Chromosome Mutations

A single base change can alter one protein, but a chromosome mutation can add, remove or rearrange hundreds of genes at once. These large scale changes cause about half of early miscarriages, Down syndrome and many developmental disorders, drive certain leukemias, and built some of the most important crops on Earth. This chapter shows how chromosomes are described, how they break and rejoin, how whole chromosomes or whole sets are gained or lost, and what each change does in meiosis and in the clinic.

Section 1Chromosome structure and karyotypes

Parts of a chromosome

Every eukaryotic chromosome is one long DNA molecule packaged with proteins into chromatin. After replication, and until anaphase, each chromosome consists of two identical sister chromatids held together at the centromere. Three elements are essential for a chromosome to be copied and passed on faithfully:

• Origins of replication, many per chromosome, so that a long molecule can be copied in the hours of S phase.
• A centromere, where the kinetochore assembles and attaches to spindle microtubules. Human centromeres are built on long arrays of a 171 bp repeat called alpha satellite DNA, but they are defined epigenetically by nucleosomes containing the histone H3 variant CENP-A.
• Two telomeres, the protective ends. Human telomeres are thousands of tandem copies of TTAGGG bound by the shelterin protein complex. Telomeres keep chromosome ends from being mistaken for broken DNA and joined together.

The importance of these elements explains much of this chapter. A chromosome fragment with no centromere (acentric) cannot attach to the spindle and is lost at division. A chromosome with two centromeres (dicentric) may be pulled toward both poles and break. A broken end without a telomere is "sticky" and tends to fuse with other broken ends, which is how many rearrangements form.

Centromere position and chromosome shape

The centromere divides a chromosome into two arms. By convention the short arm is p (for petit) and the long arm is q. Chromosomes are classified by where the centromere lies, which is measured by the arm ratio (q length divided by p length) or the centromeric index (p length divided by total length).

The tiny short arms of the five human acrocentric chromosomes carry the tandem ribosomal RNA genes, the nucleolus organizer regions. Because the same genes are repeated on all five, losing the short arms of two of them, as happens in a Robertsonian translocation, has no effect on the person.

Making and reading a karyotype

A karyotype is the complete set of chromosomes of a cell, arranged in pairs by size, centromere position and banding pattern. Dividing cells (commonly cultured white blood cells, amniocytes or bone marrow) are treated with a spindle poison such as colchicine or colcemid, which arrests them in metaphase when chromosomes are most condensed. A hypotonic solution swells the cells, they are fixed and dropped onto slides, and the chromosomes are stained.

The standard stain is G-banding: a brief trypsin treatment followed by Giemsa dye. Dark G bands tend to be AT rich, gene poor and late replicating; light bands are GC rich and gene dense. Each chromosome has a reproducible pattern of about 400 to 550 bands per haploid set in a routine preparation, and up to about 850 in high resolution prometaphase spreads. Bands are numbered outward from the centromere: 7q31.2 means chromosome 7, long arm, region 3, band 1, sub-band 2.

A band at routine resolution contains roughly 5 to 10 Mb of DNA, so a G-banded karyotype detects gains, losses and rearrangements of about that size or larger. Molecular methods see far smaller changes:

• Fluorescence in situ hybridization (FISH) uses a fluorescent DNA probe for a specific region. The number and location of signals reveal deletions, duplications and translocations, even in nondividing (interphase) cells.
• Spectral karyotyping paints each chromosome a different color, making complex translocations obvious.
• Chromosomal microarray compares the amount of patient DNA with a reference at hundreds of thousands of points across the genome. It detects copy number variants as small as about 50 to 100 kb, but it cannot detect balanced rearrangements, which change position without changing amount.

Karyotype notation

The international system for human cytogenomic nomenclature (ISCN) writes the total chromosome number, then the sex chromosomes, then any abnormality. A normal female is 46,XX and a normal male 46,XY. A plus or minus sign before a chromosome number means a whole extra or missing chromosome. Structural changes use abbreviations followed by the chromosome(s) in one set of parentheses and the breakpoints in the next.

Section 2Deletions, duplications, inversions and translocations

How rearrangements arise

Structural rearrangements begin with double strand breaks, caused by ionizing radiation, some chemicals, replication errors or the cell's own programmed cuts. If the broken ends are rejoined wrongly, or if crossing over occurs between similar sequences at the wrong positions (nonallelic homologous recombination), the gene order changes. Repeated sequences, such as segmental duplications and transposable elements, are hotspots because they provide misleading homology.

Deletions

A deletion (deficiency) removes a segment, either from the end (terminal) or from within (interstitial). In a deletion heterozygote, the normal homolog has nothing to pair with across the missing region, so it buckles out as a deletion loop during synapsis, visible in Drosophila polytene chromosomes. Two genetic effects reveal deletions. First, a deletion uncovers recessive alleles on the other homolog: a recessive allele opposite a deletion is expressed, a phenomenon called pseudodominance, which makes deletions excellent tools for mapping genes to small regions (just as Benzer used them in phage). Second, many genes are haploinsufficient: one copy does not make enough product for a normal phenotype. Large deletions are usually lethal when homozygous and often harmful when heterozygous. Deletion of part of the short arm of human chromosome 5 causes cri du chat syndrome, named for the high pitched, catlike cry of affected infants, together with intellectual disability and a small head.

Duplications

A duplication repeats a segment. In a tandem duplication the copies are adjacent and in the same orientation; in a reverse duplication they are in opposite orientation; a duplicated segment may also be inserted elsewhere. Duplications commonly arise by unequal crossing over between misaligned repeats, which creates a duplication on one chromatid and a reciprocal deletion on the other.

The Bar mutation of Drosophila is the classic case. Bar is a tandem duplication of a small region of the X chromosome that reduces the number of facets in the eye. Females with one Bar region on each X (B/B) have four copies of the region and small eyes, but females with a triple copy on one X and a single copy on the other (double Bar over wild type) also have four copies and even smaller eyes. Same dose, different phenotype: the arrangement mattered, an example of a position effect.

Duplications are also the raw material of evolution. Once a gene is duplicated, one copy can keep the original function while the other accumulates mutations and may gain a new role. The globin genes, olfactory receptor genes and Hox clusters are families that arose this way.

Inversions

An inversion cuts out a segment and reinserts it in reverse. If the segment does not include the centromere, it is a paracentric inversion; if it does, it is a pericentric inversion, which can change the arm ratio and therefore the shape of the chromosome. An inversion homozygote usually has a normal phenotype, because no genes are lost or gained, unless a breakpoint disrupts a gene or moves it next to heterochromatin. The genetic action happens in heterozygotes. To pair gene for gene, one homolog must twist into an inversion loop.

A single crossover within the loop produces unbalanced chromatids:

• In a paracentric inversion heterozygote, the crossover produces a dicentric chromatid (two centromeres) and an acentric fragment (none). At anaphase I the dicentric stretches between the poles as a dicentric bridge and breaks, while the acentric fragment is lost. Both recombinant products are missing genes.
• In a pericentric inversion heterozygote, each recombinant chromatid has one centromere but carries a duplication of one end and a deficiency of the other.

In both cases the two nonrecombinant chromatids (one normal, one inverted) are balanced. Gametes carrying the unbalanced recombinant chromatids are inviable in plants or give inviable zygotes in animals, so recombinant progeny are not recovered for genes within the inversion. Inversions therefore act as crossover suppressors, keeping groups of alleles together. Geneticists exploit this in Drosophila balancer chromosomes, which carry multiple overlapping inversions, a dominant visible marker and a recessive lethal, to maintain a mutation in a stock without it being lost by recombination.

Translocations

A translocation moves a segment to a nonhomologous chromosome. In a reciprocal translocation, two nonhomologous chromosomes exchange segments. A carrier with a balanced reciprocal translocation has all its genes, usually in normal amounts, and is typically healthy. In meiosis, however, the two normal and two translocated chromosomes pair as a cross shaped quadrivalent, and how the four chromosomes segregate determines whether gametes are balanced:

• Alternate segregation: the two normal chromosomes go to one pole and the two translocated chromosomes to the other. Both gametes are balanced (one normal, one balanced carrier).
• Adjacent-1 segregation: each pole receives one normal and one translocated chromosome, with nonhomologous centromeres going together. Both gametes are unbalanced, carrying a duplication of one segment and a deficiency of another.
• Adjacent-2 segregation: homologous centromeres go to the same pole. Both gametes are unbalanced; this is rarer.

Because adjacent and alternate segregation occur at similar frequencies in many plants, a translocation heterozygote is often semisterile, with about half its pollen or ovules aborted. In humans, balanced translocation carriers may have recurrent miscarriages or a child with an unbalanced karyotype. Translocations also produce pseudolinkage: genes near the breakpoints on the two different chromosomes behave as if linked, because only the alternate segregation products survive.

Robertsonian translocations

A Robertsonian translocation fuses the long arms of two acrocentric chromosomes at or near their centromeres, and the two tiny short arms are lost. The carrier has 45 chromosomes but is healthy, because the lost short arms carry only redundant ribosomal RNA genes. About 1 in 1,000 people carry one; rob(13;14) and rob(14;21) are the most common. The danger is to offspring.

Section 3Aneuploidy and nondisjunction

Terms for changes in chromosome number

Changes in chromosome number come in two kinds. Euploidy changes involve whole sets (the subject of Section 4). Aneuploidy is the gain or loss of individual chromosomes, so that the number is not an exact multiple of the haploid number. Using 2n for the normal diploid number:

• Nullisomy (2n − 2): both members of a pair missing.
• Monosomy (2n − 1): one chromosome missing, for example 45,X.
• Trisomy (2n + 1): one extra chromosome, for example 47,XX,+21.
• Tetrasomy (2n + 2): two extra copies of the same chromosome.
• Double trisomy (2n + 1 + 1): one extra copy each of two different chromosomes.

Aneuploidy is usually far more harmful than polyploidy, even though it changes fewer genes. The reason is gene balance: many proteins work in complexes or pathways whose parts must be made in the right proportions. Adding a whole set keeps every ratio at 1:1; adding one chromosome raises the dose of hundreds of genes by 50 percent relative to all the others. In humans, every autosomal monosomy and most autosomal trisomies are lethal before birth.

Nondisjunction

Most aneuploidy arises from nondisjunction, the failure of chromosomes to separate properly. In meiosis I nondisjunction, a pair of homologs goes to the same pole; in meiosis II nondisjunction, a pair of sister chromatids goes to the same pole. The two produce different gamete sets:

• After nondisjunction in meiosis I, all four products are abnormal: two are n + 1, carrying one copy of each homolog, and two are n − 1.
• After nondisjunction in meiosis II (in one of the two secondary cells), half the products are abnormal: one n + 1 carrying two copies of the same chromatid, one n − 1 and two normal.

That difference is the key to working out where nondisjunction occurred. If an extra chromosome carries two different alleles from one parent (both of that parent's homologs), the error was in meiosis I. If it carries two identical copies of one parental homolog, the error was most likely in meiosis II. (Crossing over complicates this for loci far from the centromere, so markers near the centromere give the cleanest answer.) Related mechanisms include anaphase lag, in which a chromosome is left behind and lost, and premature separation of sister chromatids in meiosis I.

Human aneuploidies

Only three autosomal trisomies commonly survive to birth, those of chromosomes 21, 18 and 13, which are among the most gene poor autosomes. Sex chromosome aneuploidies are far better tolerated, for two reasons: all X chromosomes beyond one are inactivated (Chapter 3), and the Y carries few genes.

About 95 percent of Down syndrome is free trisomy 21, and in roughly 90 percent of these the extra chromosome comes from the mother, most often from a meiosis I error. About 3 to 4 percent of cases are due to a Robertsonian translocation (Section 2), which matters because it can recur in a family, and 1 to 2 percent are mosaic. The risk of free trisomy rises steeply with maternal age: approximately 1 in 1,250 births at age 25, 1 in 1,000 at 30, 1 in 350 at 35, 1 in 100 at 40 and 1 in 30 at 45. A leading explanation is that human oocytes begin meiosis I before birth and remain arrested for decades; the cohesin complexes that hold homologs and sisters together slowly deteriorate, and with fewer or poorly placed crossovers, homologs separate prematurely or not at all.

Mosaicism and uniparental disomy

Nondisjunction in a mitotic division after fertilization produces a mosaic, a person with two or more cell lines of different karyotype, such as 46,XX/47,XX,+21. Mosaics are usually more mildly affected than full trisomics, in proportion to how many cells, and which tissues, are abnormal. Mosaicism confined to the placenta can confuse prenatal tests that sample chorionic villi.

A trisomic embryo can sometimes "rescue" itself by losing one of the three copies in an early mitosis. If the lost copy is the one from the parent who contributed only one, the embryo ends up with both copies from the same parent, which is uniparental disomy (UPD). UPD is harmless for most chromosomes but causes disease when the region contains imprinted genes, or when the doubled chromosome carries a recessive mutation (isodisomy). Maternal UPD of chromosome 15 causes about a quarter of cases of Prader Willi syndrome, because the paternally expressed genes in 15q11-q13 are missing.

Section 4Polyploidy

Whole extra sets

Polyploids carry more than two complete chromosome sets. Geneticists use x for the number of chromosomes in one basic set and n for the number in a gamete. In a diploid these are the same, but in polyploids they differ. Bread wheat, for instance, is 2n = 6x = 42: each body cell has six sets of 7 chromosomes, and each gamete (n) has 21. Triploids (3x), tetraploids (4x), hexaploids (6x) and octoploids (8x) are all known.

Polyploidy is common in plants (most flowering plant lineages have undergone at least one whole genome duplication in their history) and in some fish, amphibians and insects, but rare in mammals. Plants tolerate it better for several reasons: many reproduce asexually or self fertilize, they lack chromosomal sex determination that would be upset by extra sets, and their development is more flexible. Polyploid plants often have larger cells and larger organs (fruits, flowers, leaves), the "gigas" effect, which made them attractive in agriculture.

Autopolyploidy

An autopolyploid has multiple sets from the same species. It can arise when a diploid gamete (from a failure of meiosis) fuses with a normal gamete, giving a triploid, or with another diploid gamete, giving a tetraploid. In the laboratory, colchicine, which binds tubulin and prevents spindle formation, blocks anaphase so that the chromosomes of a dividing cell are not separated, doubling the chromosome number in that cell and its descendants.

Autotetraploids are fertile but segregate in unusual ratios, because each chromosome has four homologs that can pair as a quadrivalent or as two bivalents, and any two of the four go to each gamete. Triploids are almost always highly sterile. With three homologs for each chromosome, one pole gets two and the other one, at random for each chromosome type. A gamete is balanced only if it receives exactly one copy of every chromosome (n = x) or two copies of every chromosome (2x). Seedless watermelons and the Cavendish banana (a triploid, 2n = 3x = 33) owe their seedlessness to this sterility.

Allopolyploidy

An allopolyploid combines chromosome sets from different species. A hybrid between two diploid species is usually sterile: its two sets are not homologous, so the chromosomes cannot pair and meiosis produces unbalanced gametes. If the hybrid's chromosome number doubles, however, every chromosome now has a partner, meiosis proceeds normally, and the result is a fertile amphidiploid (also called an allotetraploid). Because it cannot produce fertile offspring with either parent species, it is, in effect, a new species formed in a single generation.

The first deliberately made example was Georgii Karpechenko's 1928 cross of radish (Raphanus, 2n = 18) with cabbage (Brassica, 2n = 18). The F₁ had 9 radish and 9 cabbage chromosomes and was sterile; a rare doubled form with 36 chromosomes was fertile. (Unfortunately for gardeners, it had the root of a cabbage and the leaves of a radish.)

Polyploidy in crops and in animals

Many crops are polyploid, and their histories can be read in their genomes. Bread wheat (Triticum aestivum) arose from two hybridizations: a diploid AA wheat crossed with a BB goat grass to form tetraploid AABB (the ancestor of durum wheat), which later crossed with a third species, DD goat grass, giving hexaploid AABBDD. Plant breeders have made new allopolyploids too, such as triticale, a wheat by rye hybrid doubled with colchicine.

In animals, whole genome duplication occurred twice early in vertebrate evolution, which is why we have four Hox clusters where an insect has one, and again in the ancestors of teleost fish and of salmon. Living polyploid animals include some frogs, lizards and fish, often reproducing asexually. In humans, triploidy (69 chromosomes) occurs in about 1 to 2 percent of conceptions, usually from fertilization of an egg by two sperm, and almost all are miscarried. Many human tissues contain endopolyploid cells, in which DNA has replicated without division: liver cells are often tetraploid, and megakaryocytes reach 16n or more. The giant polytene chromosomes of Drosophila salivary glands are an extreme case, with about 1,000 aligned copies of each chromatid, which makes deletions and inversions directly visible.

Section 5Chromosome mutations in human disease and cancer

How common are chromosome abnormalities?

Chromosome errors are strikingly common at conception and much rarer at birth, because most are lethal. About half of first trimester miscarriages are chromosomally abnormal. Among these, autosomal trisomies make up roughly half (trisomy 16, never seen at birth, is the single most common), followed by 45,X and triploidy. Among liveborn infants, about 1 in 150 (roughly 0.6 percent) has a chromosome abnormality, divided among aneuploidies, balanced rearrangements and unbalanced structural changes.

Microdeletion and microduplication syndromes

Some deletions are too small to see on a routine karyotype (under about 5 Mb) yet remove a group of neighboring genes, causing a recognizable contiguous gene syndrome. Many recur because they are flanked by low copy repeats that promote nonallelic homologous recombination; the same mechanism produces reciprocal duplications. They are diagnosed by FISH or chromosomal microarray.

Chromosome changes in cancer

Cancer cells are genetically unstable, and chromosome mutations are among their most characteristic features. Most solid tumors are aneuploid, often with dozens of numerical and structural changes. But some cancers are driven by one specific rearrangement, and those have taught us how chromosome mutations activate genes. Rearrangements cause cancer in three main ways.

1. Fusion genes. A translocation joins parts of two genes into a new gene encoding a hybrid protein with abnormal activity.
2. Promoter or enhancer swapping. A translocation moves a proto-oncogene next to a strongly active regulatory region, so it is overexpressed even though its protein is normal.
3. Copy number change. Amplification of an oncogene raises its dose, and deletion removes a tumor suppressor gene.

The first consistent cancer rearrangement found was the Philadelphia chromosome, a small chromosome 22 seen in about 95 percent of cases of chronic myeloid leukemia (CML). It results from the reciprocal translocation t(9;22)(q34;q11). The breakpoints fall within the ABL1 gene on 9q34 and the BCR gene on 22q11, and the derivative chromosome 22 carries a BCR-ABL1 fusion gene. Its protein is a tyrosine kinase that is always switched on, driving proliferation of myeloid cells. That discovery led to imatinib, a drug that blocks the ATP binding site of the BCR-ABL1 kinase and transformed CML from a fatal disease into a manageable one.

Amplified genes appear cytologically in two forms: double minutes, small paired acentric chromatin bodies, and homogeneously staining regions, long stretches of a chromosome that lack normal bands because they consist of repeated copies of the amplicon. Some tumors also show chromothripsis, in which one or a few chromosomes shatter into many pieces in a single catastrophic event and are stitched back together in random order.

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