 21 shown on the composite figure: nuchal thickening, mild ventricular dilatation, brachycephaly, hypoplastic nose, echogenic intracardiac focus, clinodactyly, hyperechoic bowel, pyelectasis, widened pelvis, sandal gap, shortened limbs.

Non-disjunction in aneuploidy

  • Non-disjunction is failure of homologous chromosomes to separate in anaphase I, or failure of sister chromatids to separate at meiosis II.
  • It gives rise to nullisomic and disomic gametes.
  • Meiosis I non-disjunction (with meiosis II normal) produces gametes of n+1, n+1, n-1, n-1.
  • Meiosis II non-disjunction (after a normal meiosis I, affecting one of the two cells) produces gametes of n+1, n-1, n, n.
  • The Turner non-disjunction flowchart, starting from a cell with two X chromosomes and branching through normal separation or non-disjunction at meiosis I and again at meiosis II, ends in gametes of 24, 22, 23, 23, 24, 24, 22, 22 chromosomes.

Down syndrome

Diagnosis can be made by four techniques:

  1. Metaphase karyotype (chromosome 21 present in three copies)
  2. Interphase FISH (three probe signals in the nucleus)
  3. Chromosomal microarray (elevated log R fluorescence ratio over chromosome 21)
  4. Whole genome sequencing (elevated normalised sequence representation for chromosome 21)

Genetic basis:

  • 96%: meiotic non-disjunction, giving free-standing trisomy 21. 95% occur in the egg. Recurrence risk is 1% until the mother’s own age-related risk exceeds 1% at age 40, after which it increases according to maternal age.
  • 3 to 4%: translocation (Robertsonian), usually with one chromosome 21 attached to chromosome 14, 21 or 22. For the 14/21 translocation, one third of patients have a parent who carries it with a balanced karyotype. Of those, 90% have the mother as carrier, with a recurrence chance of 10 to 15%; 10% have the father as carrier, with a recurrence chance of 2 to 5%.
  • 1 to 2%: mosaicism, arising post-zygotically. The number of affected cells varies between individuals and clinical findings vary widely.

Robertsonian (centric fusion) translocations:

  • Chromosomes 13 to 15, 21 and 22 are acrocentric, with no important DNA in the p arm.
  • They arise from breaks at or near the centromere in chromosome 21 and another acrocentric chromosome (frequently 14), with cross-fusion of the two products to give a derivative such as der(14;21) plus a small residual fragment.
  • Problems arise at gametogenesis.
  • There is no maternal age effect, and there is a risk of recurrence.
  • Meiosis in a carrier: carriers are asymptomatic but often produce unbalanced gametes which can result in a monosomic or trisomic zygote. From a carrier cell containing chromosome 14, the 14/21 fusion chromosome and chromosome 21, fertilisation by a normal gamete gives zygotes that are normal, balanced carrier, (trisomy 14), (monosomy 14), (monosomy 21), or trisomy 21. The bracketed monosomic and trisomic zygotes would not develop to term.

Multisystem management: there are well-defined guidelines for management of people with Down syndrome, covering

  • Neurodevelopmental: intellectual disability, developmental delay, language disorders, cerebellar hypoplasia
  • Psychiatric: anxiety and depression, behavioural disturbance
  • Neurological: Alzheimer disease, epilepsy
  • Craniofacial: small low-set ears, epicanthic folds, flat nasal bridge, flat occiput, small mouth, upslanting palpebral fissures
  • Sensory: conductive and sensorineural hearing loss, refractive errors, cataracts, keratoconus and amblyopia
  • Cardiovascular: congenital heart defects, especially AVSD
  • Respiratory: obstructive sleep apnoea, respiratory tract infections
  • Musculoskeletal: atlantoaxial instability, small stature, short fingers, hypotonia
  • Autoimmune: thyroid disease, coeliac disease, alopecia, type 1 diabetes mellitus, psoriasis
  • Other: haematological disorders, immune dysfunction, obesity, bowel dysfunction, gastrointestinal structural defects, male infertility

Prenatal screening for Down syndrome

  • Population incidence: 1:700.
  • Maternal age effect: incidence rises sharply with maternal age, approximately 1/2300 at 20, 1/1600 at 24, 1/1200 at 28, 1/880 at 32, 1/299 at 37, 1/100 at 42 and 1/46 at 47.

Important

Despite the steep maternal age curve, 70% of Down syndrome occurs in mothers under 35, so screening cannot be confined to older mothers.

  • Current screening programme: late first or early second trimester ultrasound (nuchal translucency, NT) plus maternal serum markers (2 or 4) at 10 to 15 weeks. The result is reported as “increased risk” or “low risk”, and increased risk leads to amniocentesis.
  • New technology: non-invasive prenatal screening for genetic anomalies.
  • Cell-free placental DNA in maternal blood: fetal (placental) DNA fragments circulate in maternal blood alongside maternal DNA.
    • Fragments are isolated from maternal serum and subjected to high throughput DNA sequencing.
    • Their chromosome source is identified and the number of fragments is counted.
    • Stupendous numbers of fragments are sequenced (>1x10
    • An excess of chromosome 21-derived sequences indicates an aneuploid pregnancy. In the cell-free fetal DNA fraction the fetal portion is visibly larger in a T21 pregnancy than in a normal one, whereas total DNA in maternal blood shows comparatively little difference.
    • It still requires corroborative invasive testing, i.e. amniocentesis.

Recurrent miscarriage and structural rearrangements (indication III)

  • The underlying problem is carriage of a chromosomal rearrangement that can give rise to gametes with unbalanced chromosomal configurations.
  • Clinical consequences: recurrent miscarriages, relative infertility, malformations or intellectual disability in relatives.

Reciprocal translocations:

  • Definition: the balanced transfer of material from one chromosome to another. A break occurs in each of two chromosomes and the terminal segments are exchanged and rejoined, giving a balanced translocation.
  • Frequency: 1 in 600.
  • Most have no effect in the balanced state.
  • A small minority may affect the health of a carrier in the balanced state.
  • Some unbalanced configurations can be viable to term, usually with severe phenotypic consequences.
  • Segregation: a parent’s cell carrying a reciprocal translocation, together with a parent’s cell with normal chromosomes, produces gametes that on fertilisation give four zygote types: normal and translocation carrier (both a healthy phenotype), and two duplication-deletion outcomes (phenotypically abnormal or miscarriage).

Keys to thinking about reciprocal translocations:

  • A history of recurrent miscarriages
  • Think wider than the nuclear family: “skipped” individuals
  • A positive family history of delayed and/or malformed relatives, not necessarily a first degree relative
  • Check for carrier status using karyotype
  • Check for imbalance with chromosomal microarray

Warning

On the accompanying pedigree, a circled numeral “3” marks one symbol and is not explained by any on-slide legend. By conventional pedigree notation this typically denotes multiple pregnancy losses at that position, but the slide itself does not state this.

Consequences of chromosomal imbalance

  • Imbalances are usually private: translocations are almost always unique, with the notable exception of Robertsonian translocations.
  • Balanced reciprocal translocations can have multiple different outcomes.
  • Monosomy is usually more severe than trisomy.
  • The length of imbalance is inversely correlated with viability.
  • Cognitive function is almost always affected.
  • Phenotypes can “skip” generations.
  • Meiosis in a carrier produces gametes that, on fertilisation by a normal gamete, give zygotes that are normal, balanced carrier, partial trisomy plus partial monosomy, or partial monosomy plus partial trisomy.

Summary

  • Chromosomes can be assessed at high resolution.
  • Chromosomal anomalies can arise pre-conceptually or post-conception.
  • Chromosomal microarray is the main technique used to analyse chromosomal balance.
  • Karyotypes retain some utility in diagnosis, specifically for mosaicism and for identifying carriers of balanced rearrangements.
  • Sex chromosome aneuploidies can lead to disparate and complex phenotypes.
  • Down syndrome can arise from different mechanisms.
  • Non-invasive prenatal detection of Down syndrome is becoming widespread.
  • Balanced translocations can give rise to familial presentations of chromosomal disorders in offspring.

Self-test

  1. Define G-banding and state what the dark and light bands represent.
  2. Distinguish acrocentric from metacentric chromosomes, and state what the short arms of acrocentric chromosomes contain.
  3. Explain how chromosome bands are numbered and what p and q stand for, and describe what a full human karyogram displays.
  4. Describe what DNA replication does to a pair of homologous chromosomes, using the terms centromere and sister chromatids.
  5. Decode the karyotypes 47,XY,+21 and 46,XX,+13,der(13;21)(q10;q10).
  6. Decode the karyotypes 46,XY,t(5;10)(p13;q25) and 46,XX,dup(2)(p13p22).
  7. State the appropriate gene dosage for autosomal genes and for sex chromosome genes, and explain how males and females each achieve the sex chromosome dosage.
  8. List the proportions of spontaneously aborted fetuses, stillborn babies and liveborn babies that carry chromosomal abnormalities.
  9. Explain what the microarray study of 3400 healthy children showed and what interpretive challenge it creates.
  10. List the four clinical indications for assessing chromosomal structure.
  11. Describe the presenting features and incidence of Turner syndrome, and describe what the growth chart on the slide shows.
  12. Describe the chromosomal causes of Turner syndrome, with their proportions and the structural forms involved.
  13. Explain why the recurrence risk for whole chromosomal monosomy X is negligible.
  14. Explain why the clinical phenotype of Turner syndrome is often not predictable.
  15. Describe the process of X-inactivation as the slide depicts it, and state what the accompanying X chromosome gene figure shows.
  16. Describe the karyotype, clinical features, incidence, management and cause of Klinefelter syndrome.
  17. Define non-disjunction, and state the gamete chromosome counts produced when it occurs at meiosis I versus meiosis II.
  18. Which whole aneuploidies does the lecture name as survivable to term, and how is that claim qualified?
  19. List the prenatal ultrasound features of trisomy 21 shown on the slide.
  20. List the four techniques by which Down syndrome can be diagnosed, and state what each shows for chromosome 21.
  21. Give the chromosomal bases of Down syndrome with their approximate percentages, and the recurrence risk attached to free-standing trisomy 21.
  22. Describe how a Robertsonian translocation arises, which chromosomes are involved, and why there is a recurrence risk but no maternal age effect.
  23. Predict the zygote outcomes when a 14/21 Robertsonian carrier’s gametes are fertilised by a normal gamete, and state which are not viable to term.
  24. A woman aged 29 is pregnant. Explain why she is still offered Down syndrome screening despite her age, and outline the current screening pathway.
  25. Describe how cell-free placental DNA screening detects trisomy 21, and state its limitation.
  26. State the frequency of reciprocal translocations and describe the possible consequences for a carrier in the balanced state.
  27. A woman presents with three miscarriages and a cousin with intellectual disability. Explain what should raise your suspicion and which two tests the lecture recommends, saying what each is for.
  28. List the generalisations the lecture gives about the consequences of chromosomal imbalance.
  29. Integrative question: according to the summary, which technique is the main one for analysing chromosomal balance, and what utility do karyotypes retain? Relate each to a scenario covered earlier in the lecture.

Answers

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