Cytogenetics

Question 1

An exact multiple of the haploid chromosome number (n) is termed [], and any other chromosome number is termed [].

Answer 1

An exact multiple of the haploid chromosome number (n) is termed euploid, and any other chromosome number is termed .

Question 2

The most common mechanism by which aneuploidy occurs is [].

Answer 2

The most common mechanism by which aneuploidy occurs is meiotic nondisjunction.

Question 3

Often, chromosome abnormalities are the result of structural rearrangements rather than differences in chromosome number. Such rearrangements are defined as[] if the resultant chromosome has the normal complement of genetic material or [], if there is additional or missing genetic information.

Answer 3

Often, chromosome abnormalities are the result of structural rearrangements rather than differences in chromosome number. Such rearrangements are defined as balanced if the resultant chromosome has the normal complement of genetic material or unbalanced, if there is additional or missing genetic information.

Question 4

Unbalanced rearrangements cause [] and [] involving part or all of a chromosome. Balanced rearrangements include[] (either [] or []), and [].

Answer 4

Unbalanced rearrangements cause deletions and duplications involving part or all of a chromosome. Balanced rearrangements include inversions (either paracentric or pericentric), and translocations.

•Balanced rearrangements usually result in no structural or functional disability in the affected individual, but they can result in unbalanced chromosome complements in offspring. Translocations and large pericentric inversions are particularly liable to result in aberrant reproductive outcome.

Question 5

A [] is a pictorial representation of the chromosomes. Human chromosomes are classified according to 3 characteristics: (1) [] (2) [] and (3) [].

Answer 5

A karyotype is a pictorial representation of the chromosomes. Human chromosomes are classified according to 3 characteristics: (1) centromere position (2) size and (3) banding pattern

Question 6

Metacentric…

Answer 6

…chromosomes have a central centromere.

Question 7

Submetacentric…

Answer 7

…chromosomes have an off center centromere.

Question 8

Acrocentric…

Answer 8

…chromosomes are recognizable by the centromere being near one end.

•The human acrocentric chromosomes (13, 14, 15, 21, and 22) have small masses of chromatin known as satellites attached to their short arms by narrow stalks. The stalks of these five chromosome pairs contain the genes for 18S and 28S ribosomal RNA (rRNA). (Since this genetic material is duplicated elsewhere in the genome, deletion of the satellites and stalks of one pair of acrocentric chromosomes has no effects on the phenotype).

Question 9

Standard Banding

Answer 9

• initially treated with an agent such as trypsin, which partially “digests” the protein of the chromosome
• secondary stain is then applied which allows for differential banding to be visualized. The most commonly used banding technique is known as G-banding, so-called because Giemsa stain is applied following treatment of the chromosome with trypsin
• the chromosomes appear as a distinctive series of light and dark bands
• darkly staining bands tend to be condensed, AT-rich and nontranscribed heterochromatin, while the light staining bands tend to be elongated, GC-rich and transcribed euchromatin

Question 10

High Resolution Banding

Answer 10

• A technique that allows identification of greater detail in the karyotype.
• With this technique, chromosomes that have been prepared at an earlier stage of mitosis (prophase or prometaphase), when they are still in a relatively uncondensed state, are stained and analyzed.
• Prometaphase and prophase chromosomes reveal 550-850 bands or even more in a haploid set in comparison to standard metaphase preparations which show only about 400 bands.

Question 11

Fluorescence in-situ hybridization (FISH)

Answer 11

• Allows a focused search for the absence (deletion) or excess (duplication) of specific chromosomal regions.
• In this technique, specific DNA segments that are complementary to segments of interest in affected individuals are constructed and “tagged” with a fluorescent label. These specific segments are then hybridized to a standard metaphase chromosome preparation from an affected or potentially affected individual. If the segment of interest is present, then there will be hybridization of the fluorescent probe and it can be detected under the fluorescent microscope.
• Through this technique, clinical detection of both numerical and structural chromosome abnormalities can be accomplished for selected disorders. There are three classes of FISH probes used for these purposes:
• Locus-specific
• Alphoid or centromeric probes
• Chromosome-sepcific painting probes

Question 12

Locus-specific FISH probes

Answer 12

• used to detect specific genetic disorders for which the disease-causing sequence is known. This is generally used to see if both copies of a particular sequence are present.
• There is a long list of disorders to which this technology can be applied, including the 22q11 deletion syndrome and Prader-Willi syndrome. In the normal individual there will be 2 fluorescent signals; whereas in the person with that segment deleted, only one signal will be present.

Question 13

Alphoid or centromeric probes

Answer 13

• Used to detect abnormalities in chromosome number.
• These probes take advantage of the fact that most chromosome pairs have a unique sequence near the centromere that distinguishes this chromosome pair from the other 22 pairs.

-For example, a chromosome 21 specific centromere probe would detect 2 signals from a normal person with 2 copies of chromosome #21 and 3 copies from an individual with trisomy 21/Down syndrome.

• This type of FISH analysis can be used when there is a need for a rapid diagnosis of trisomies, which might include pregnancies in which one of the trisomies is likely because of characteristic prenatal ultrasound features; or, when there is a critical need for diagnosis of a newborn with life-threatening birth defects and a trisomy is suspected.
• For routine diagnosis of trisomies, however, standard chromosome analysis is adequate and will readily detect the extra chromosome.

Question 14

Chromosome-specific painting probes

Answer 14

• Used to detect rearrangements suspected by standard cytogenetic techniques.
• In this technique the whole chromosome is “painted” with a cocktail of unique sequences to give fluorescence of essentially the whole chromosome. This may be used in the case when there is extra chromosomal material present but the material is of unknown origin. This FISH technique can identify the origin of the abnormal chromosomal material.

Question 15

Array comparative genomic hybridization (aCGH)

Answer 15

•Also known as chromosome microarray (CMA) represents a second generation of molecular cytogenetic techniques that can detect chromosome deletions or duplications as small as a few hundred base pairs.

Question 16

Massively parallel/Next generation DNA sequencing

Answer 16

•There are a few varieties of these next generation sequencing (NGS) technologies, but all rely on sequencing each base of DNA multiple (2-1000) times to overcome inherently high error rates. Essentially, the methods simultaneously perform millions of sequencing reactions and read the sequencing from each using a detector connected to a computer. Each DNA sequence read is typically 50-200 bases long and significant computational resources are then used to map these short reads back to a reference human genome.

Question 17

Methylation Studies

Answer 17

•The methylation state of a specific region of DNA can be assessed with methylation-sensitive restriction enzymes. Essentially, these will only cut methylated (or unmethylated) sequences, while the alternative methylation state will remain uncut. After attempted cleavage, patient DNA can be separated by electrophoresis and the sizes of bands analyzed by Southern blotting. Whether the source DNA was or was not methylated can then be inferred by the sizes of the resulting bands. Methylation state can also be determined treating DNA with sodium bisulfite that converts cytosine to uracil, but leaves 5-methylcytosine residues unaffected. Amplifying the resulting sequencing with specific primers, direct sequencing, or methylationspecific microarray can then allow determination of the underlying methylation state of a sequence or a small region.

Question 18

In general, people with autosomal chromosome abnormalities have the triad of [], [] and [].

Answer 18

In general, people with autosomal chromosome abnormalities have the triad of growth retardation, intellectual disability (previously referred to as mental retardation) and birth defects.

Question 19

Sex chromosome abnormalities medical problems primarily involve [] and [].

Answer 19

Sex chromosome abnormalities medical problems primarily involve abnormalities of stature and sexual maturation.

Question 20

Nondisjunction

Answer 20

• In the normal situation during meiosis, the 2 members of each homologous chromosome pair disjoin, with one chromosome going to each daughter cell. When these 2 chromosomes fail to disjoin, it is known as nondisjunction.
• Nondisjunction may produce either a trisomic or a monosomic line of cells for the involved chromosome (commonly 21, 18 or 13).
• An autosomal monosomic cell line (45 chromosomes) generally does not persist, but the trisomic cell line may survive. Trisomic (47 chromosomes) live births are not rare, although postnatal survival may be compromised by multiple malformations.
• The rate at which autosomal nondisjunction occurs increases with advancing maternal age.
• There are three clinically important autosomal trisomies: trisomy 21, 18 and 13. For the most part, these are the only autosomal trisomies that are seen in liveborn children, since trisomies for other autosomes result in very low viability and spontaneous abortion (miscarriage).
• Autosomal monosomies are uniformly lethal prenatally or in very rare circumstances, soon after birth.

Question 21

Trisomy 21

Answer 21

• Down Syndrome
• About 95% of children with Down syndrome have what is known as free 21 trisomy, which is designated 47,XY,+21 or 47,XX,+21 and is the result of nondisjunction. There is about a 1% risk for having additional children with an autosomal trisomy in the parents of children with free 21 trisomy. Parent karyotypes are not recommended when the child has free 21 trisomy.
• However, about 4% of children have a Robertsonian translocation leading to Down syndrome. In these children, one of the parents may be a translocation carrier, so parent karyotypes are recommended. If one of the parents is found to carry a balanced translocation, then they have about a 10% risk of having another child with Down syndrome.
• About 1% of children with Down syndrome have mosaicism. Mosaicism means that there are two or more cell lines in one individual. In the case of Down syndrome the karyotype designation is 46,XY/47,XY,+21 in the case where one cell line is normal and the other contains a trisomy 21 cell line. Some children with mosaicism may have a milder phenotype as a result of the mitigating effect of the normal cell line.

Question 22

Trisomy 18

Answer 22

• Children with trisomy 18 have low birthweight, small mouth, prominent occiput, short sternum, cardiac defects in 80% or more and decreased viability.
• While the median life expectancy is around one week, many patients can live much longer.

Question 23

Trisomy 13

Answer 23

• Children with trisomy 13 are also characteristic, with frequent features being holoprosencephaly (a severe abnormality of brain development), cleft lip and palate, extra digits and decreased viability.
• Median life expectancy is similar to children with trisomy 18.

Question 24

Deletions

Answer 24

•Deletions are losses of chromosome material and may be terminal deletions, occurring at the end of one of the chromosome arms; or interstitial deletions, occurring between the centromere and telomere and removing an intervening sequence.

Question 25

cri-du-chat syndrome

Answer 25

• terminal deletion
• short arm of 5th chromosome

Question 26

DiGeorge syndrome

Answer 26

• aka velocardiofacial syndrome
• deletion of the 22q11.2 band
• Affected individuals have a variable phenotype including cleft palate and conotruncal heart defects (abnormalities of the ventricular outflow tract), as well as abnormalities of the thymus. 22q11 deletions are now usually detected through the application of FISH (fluorescence in-situ hybridization) analysis

Question 27

Duplications

Answer 27

•Duplications are gains of chromosome material, and like deletions, they may be terminal (at the ends of chromosome arms) or interstitial (within a chromosome arm). There are few well described duplication syndromes in humans. An inversion occurs when a single chromosome undergoes two breaks and is reconstituted with the segment between the breaks inverted.

Question 28

Translocations

Answer 28

•Translocations are structural rearrangements consisting of chromosomal segments transferred between nonhomologous chromosomes. The translocation may be balanced (no genetic material lost and the individual is phenotypically normal), or unbalanced (genetic material in excess and/or deficit with significant medical consequences). Two major types of translocations include:

• Reciprocal
• Robertsonian

•Translocations are especially important clinically because a balanced carrier may transmit an unbalanced form of the rearrangement to offspring. Translocations may be familial, in which case there are important counseling implications for a couple’s reproductive expectations. De novo translocations may also occur as an error in a single germ cell of the parent rather than being familial and carried in all germ cells.

Question 29

Reciprocal Translocations

Answer 29

• Reciprocal translocations (mutual segment exchange between any two chromosomes).
• Balanced state has no net loss or gain of material, whereas the unbalanced state usually has deleted material from one chromosome and duplicated material from another chromosome.
• Regardless of whether the translocation is balanced or unbalanced, there are 46 chromosomes present.

Question 30

Robertsonian Translocations

Answer 30

• confined to acrocentric chromosomes 13-15 and 21-22
• In this type of translocation the breaks are at the centromere level, and the resultant rearrangement is a centric fusion of long arms. The acrocentric short arms are lost in subsequent cell divisions because they lack centromeres. The balanced state has 45 chromosomes; the unbalanced has 46.

Question 31

Imprinting Defects

Answer 31

• A small percentage of human genes are imprinted by being methylated during meiosis. Differential methylation of maternal and paternal gene copies is called imprinting and it can cause only the gene inherited from a specific parent to be active in a person.
• Prader - Willi
• Angelman

Question 32

Angelman Syndrome

Answer 32

• chromosome 15q
• Generally, the paternal copy of the UBE3A gene is imprinted and thus silenced. When the maternal copy of the UBE3A gene is lost, then there is no paternal copy to make an important ubiquinating protein product and the child has Angelman syndrome. Symptoms of Angelman syndrome include significant developmental delay, seizure, lack of speech, and odd ‘puppet-like’ movements.
• Loss of the maternal gene (causing Angelman syndrome) or paternal gene (causing Prader-Willi syndrome) can happen because of microdeletion, mutation, or uniparental disomy.

Question 33

Prader-Willi Syndrome

Answer 33

• chromosome 15q
• the SNRPN gene is found nearby on chromosome 15q and the maternally-derived copy of this gene is generally silenced.
• When the paternal SNRPN copy is lost, children have Prader-Willi syndrome with hyperphagia, developmental delay, and decreased muscle tone.
• Loss of the maternal gene (causing Angelman syndrome) or paternal gene (causing Prader-Willi syndrome) can happen because of microdeletion, mutation, or uniparental disomy.

Question 34

Uniparental Disomy

Answer 34

•Uniparental disomy happens when two copies of a chromosome both come from the same parent and can occur from a random event during meiosis making egg or sperm cells, or from a mitotic error during early fetal development.

Question 35

Turner Syndrome

Answer 35

• Individuals with a 45,X karyotype have a female phenotype with a very variable set of clinical characteristics.
• While many if not most of girls with this karyotype will generally appear normal, some will have a characteristic phenotype described by Turner in 1939. This include short stature, absent secondary sex characteristics, excess nuchal skin, broad chest, increased carrying angle of the arms and peripheral edema.
• While 45,X is the most readily recognized karyotype in girls with all or some of these physical features, a number of other karyotypes can also be associated with these findings including partial deletions and mosaic karyotypes. The 45,X karyotype is very common in human conceptions, representing up to 1% or so of all conceptions. However, it is present in only about 1 in 2500 liveborn infants, which means that over 99% of all 45,X conceptuses are spontaneously aborted. This is especially surprising, because women with Turner syndrome are generally healthy and of normal intelligence.
• Typically, girls with Turner syndrome have primary amenorrhea, which means that they never menstruate; and, they are infertile. These features are a consequence of ovarian dysgenesis, in which the ovaries involute during fetal life. The infertility is the result of absence of oocytes, while the amenorrhea is the result of absence of estrogen-producing cells within the ovary.
• Girls with Turner syndrome also have short stature, with final adult height in the range of 4’8” to 4’10”. The FDA has approved the use of growth hormone for girls with Turner syndrome, which offers the possibility of improving or normalizing adult height. Girls with Turner syndrome are also provided with estrogen replacement therapy to promote development of secondary sex characteristics and to prevent osteoporosis.
• Women with Turner syndrome have borne children through the process of oocyte donation and in vitro fertilization where the embryo is implanted, and the intrauterine milieu is manipulated with hormonal treatment to allow for establishment of the pregnancy.

Question 36

Klinefelter Syndrome

Answer 36

• In 1938 Klinefelter described a group of boys with tall stature, developmental disabilities, gynecomastia (breast development), small testes and infertility. We now recognize that this phenotype is the result of an extra X chromosome.
• While some boys with a 47,XXY karyotype will definitely have a phenotype like that described by Klinefelter, most affected boys are not so striking. Affected boys are less intelligent than expected when compared to their chromosomally normal siblings, but few of them function in the intellectually disabled range. The meiotic nondisjunction event that produces the 47,XXY genotype appears to occur equally in both males and females. While boys with XXY are infertile, they have normal genitalia and normal sexual functioning, although they do benefit by testosterone supplementation to improve libido. Testosterone is low in affected boys due to involution of the testicular Leydig cells that produce testosterone.