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What you’ll learn to do: Examine karyotypes and identify the effects of significant changes in chromosome number
We previously learned how errors in mitosis can potentially lead to cancer. What could errors in meiosis result in? In this outcome, we’ll learn what happens when errors occur in chromosome number.
- Identify a karyotype and describe its uses in biology
- Identify common errors that can create an abnormal karyotype
- Identify syndromes that result from a significant change in chromosome number
The isolation and microscopic observation of chromosomes forms the basis of cytogenetics and is the primary method by which clinicians detect chromosomal abnormalities in humans. A karyotype is the number and appearance of chromosomes, and includes their length, banding pattern, and centromere position. To obtain a view of an individual’s karyotype, cytologists photograph the chromosomes and then cut and paste each chromosome into a chart, or karyogram, also known as an ideogram (Figure 1). The simplest use of a karyotype (or its karyogram image) is to identify abnormal chromosomal numbers.
In a given species, chromosomes can be identified by their number, size, centromere position, and banding pattern. In a human karyotype, autosomes or “body chromosomes” (all of the non–sex chromosomes) are generally organized in approximate order of size from largest (chromosome 1) to smallest (chromosome 22). The X and Y chromosomes are not autosomes. However, chromosome 21 is actually shorter than chromosome 22. This was discovered after the naming of Down syndrome as trisomy 21, reflecting how this disease results from possessing one extra chromosome 21 (three total). Not wanting to change the name of this important disease, chromosome 21 retained its numbering, despite describing the shortest set of chromosomes. The chromosome “arms” projecting from either end of the centromere may be designated as short or long, depending on their relative lengths. The short arm is abbreviated p (for “petite”), whereas the long arm is abbreviated q (because it follows “p” alphabetically). Each arm is further subdivided and denoted by a number. Using this naming system, locations on chromosomes can be described consistently in the scientific literature.
Although Mendel is referred to as the “father of modern genetics,” he performed his experiments with none of the tools that the geneticists of today routinely employ. One such powerful cytological technique is karyotyping, a method in which traits characterized by chromosomal abnormalities can be identified from a single cell. To observe an individual’s karyotype, a person’s cells (like white blood cells) are first collected from a blood sample or other tissue. In the laboratory, the isolated cells are stimulated to begin actively dividing. A chemical called colchicine is then applied to cells to arrest condensed chromosomes in metaphase. Cells are then made to swell using a hypotonic solution so the chromosomes spread apart. Finally, the sample is preserved in a fixative and applied to a slide.
The geneticist then stains chromosomes with one of several dyes to better visualize the distinct and reproducible banding patterns of each chromosome pair. Following staining, the chromosomes are viewed using bright-field microscopy. A common stain choice is the Giemsa stain. Giemsa staining results in approximately 400–800 bands (of tightly coiled DNA and condensed proteins) arranged along all of the 23 chromosome pairs; an experienced geneticist can identify each band. In addition to the banding patterns, chromosomes are further identified on the basis of size and centromere location. To obtain the classic depiction of the karyotype in which homologous pairs of chromosomes are aligned in numerical order from longest to shortest, the geneticist obtains a digital image, identifies each chromosome, and manually arranges the chromosomes into this pattern (Figure 1).
At its most basic, the karyogram may reveal genetic abnormalities in which an individual has too many or too few chromosomes per cell. Examples of this are Down Syndrome, which is identified by a third copy of chromosome 21, and Turner Syndrome, which is characterized by the presence of only one X chromosome in women instead of the normal two. Geneticists can also identify large deletions or insertions of DNA. For instance, Jacobsen Syndrome—which involves distinctive facial features as well as heart and bleeding defects—is identified by a deletion on chromosome 11. Finally, the karyotype can pinpoint translocations, which occur when a segment of genetic material breaks from one chromosome and reattaches to another chromosome. Translocations are implicated in certain cancers, including chronic myelogenous leukemia.
During Mendel’s lifetime, inheritance was an abstract concept that could only be inferred by performing crosses and observing the traits expressed by offspring. By observing a karyogram, today’s geneticists can actually visualize the chromosomal composition of an individual to confirm or predict genetic abnormalities in offspring, even before birth.
Of all of the chromosomal disorders, abnormalities in chromosome number are the most obviously identifiable from a karyogram. Disorders of chromosome number include the duplication or loss of entire chromosomes, as well as changes in the number of complete sets of chromosomes. They are caused by nondisjunction, which occurs when pairs of homologous chromosomes or sister chromatids fail to separate during meiosis. Misaligned or incomplete synapsis, or a dysfunction of the spindle apparatus that facilitates chromosome migration, can cause nondisjunction. The risk of nondisjunction occurring increases with the age of the parents.
Nondisjunction can occur during either meiosis I or II, with differing results (Figure 2). If homologous chromosomes fail to separate during meiosis I, the result is two gametes that lack that particular chromosome and two gametes with two copies of the chromosome. If sister chromatids fail to separate during meiosis II, the result is one gamete that lacks that chromosome, two normal gametes with one copy of the chromosome, and one gamete with two copies of the chromosome.
Which of the following statements about nondisjunction is true?
- Nondisjunction only results in gametes with n+1 or n–1 chromosomes.
- Nondisjunction occurring during meiosis II results in 50 percent normal gametes.
- Nondisjunction during meiosis I results in 50 percent normal gametes.
- Nondisjunction always results in four different kinds of gametes.
[reveal-answer q=”235653″]Show Answer[/reveal-answer]
[hidden-answer a=”235653″]Answer b is true.[/hidden-answer]
An individual with the appropriate number of chromosomes for their species is called euploid; in humans, euploidy corresponds to 22 pairs of autosomes and one pair of sex chromosomes. An individual with an error in chromosome number is described as aneuploid, a term that includes monosomy (loss of one chromosome) or trisomy (gain of an extraneous chromosome). Monosomic human zygotes missing any one copy of an autosome invariably fail to develop to birth because they lack essential genes. This underscores the importance of “gene dosage” in humans. Most autosomal trisomies also fail to develop to birth; however, duplications of some of the smaller chromosomes (13, 15, 18, 21, or 22) can result in offspring that survive for several weeks to many years. Trisomic individuals suffer from a different type of genetic imbalance: an excess in gene dose. Individuals with an extra chromosome may synthesize an abundance of the gene products encoded by that chromosome. This extra dose (150 percent) of specific genes can lead to a number of functional challenges and often precludes development. The most common trisomy among viable births is that of chromosome 21, which corresponds to Down Syndrome. Individuals with this inherited disorder are characterized by short stature and stunted digits, facial distinctions that include a broad skull and large tongue, and significant developmental delays. The incidence of Down syndrome is correlated with maternal age; older women are more likely to become pregnant with fetuses carrying the trisomy 21 genotype (Figure 3).
An individual with more than the correct number of chromosome sets (two for diploid species) is called polyploid. For instance, fertilization of an abnormal diploid egg with a normal haploid sperm would yield a triploid zygote. Polyploid animals are extremely rare, with only a few examples among the flatworms, crustaceans, amphibians, fish, and lizards. Polyploid animals are sterile because meiosis cannot proceed normally and instead produces mostly aneuploid daughter cells that cannot yield viable zygotes. Rarely, polyploid animals can reproduce asexually by haplodiploidy, in which an unfertilized egg divides mitotically to produce offspring. In contrast, polyploidy is very common in the plant kingdom, and polyploid plants tend to be larger and more robust than euploids of their species (Figure 4).
Sex Chromosome Nondisjunction in Humans
Humans display dramatic deleterious effects with autosomal trisomies and monosomies. Therefore, it may seem counterintuitive that human females and males can function normally, despite carrying different numbers of the X chromosome. Rather than a gain or loss of autosomes, variations in the number of sex chromosomes are associated with relatively mild effects. In part, this occurs because of a molecular process called X inactivation. Early in development, when female mammalian embryos consist of just a few thousand cells (relative to trillions in the newborn), one X chromosome in each cell inactivates by tightly condensing into a quiescent (dormant) structure called a Barr body. The chance that an X chromosome (maternally or paternally derived) is inactivated in each cell is random, but once the inactivation occurs, all cells derived from that one will have the same inactive X chromosome or Barr body. By this process, females compensate for their double genetic dose of X chromosome.
In so-called “tortoiseshell” cats, embryonic X inactivation is observed as color variegation (Figure 5). Females that are heterozygous for an X-linked coat color gene will express one of two different coat colors over different regions of their body, corresponding to whichever X chromosome is inactivated in the embryonic cell progenitor of that region.
An individual carrying an abnormal number of X chromosomes will inactivate all but one X chromosome in each of her cells. However, even inactivated X chromosomes continue to express a few genes, and X chromosomes must reactivate for the proper maturation of female ovaries. As a result, X-chromosomal abnormalities are typically associated with mild mental and physical defects, as well as sterility. If the X chromosome is absent altogether, the individual will not develop in utero.
Several errors in sex chromosome number have been characterized. Individuals with three X chromosomes, called triplo-X, are phenotypically female but express developmental delays and reduced fertility. The XXY genotype, corresponding to one type of Klinefelter syndrome, corresponds to phenotypically male individuals with small testes, enlarged breasts, and reduced body hair. More complex types of Klinefelter syndrome exist in which the individual has as many as five X chromosomes. In all types, every X chromosome except one undergoes inactivation to compensate for the excess genetic dosage. This can be seen as several Barr bodies in each cell nucleus. Turner syndrome, characterized as an X0 genotype (i.e., only a single sex chromosome), corresponds to a phenotypically female individual with short stature, webbed skin in the neck region, hearing and cardiac impairments, and sterility.
Duplications and Deletions
In addition to the loss or gain of an entire chromosome, a chromosomal segment may be duplicated or lost. Duplications and deletions often produce offspring that survive but exhibit physical and mental abnormalities. Duplicated chromosomal segments may fuse to existing chromosomes or may be free in the nucleus. Cri-du-chat (from the French for “cry of the cat”) is a syndrome associated with nervous system abnormalities and identifiable physical features that result from a deletion of most of 5p (the small arm of chromosome 5) (Figure 6). Infants with this genotype emit a characteristic high-pitched cry on which the disorder’s name is based.
Check Your Understanding
Answer the question(s) below to see how well you understand the topics covered in the previous section. This short quiz does not count toward your grade in the class, and you can retake it an unlimited number of times.
Use this quiz to check your understanding and decide whether to (1) study the previous section further or (2) move on to the next section.
Gamete binning: chromosome-level and haplotype-resolved genome assembly enabled by high-throughput single-cell sequencing of gamete genomes
Generating chromosome-level, haplotype-resolved assemblies of heterozygous genomes remains challenging. To address this, we developed gamete binning, a method based on single-cell sequencing of haploid gametes enabling separation of the whole-genome sequencing reads into haplotype-specific reads sets. After assembling the reads of each haplotype, the contigs are scaffolded to chromosome level using a genetic map derived from the gametes. We assemble the two genomes of a diploid apricot tree based on whole-genome sequencing of 445 individual pollen grains. The two haplotype assemblies (N50: 25.5 and 25.8 Mb) feature a haplotyping precision of greater than 99% and are accurately scaffolded to chromosome-level.
Numerical aberrations are generally caused by a failure in chromosome division during meiosis that results in gametic cells with an extra chromosome or a deficiency in the number of chromosomes. Variation in chromosome number involves
1) addition or loss of one or more chromosomes (Aneuploidy)
2) addition or loss of one or more haploid sets of chromosomes (Euploidy)
1- Aneuploidy ( Greek, aneu= uneven, ploids= units)
When an organism gains or loses one or more chromosomes, but not a complete set, this condition is called aneuploidy. It leads to the variation in the number of chromosomes but not involves the whole set of chromosomes. The nuclei of aneuploids contain chromosomes whose number is not the true multiple of basic number (n).
Examples: are Down syndrome (which has 47 chromosomes instead of 46) and Turner syndrome (45 chromosomes instead of 46). Here the number of chromosomes in the individual is not a true multiple of basic number n (n=23).
Type of aneuploidy
i. Monosomy: The loss of one chromosome produces a monosomic (2n-1) and the condition is known as monosomy. Its example is Turner syndrome (2n-1 = 45 chromosomes in humans)
ii. Trisomy: The gain of one extra chromosome produces trisomic (2n+1) and the condition is called trisomy. Example are
- Down syndrome (2n+1= 47 chromosomes in humans).
- Trisomy 18 (Edwards syndrome) have an additional copy of chromosome 18
- Trisomy 13 (Patau syndrome) have an additional copy of chromosome 13
- Trisomy 8 (Warkany syndrome 2) have an additional copy of chromosome 8
iii. Tetrasomy: The gain of two extra chromosomes produces tetrasomic (2n+2) individuals and the condition is called Tetrasomy. Examples are
- XXXY syndrome (Klinefelter’s syndrome)
- XXXX syndrome ( 48 chromosomes)
- XXYY syndrome ( 48 chromosomes)
iv. Pentasomy: The gain of three extra chromosomes produces pentasomic (2n+3) individuals and the condition is called Pentasomy.
Penta X Syndrome: (49, XXXXX), female has five X chromosomes instead of the normal two. Signs are intellectual disability, short height, less muscle tone, and delay in development.
V. Nullisomy: It is a condition in which a pair of homologous chromosomes is completely lost. ( 2n-2). Humans with this disorder will not survive.
Example 2 : About 21 nullisomics of the allohexaploid Triticum aestivum have been made, they differ in appearance from normal hexaploids and are have less vigor.
2- Euploidy ( Greek, ae= even or true, ploids= units)
When one or more complete haploid set of chromosomes are involved in the aberration, the resulting abnormality is called Euploidy. It is more tolerated in plants rather than animals. For example, if there is a human cell that has an extra set of 23 chromosomes it will have Euploidy.
Types of Euploidy
Ploidy refers to the number of homologous sets of chromosomes in the genome of a cell or an organism. Each set is designated by n.
Monoploidy: The state of having a single set of chromosomes is called monoploidy and is represented by 1n. The cell or organism with a single set of chromosomes is called a monoploid. Monoploidy is lethal in animals but in the case of plant species, this can be more tolerated.
In most animal species this could mean death but there are few animal species where monoploidy is a normal part of the life cycle, such as male wasps, ants, and bees. The offsprings that have arisen from monoploidy are those that have developed from unfertilized eggs.
The condition in which a normally diploid cell or organism acquires one or more additional sets of chromosomes is called polyploidy. In other words, the polyploid cell or organism has three or more times the number of haploid chromosomes. Polyploidy arises as a result of the total nondisjunction of the chromosomes during mitosis or meiosis.
Polyploidy in plants:
Polyploidy is common among plants and has, in fact, been an important source of speciation in angiosperms. Particularly important is allopolyploidy, which involves the duplication of chromosomes in a hybrid plant.
Typically, a diploid hybrid is sterile because it does not have the homologous chromosome pairs necessary for successful gamete formation during meiosis. However, in the case of tetra polyploids, the plant duplicates the set of chromosomes inherited from each parent, meiosis can occur, because each chromosome will have a homolog derived from its duplicate set. Thus, Tetrapolyploidy confers fertility on the previously sterile hybrid, which therefore attains the status of a complete species distinct from either parent.
Up to half of the known angiosperm species have been estimated to have arisen through polyploidy, including some of the species most prized by man. Plant breeders use this process, treating desirable hybrids with chemicals, such as colchicine, which are known to induce polyploidy.
Polyploid animals are much less common, and the process appears to have had little effect on animal speciation.
B- Structural aberrations
These occur due to a loss of genetic material, or a reorganization in the location of the genetic material. They include deletions, duplications, inversions, ring formations, and translocations.
Unbalanced rearrangements include deletions, duplications, or insertions of a chromosomal segment. Balanced rearrangements include inverted or translocated chromosomal regions. Since the full complement of DNA material is still present, balanced chromosomal rearrangements can go unnoticed because they may not cause disease. 3
The disease can arise as a result of a balanced rearrangement if breaks in the chromosomes occur in one gene, resulting in a missing or non-functional protein, or if the fusion of chromosomal segments results in a hybrid of two genes, producing a new protein product whose function is detrimental to the cell. 3
Types of structural chromosomal aberrations
Types of structural aberrations
Deletions: A part of the chromosome is missing or removed. Known disorders include Wolf-Hirschhorn syndrome, which is caused by partial removal of the short arm from chromosome 4 and Jacobsen syndrome, also called 11q terminal deletion disorder, caused by the terminal removal of the q arm of chromosome 11.
Duplications: A part of the chromosome is duplicated, resulting in additional genetic material. Known disorders include Charcot-Marie-Tooth disease type 1A, which can be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17.
Translocations: When a part of a chromosome is transferred to another chromosome. There are two main types of translocations.
- In a reciprocal translocation, segments of two different chromosomes have been exchanged.
- In a Robertsonian translocation, one complete chromosome has joined another in the centromere These only occur with chromosomes 13, 14, 15, 21, and 22.
Inversions: a part of the chromosome has been broken, upside down, and reattached, therefore, the genetic material is inverted.
Insertions: A portion of one chromosome has been deleted from its normal place and inserted into another chromosome.
Rings: Ring chromosomes can result when one chromosome undergoes two breaks and the broken ends are fused into a circular chromosome. This can happen with or without loss of genetic material.
Isochromosome: An isochromosome can form when one arm of the chromosome is missing and the remaining arm is duplicated.
How chromosomal aberrations or abnormalities occur?
Chromosomal abnormalities can occur as an accident when the egg or sperm forms or during the early stages of fetus development. The mother’s age and certain environmental factors may play a role in the occurrence of genetic errors.
Most chromosomal abnormalities occur as an accident in the egg or sperm and are therefore not inherited. The abnormality is present in all cells of the body however, some abnormalities can occur after conception, resulting in mosaicism (where some cells have the abnormality and others do not).
Prenatal exams and tests can be done to examine the fetus’s chromosomes and detect some, but not all, types of chromosomal abnormalities.
Interphase chromosome positioning in proliferating and nonproliferating cells
To determine the nuclear location of specific chromosomes, human dermal fibroblasts (HDFs) were harvested and fixed for standard 2D-fluorescence in situ hybridization (FISH). Representative images of chromosome territories in proliferating cells are displayed in Figure 1a-d. Digital images were subjected to erosion analysis [4–6, 8, 9], whereby the images of 4',6-diamidino-2-phenylindole (DAPI)-stained flattened nuclei are divided into five concentric shells of equal area, and the intensity of the DAPI signal and probe signal is measured in each shell. The chromosome signal is then normalized by dividing it by the percentage of DAPI signal. The data for each chromosome are then plotted as a histogram with error bars, with the x-axis displaying the nuclear shells from 1 to 5, representing the nuclear periphery to the nuclear interior, respectively (Figure 1e-h).
Chromosome positioning in proliferating interphase nuclei. Proliferating human dermal fibroblasts (HDFs) cultures were subjected to 2D- or 3D-fluorescence in situ hybridization (FISH) to delineate and analyze the nuclear location of chromosomes 10, 13, 18, and X. In panels (a-d), the chromosome territories are revealed in green with a single chromosome territory for chromosome X, because the HDFs are male in origin. The red antibody staining is the nuclear distribution of the proliferative marker anti-pKi-67, the presence of which denotes a cell in the proliferative cell cycle. DAPI (4',6-diamidino-2-phenylindole) in blue is a DNA intercalator dye and reveals the nuclear DNA. Scale bar = 10 μm. The histograms in panels (e-h) display the distribution of the chromosome signal in 50 to 70 nuclei for each chromosome for 2D FISH, as analyzed with erosion analysis. This analysis divides each nucleus into five concentric shells of equal area, with shell 1 being the most peripheral shell, and shell 5 being the most interior shell [4–6, 9]. The percentage of chromosome signal measured in each shell was divided by the percentage of DAPI signal in that shell. Bars represent the mean normalized proportion (percentage) of chromosome signal for each human chromosome. Error bars represent the standard error of the mean (SEM). Panels i and j display 3D projections of 0.2-μm optical sections through 3D preserved nuclei subjected to 3D-FISH and imaged with confocal laser scanning microscopy. The chromosome territories are displayed in red, and proliferating cells also were selected with positive anti-pKi-67 staining (not shown in reconstruction). Scale bar = 10 μm. The line graph in panel (k) displays a frequency distribution of micrometers from the geometric center of the chromosome territories to the nearest nuclear periphery, as defined by DAPI staining. Images for 20 nuclei were analyzed.
In young proliferating fibroblasts, interphase chromosomes are positioned nonrandomly in a radial pattern within nuclei . In our 2D studies, we consistently found gene-poor chromosomes, such as chromosomes X, 13, and 18, located at the nuclear periphery [5, 9], which fits with their having more lamina-associated domains than gene-poor chromosomes (see ). In this study, we recapitulated the interphase chromosome positioning with our present cultures and demonstrated that these chromosomes are located at the nuclear periphery in young proliferating cells (Figure 1b-d, f-h). Proliferating cells within the primary cultures were identified by using the proliferative marker, anti-pKi-67, which is distributed in a number of different patterns within proliferating human fibroblasts . Its distribution is mainly nucleolar and is shown in red (Figure 1a-d). Figure 1a and e demonstrate the nuclear location of chromosome 10, unlike chromosomes 13, 18, and X it is found in an intermediate position in proliferating fibroblasts. The relative interphase positions of chromosomes 10 and X have been confirmed in 3D-FISH analyses (Figure 1i-k), whereby HDFs were fixed to preserve their three-dimensionality with 4% paraformaldehyde and subjected to 3D-FISH . Measurements in micrometers from the geometric center the chromosome territories to the nearest nuclear periphery, as determined by the DAPI staining, were taken in at least 20 nuclei. The data were not normalized for size measurements, so that actual measurements in micrometers can be seen. However, all data were normalized by a size measurement, and this not does alter the relative positioning of the chromosomes.
We have evidence from prior studies that chromosomes such as chromosomes 13  and 18 [5, 9] alter their nuclear position when primary fibroblasts exit the proliferative cell cycle and that chromosome X remains at the nuclear periphery . However, this is only two chromosomes of 24, and so to determine which other chromosomes reposition after cell-cycle exit into quiescence (G0), elicited through serum removal, we positioned all human chromosomes in G0 cells (Figures 2 and 3).
Chromosome positioning in quiescent interphase nuclei. Representative images displaying nuclei prepared for fluorescence in situ hybridization (2D-FISH), with whole-chromosome painting probes (green), and nuclear DNA is counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). The cells were subjected to indirect immunofluorescence with anti-pKi-67 antibodies, and negative cells were selected. Cells were placed in low serum (0.5%) for 7 days, before fixation with methanol:acetic acid (3:1). The numbers (or letters) on the left side of each panel indicate the chromosome that has been hybridized. Scale bar = 10 μm.
Analysis of radial chromosome positioning in quiescent cell nuclei. Histograms displaying chromosome positions in primary human quiescent fibroblast nuclei. The 50 to 70 nuclei per chromosome were subjected to erosion analysis, which divides each nucleus into five concentric shells of equal area, with shell 1 being the most peripheral shell, and shell 5 being the most interior shell [4–6, 9]. The percentage of chromosome signal measured in each shell was divided by the percentage of 4',6-diamidino-2-phenylindole (DAPI) signal in that shell. Bars represent the mean normalized proportion (percentage) of chromosome signal for each human chromosome. Error bars represent the standard error of mean (SEM).
To make cells quiescent, young, HDFs were grown in 10% NCS for 48 hours, and then the cells were washed twice with serum-free medium and placed in 0.5% NCS medium for 168 hours (7 days). However, when the positioning analysis was performed on the quiescent nuclei, we found that certain chromosomes were in very different positions from those in which they were found in proliferating nuclei, that is, chromosomes 1, 6, 8, 10, 11, 12, 13, 15, 18, and 20 (Table 1).
The data demonstrated in Figure 3 and Table 1 reveal that a number of chromosomes alter their nuclear positions when cells become quiescent as shown before, both chromosomes 13 and 18 move from a peripheral nuclear location to an interior location (Figure 3m and r). Chromosome 10 is one of a number of chromosomes that move from an intermediate nuclear location to the nuclear periphery (Figure 3j, Table 1), whereas chromosome X does not change its location at the nuclear periphery (Figure 3w), and chromosomes such as 17 and 19 do not change their interior location (Figure 3q and s, respectively).
It certainly appears that the chromosome positioning in quiescent G0 cells is correlated with size. However, it is not clear why a repositioning of chromosomes occurs after serum removal and when and how it is elicited.
The movement of chromosomes when normal fibroblasts exit the cell cycle is rapid, active, and requires myosin and actin
To determine when the genome is reorganized on exit from the cell cycle and the speed of the response to the removal of growth factors, we took actively proliferating young cultures of primary HDFs and replaced 10% NCS medium with 0.5% NCS medium. Samples were taken at 0, 5, 10, 15, and 30 minutes after serum starvation for fixation, and chromosome position in interphase was determined with 2D-FISH and erosion analysis (Figure 4 and Additional file 1). Chromosomes 13 and 18 relocated from the nuclear periphery to the nuclear interior within 15 minutes (Figure 4h and l), with an intermediate-type nuclear positioning visible in the intervening time points (5 and 10 minutes Figure 4f, g, j, and k). In addition, chromosome 10 moved from an intermediate location to a peripheral location in the same time window (15 minutes Figure 4d). Chromosome X did not relocalize at all, as was reported previously  (Figure 4m-o), apart from some slight difference at 15 minutes (Figure 4p).
Rapid repositioning of chromosomes after removal of serum. Chromosomes move rapidly in proliferating cells placed in low serum. The nuclear locations of human chromosomes 10 (a-d), 13 (e-h), 18 (i-l), and X (m-p) were analyzed in normal fibroblast cell nuclei fixed for 2D-FISH (fluorescence in situ hybridization) after incubation in medium containing low serum (0.5%) for 0, 5, 10, and 15 minutes. The filled-in squares indicate significance difference (P < 0.05) when compared with control proliferating fibroblast cell nuclei.
In a previous study, we demonstrated that relocation of chromosome 18 from the nuclear interior in G0 cells to the nuclear periphery in serum-restimulated cells took 30+ hours and appeared to require cells to rebuild their nuclear architecture after a mitotic division . We showed here that the same is true for chromosome 10, with a return to an intermediate nuclear location 24 to 36 hours after restimulation of G0 cells with 10% NCS (Figure 5d-f). We again showed that chromosome 18 requires similar times to return to the nuclear periphery (that is, 36 hours Figure 5l). Although chromosome X remains at the nuclear periphery, a slight change in the distribution of chromosome X occurs at 32 to 36 hours (Figure 5q-r). From these data, it seems that a rapid response to the removal of growth factors reorganizes the whole genome within the interphase nucleus, and this reorganization is corrected in proliferating cells only after 24+ hours in high serum, presumably after the cells have passed through mitosis, as indicated by the peak of mitotic cells at 24 to 36 hours after serum restimulation (0 hours, none 8 hours, none 24 hours, 0.3% 32 hours, 2.6% and 36 hours, 1.2%).
Restoration of proliferative chromosome position after restimulation of G 0 cells. The relocation of chromosomes to their proliferative nuclear location takes 24+ hours for chromosome 10 and 36 hours for chromosome 18. Proliferating cells (a, g, m) were placed in low serum (0.5%) for 7 days (b, h, n) and then restimulated to enter the proliferative cell cycle with the readdition of high serum. Samples were taken at 8 hours (c, i, o), 24 hours (d, j, p), 32 hours (e, k, q), and 36 hours (f, l, r) after restimulation. The graphs display the normalized distribution of chromosome signal in each of the five shells. Shell 1 is the nuclear periphery, and shell 5 is the innermost region of the nucleus. The solid squares represent a significant difference (P < 0.05) for that shell when compared with the equivalent shell for the time 0 data (G0 data) for the erosion analysis.
Such rapid movement of large regions of the genome in response to low serum implies an active process, perhaps requiring ATP/GTP. When inhibitors of ATPase and/or GTPase, ouabain, and AG10, were incubated with proliferating cell cultures in combination with low serum, chromosome 10 did not change nuclear location (Figure 6a-d, and see Additional file 3). The relocation to the nuclear interior of chromosome 18 territories after incubation of cells in low serum also was perturbed by these inhibitors (Figure 6a-d). The control chromosome, chromosome X, remained at the nuclear periphery (Figure 6 and Additional file 3). Because other studies suggest that nuclear motors move genomic regions around the nucleus by actin and/or myosin [42, 44] we decided to use inhibitors of actin and myosin polymerization to attempt to block any chromosome movement elicited by these nuclear motors when serum was removed. Latrunculin A, an inhibitor of actin polymerization, inhibited the movement of both chromosomes 10 and 18 when cells were placed in low serum (Figure 7a and Additional file 3). In contrast, phalloidin oleate, another inhibitor of actin polymerization did not prevent relocalization of either chromosome 10 or 18, when cells were placed in low serum (Figure 7b and Additional file 3). However, two inhibitors of myosin polymerization (BDM) and function (Jasplakinolide also affects actin polymerization) did inhibit movements of both these chromosomes upon serum removal (Figure 7c, d, and Additional file 3). Figure 7e provides a comparison for the rapid change in chromosome position when no inhibitors are used. These data imply that rapid chromosome movement observed in cells as they respond to removal of growth factors is due to an energy-driven process involving a nuclear actin:myosin motor function.
Chromosome repositioning requires energy. The relocation of human chromosomes 10 and 18 after incubation in low serum is energy dependent. The nuclear location of human chromosomes 10, 18, and X in were determined in normal human proliferating cell nuclei treated with ouabain (ATPase inhibitor) (a), AG10 (GTPase inhibitor) (b), or a combination of both (c) before and during incubation in low serum for 15 minutes. Normal control analysis data without any treatment is displayed in (d). The error bars show the standard error of the mean. The stars indicate a significant difference (P < 0.05) from cells treated only with the inhibitor.
Chromosome repositioning requires nuclear myosin and actin. The relocation of human chromosomes 10 and 18 after incubation in low serum is myosin and actin dependent. The nuclear locations of chromosomes 10, 18, and X were determined in normal human proliferating cell nuclei treated with latrunculin A and phalloidin oleate (inhibitors of actin polymerisation) (a, b) and BDM and jasplakinolide (inhibitors of myosin polymerization) (c, d) before and during incubation in low serum for 15 minutes. The error bars show the standard error of the mean. The stars indicate a significant difference (P < 0.05) from cells treated only with the inhibitor. Normal control analysis data without any treatment is displayed in (e).
Nuclear myosin 1β is required for chromosome territory repositioning in HDFs placed in low serum
In an effort to elucidate which myosin isoform was involved in chromosome movement after serum removal in culture, we used suppression by RNA interference with small interfering RNAs (siRNAs). An siRNA pool for the gene MYO1C was selected because it encodes for a cytoplasmic myosin 1C and the nuclear isoform nuclear myosin 1β, a major candidate myosin for chromatin relocation [39, 49]. mRNA analysis had revealed insufficient differences in sequence for suppression of myosin 1β alone (data not shown). With a double transfection of the siRNA, we observed 93% of cells displaying no nuclear myosin staining at all (Figure 8k, q, and s) but still with some cytoplasmic staining, whereas in the control cells and the cells transfected with the control construct, >95% of cells displayed a nuclear distribution of anti-nuclear myosin 1β, which was distributed in proliferating cells as accumulations at the inner nuclear envelope, the nucleoli, and throughout the cytoplasm (Figure 8g-j, m-p). These numbers did not change significantly after serum removal for 15 minutes, as per the chromosome-movement assay (data not shown).
Suppression of nuclear myosin expression by short interference RNAs (siRNAs). Normal human dermal fibroblasts (HDFs) were transfected with negative control or MYO1C targeting siRNA (double transfection) and samples for immunofluorescence staining and 2D-FISH (fluorescence in situ hybridization) were fixed 48 hours after the final transfection. Representative images of nuclei stained for anti-NMIβ (red) in control (g, h, m, n) cells transfected with negative control siRNA (i, j, o, p) and in cells transfected with MYO1C siRNA (k, l, q, r) after 0 and 15 minutes of serum starvation are displayed. The percentage of nuclei that are positive for NM1β in controls, in cells transfected with negative control siRNA, and in cells transfected with MYO1C siRNA are displayed in the adjacent table (s).
After siRNA suppression of nuclear myosin, the chromosome-movement assay was repeated by placing the double-transfected cells into low serum for 15 minutes. The graphs in Figure 9 show that chromosomes 10, 18, and X behave as expected after removal of serum in the control cells (Figure 9a-f) and in the cells transfected with the control construct (Figure 9g-l), with chromosome 10 becoming more peripheral, chromosome 18 becoming more interior, and chromosome X remaining at the nuclear periphery. However, in the cells that had been transfected with MYO1C-targeting siRNA, chromosome movement was much less dramatic, with the chromosomes still residing in similar nuclear compartments before and after the serum removal (Figure 9m-r).
Chromosome repositioning is inhibited by short interference RNA (siRNA) that suppresses nuclear myosin1β. Chromosome positioning was determined with 2D-FISH (fluorescence in situ hybridization) and erosion analysis, and the normalized position data plotted as histograms in control cells, in cells transfected with the negative control, and in cells transfected with the MYO1C siRNA construct. In control human dermal fibroblasts (HDFs) and in HDFs transfected with negative control, siRNA chromosome 10 is repositioned from an intermediate nuclear location (a and g, respectively) to the nuclear periphery (d, j) after 15 minutes of incubation in low serum. Chromosome 18 territories, conversely, are repositioned from the nuclear periphery (b, h) to the nuclear interior (e, k) after 15 minutes of incubation in low serum in control HDFs and in HDFs transfected with negative control siRNA. In HDFs transfected with the MYO1C siRNA construct, chromosomes 10 (m, p) and 18 (n, q) do not show repositioning after 15 minutes of incubation in low serum. Chromosome X remains at the nuclear periphery at all times (c, f, i, l, o, r). Unpaired, unequal variances two-tailed Students t tests were performed to assess statistical differences. The solid squares indicate a significant difference (P < 0.05) from cells not incubated in, and the solid circles indicate a significant difference (P < 0.05) from control HDFs.
The distribution of the nuclear myosin 1β is very interesting in these cells, because it gives a nuclear envelope distribution, a nucleolar distribution, and a nucleoplasmic distribution (Figure 10a-c). These distributions, although revealing, are not so surprising, because nuclear myosin has a binding affinity for the integral nuclear membrane protein emerin  and is involved in RNA polymerase I transcription [37, 40, 51]. The distribution in quiescent cells is quite different, with large aggregates of NM1β within the nucleoplasm and is missing from the nuclear envelope and nucleoli. This distribution is similar to that observed in senescent human dermal fibroblasts (Mehta, Kill, and Bridger, unpublished data). With respect to chromosome movement back to a proliferating position after incubation in low serum, we showed that it does not occur until 24 to 36 hours after repeated addition of serum to a quiescent culture (Figure 5) . Correlating with this is the rebuilding of daughter nuclei after mitosis and the return of a proliferating distribution of NM1β to the nuclear envelope, nucleoli, and nucleoplasm (Figure 10g, j, p).
Anti-nuclear myosin 1b (NM1β) staining patterns in proliferating cells, quiescent cells, and after restimulation. Normal 2DD human dermal fibroblasts (HDFs) were serum starved for 7 days to induce quiescence. The cells were then restimulated with fresh serum, and samples were collected at 0, 24, 36 and 48 hours after serum restoration. Samples were also collected before serum withdrawal (proliferating cells). The samples were then fixed with methanol/acetone (1:1), and the distribution of NMIβ was assessed by performing an indirect immunofluorescence assay for NMIβ. Images in (a, c) display the distribution of NMIβ in proliferating cells, whereas those in (d and f) show the distribution of NMIβ after 0, 24, 36 and 48 hours after restimulation of quiescent fibroblasts. The table (p) displays the percentage of cells displaying various patterns of NMIβ staining after restimulation. Error is indicated by standard deviation. Scale bar = 10 μm.
The chromEvol software implements a variety of models depicting the evolution of chromosome numbers thereby providing users with a statistical tool to elucidate the pattern of chromosome number change along a phylogeny. In the new version of the program presented here, the base number of a group of interest can be inferred in a statistical framework. The concept of the base number is regularly taken to represent “the haploid number present in the initial population of a monophyletic clade” ( Guerra 2008). Because this ancestral number is frequently, although not always, equal to the monoploid number of the polyploid series observed in the group, the two terms have been used somewhat intermingly. In chromEvol, we explicitly separated these two concepts. Although the ancestral root number can be computationally inferred or be set by the user, the addition by any multiplication of the monoploid number is now an integral part of the model using the introduced β parameter ( eq. 2). Several complexities should be noted regarding this parameter. First, although a single monoploid number may define a large clade, it is also possible that, due to dysploidy, each subclade in an analyzed phylogeny will exhibit a unique polyploid series with the respective unique monoploid number ( Guerra 2008). Here, we treated the monoploid number as a single possible value. Allowing for several distinct monoploid numbers can easily be integrated into our models but will come at the expense of additional free parameters that may be warrant only when large clades are considered. Second, in the current implementation, we treated additions by any multiplier of β as equally likely. Other scenarios whereby, for example, single β additions are more likely than higher multiplications can be integrated into the model (but again, increasing model complexity). Finally, although the β parameter was initially included in our models as a means to infer the base number of the group examined, we note that practically its optimized value is not necessarily so, particularly when additional parameters modeling other polyploidization events (i.e., ρ and μ) are considered. For example, in the Hordeum data set the best-supported model was M10 with β = 14 and a high rate of WGDs ( table 1). Notably, most duplications involved transitions from 7 to 14 via the ρ parameter. Although seven is an obvious base number of this genus, the β parameter allowed for several 7 → 21 transitions, which otherwise must have been explained by multiple transitions.
The chromEvol program allows users to estimate the branches in which ploidy transitions most probably occurred. However, these estimates cannot trivially be used to determine which lineages are polyploid and which are diploid. To this end, we introduced a simulation-based approach to determine the ploidy levels of extant taxa. This approach can further be used to pinpoint the lineages in which ploidy estimates are deemed unreliable based on the current chromosome number distribution. Importantly, in the current implementation, chromEvol ignores possible association between transitions in chromosome numbers and transitions in ploidy levels. For example, it is possible that due to diploidization processes ( Wolfe 2001), the rate of descending dysploidy will be higher in a polyploid compared with a diploid background. Furthermore, polyploidy is known to have a profound impact on rates of diversification ( Fawcett et al. 2009 Soltis et al. 2009 Mayrose et al. 2011), which could confound estimation of chromosome number transition rates and ancestral state reconstruction ( Maddison 2006 Goldberg and Igic 2008). One may envision a covarion-like process ( Galtier 2001), in which the evolution of chromosome numbers and ploidy levels is jointly modeled. Accordingly, rather than assuming a constant pattern of chromosome number change across the phylogeny, different lineages may evolve under different evolutionary patterns, dictated by their ploidy levels. Such a combined model could also be integrated within a Bayesian framework (i.e., by using a Markov chain Monte-Carlo sampling strategy Hastings 1970), thereby accounting for uncertainty in parameter estimation and phylogeny reconstruction. Using this formulation, chromEvol may further be extended to allow chromosome number or ploidy levels to influence rates of speciation and extinction under the Binary State Speciation and Extinction (BiSSE) framework ( Maddison et al. 2007). Such possible extensions will come at the expense of additional free parameters and modeling complexities that may only be justified when large trees are considered. However, such large trees may be particularly unrealistic for the time-homogeneity assumption (i.e., a single transition matrix across the whole phylogeny). Certainly, exploring the association between patterns of chromosome number change and ploidy levels as well as the range of complexities that can be explored using the chromEvol model are important future directions.
Errors at the start of life
Only one in three fertilizations leads to a successful pregnancy. Many embryos fail to progress beyond early development. Cell biologists at the Max Planck Institute (MPI) for Biophysical Chemistry in Göttingen (Germany), together with researchers at the Institute of Farm Animal Genetics in Mariensee and other international colleagues, have now developed a new model system for studying early embryonic development. With the help of this system, they discovered that errors often occur when the genetic material from each parent combines immediately after fertilization. This is due to a remarkably inefficient process.
Human somatic cells typically have 46 chromosomes, which together carry the genetic information. These chromosomes are first brought together at fertilization, 23 from the father's sperm, and 23 from the mother's egg. After fertilization, the parental chromosomes initially exist in two separate compartments, known as pronuclei. These pronuclei slowly move towards each other until they come into contact. The pronuclear envelopes then dissolve, and the parental chromosomes unite.
The majority of human embryos, however, end up with an incorrect number of chromosomes. These embryos are often not viable, making erroneous genome unification a leading cause of miscarriage and infertility.
"About 10 to 20 percent of embryos that have an incorrect number of chromosomes result from the egg already containing too few or too many chromosomes prior to fertilization. This we already knew," explains Melina Schuh, director at the MPI for Biophysical Chemistry. "But how does this problem arise in so many more embryos? The time immediately after the sperm and egg unite -- the so-called zygote stage -- seemed to be an extremely critical phase for the embryo's development. We wanted to find out why this is the case."
Insights from a new model system
For their investigations, the scientists analyzed microscopy videos of human zygotes that had been recorded by a laboratory in England. They additionally set out to find a new model organism suitable for studying early embryonic development in detail. "Together with our collaboration partners at the Institute of Farm Animal Genetics, we developed methods for studying live bovine embryos, which closely resemble human embryos," explains Tommaso Cavazza, a scientist in Schuh's department. "The timing of the first cell divisions is comparable in human and bovine embryos. Furthermore, the frequency of chromosomes distributing incorrectly is about the same in both systems." Another advantage of this model system is: The scientists obtained the eggs from which the bovine embryos developed from slaughterhouse waste, so no additional animals had to be sacrificed.
Schuh's team fertilized the bovine eggs in vitro and then used live-cell microscopy to track how the parental genetic material unites. They found that the parental chromosomes cluster at the interface between the two pronuclei. In some zygotes, however, the researchers noticed that individual chromosomes failed to do so. As a result, these chromosomes were 'lost' when the parental genomes united, leaving the resulting nuclei with too few chromosomes. These zygotes soon showed developmental defects.
"The clustering of chromosomes at the pronuclear interface seems to be an extremely important step," Cavazza explains. "If clustering fails, the zygotes often make errors that are incompatible with healthy embryo development."
Dependent on an inefficient process
But why do parental chromosomes often fail to cluster correctly? The Max Planck researchers were able to uncover that as well, as Cavazza reports: "Components of the cytoskeleton and the nuclear envelope control chromosome movement within the pronuclei. Intriguingly, these elements also steer the two pronuclei towards each other. So we are dealing with two closely linked processes that are essential, but often go wrong. Thus, whether an embryo will develop healthily or not depends on a remarkably inefficient process."
The scientists' findings are also relevant for in vitro fertilization in humans. It has been discussed for some time whether the accumulation of the so-called nucleoli at the pronuclear interface in human zygotes could be used as an indicator for the chance of successful fertilization. Zygotes in which these pronuclear components all cluster at the interface have a better chance of developing successfully, and could therefore be preferentially used for fertility treatment. "Our observation that chromosomes need to cluster at the interface to guarantee healthy embryo development supports this selection criterion," Schuh says.
What happens if you have too many chromosomes?
If one of the original cells had an extra chromosome, the person will have trisomy. This is a type of aneuploidy. People with trisomies have three copies of a particular chromosome (instead of two). This means these individuals have a total of 47 chromosomes (n+1).
Trisomies are named after the chromosome pair that gets the extra chromosome. Trisomy 21 is a fairly common aneuploidy that involves an extra chromosome 21. This is also called Down syndrome. It affects about one in every 750 babies born in Canada. Children with Trisomy 21 may experience delays when learning to crawl, walk and speak. As they get older, they may have trouble with reasoning and understanding.
Two other examples are Trisomy 13 (Patau syndrome) and Trisomy 18 (Edward’s syndrome). They can both cause serious brain, heart and spinal cord defects. Many babies born with these syndromes only live a few days.
An analysis of the distribution of hetero- and isodisomic regions of chromosome 7 in five mUPD7 Silver-Russell syndrome probands
Silver-Russell syndrome (SRS) shares common features of intrauterine growth retardation (IUGR) and a number of dysmorphic features including lateral asymmetry in about 50% of subjects. Its genetic aetiology is complex and most probably heterogeneous. Approximately 7% of patients with SRS have been found to have maternal uniparental disomy of chromosome 7 (mUPD7). Genomic DNA samples from five SRS patients with mUPD7 have been analysed for common regions of isodisomy using 40 polymorphic markers distributed along the length of chromosome 7. No regions of common isodisomy were found among the five patients. It is most likely that imprinted gene(s) rather than recessive mutations cause the common phenotype. Heterodisomy of markers around the centromere indicated that the underlying cause of the mUPD7 is a maternal meiosis I non-disjunction error in these five subjects.
7.9: Errors in Chromosome Number - Biology
To detect chromosome abnormalities, thus to help diagnose genetic diseases, some birth defects, and certain disorders of the blood and lymphatic system
When pregnancy screening tests are abnormal whenever signs of a chromosomal abnormality-associated disorder are present as indicated to detect chromosomal abnormalities in a person and/or detect a specific abnormality in family members sometimes when a person has leukemia, lymphoma, myeloma, myelodysplasia or another cancer and an acquired chromosome abnormality is suspected
A blood sample drawn from a vein in your arm a sample of amniotic fluid or chorionic villus from a pregnant woman a bone marrow or tissue sample
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Chromosome analysis or karyotyping is a test that evaluates the number and structure of a person's chromosomes in order to detect abnormalities. Chromosomes are thread-like structures within each cell nucleus and contain the body's genetic blueprint. Each chromosome contains thousands of genes in specific locations. These genes are responsible for a person’s inherited physical characteristics and they have a profound impact on growth, development, and function.
Humans have 46 chromosomes, present as 23 pairs. Twenty-two pairs are found in both sexes (autosomes) and one pair (sex chromosomes) is present as either XY (in males) or XX (in females). Normally, all cells in the body that have a nucleus will contain a complete set of the same 46 chromosomes, except for the reproductive cells (eggs and sperm), which contain a half set of 23. This half set is the genetic contribution that will be passed on to a child. At conception, half sets from each parent combine to form a new set of 46 chromosomes in the developing fetus.
Chromosomal abnormalities include both numerical and structural changes. For numerical changes, anything other than a complete set of 46 chromosomes represents a change in the amount of genetic material present and can cause health and development problems. For structural changes, the significance of the problems and their severity depends upon the chromosome that is altered. The type and degree of the problem may vary from person to person, even when the same chromosome abnormality is present.
Identification of chromosomal regions
The selection of the 14 gene families used in this study was based on the location of family members close to the genes coding for NPY family peptides in fugu, Takifugu rubripes and zebrafish, Danio rerio. The gene families included have genes located on at least two of the human chromosomes 2, 3, 7, 12 and 17. Chromosome 3 is a non-Hox-bearing chromosome but earlier studies have suggested that it is a part of the Hox-paralogon [7,8,75]. The two gene families IGFBP and SLC4A were included because they were already known to have copies on several of the chromosomes in the paralogon and were located near the human NPY-family genes.
The protein family predictions in Ensembl database version 43 http://www.ensembl.org/ where used together with relevant literature and BLAST  to identify additional sequences not included in the Ensembl protein families. All amino acid sequences representing the longest transcripts of every member of the 14 gene families in Tetraodon nigroviridis, Takifugu rubripes, Danio rerio, Mus musculus and Homo sapiens were obtained in order to obtain information of the repertoires in the common ancestor of actinopterygians and sarcopterygians using the Ensembl database version 43. This allows for the identification of shared and lineage specific in teleosts and tetrapods, respectively. For a full set of Ensembl IDs see Additional file 3.
Alignments and phylogenetic analyses
Protein domains in each sequence were identified by searches against the Pfam database http://www.sanger.ac.uk/Software/Pfam/ and aligned using the Windows version of Clustal × 1.81 [88,89]. Sequences lacking described domains, or incomplete domains, were removed from the alignment (for description of which criteria that were used for each family see figure legends in supplementary file 1 and 2). Alignments were thereafter manually inspected and edited to remove poorly aligned sequences. Phylogenetic trees were constructed using the neighbor-joining (NJ) method with standard settings (Gonnet weight matrix, gap opening penalty 10.0 and gap extension penalty 0.20) as implemented in the windows version of Clustal W 1.81  with 1000 bootstrap replicates. Bootstrap values below 50% were considered non-supportive. Sequences from Drosophila melanogaster and Ciona intestinalis (or Ciona savignyi or Branchiostoma floridae) were used as outgroups in the phylogenetic analyses in order to relatively date duplications. Quartet puzzling maximum-likelihood trees were constructed using Tree-Puzzle 5.2  (for Tree-puzzle settings in each analysis, see supplementary file 2).
Based on the phylogenetic analyses, positional information from gene family members duplicated after the split of urochordates and the rest of the chordates and before the split of sarcopterygians and actinopterygians was used to draw chromosomal maps. This allowed for identification gene families that most likely were syntenic in the ancestor of all vertebrates.