Principles of Clinical Cytogenetics

Question 1

Clinical Cytogenetics

Answer 1

It is the study of abnormalities of chromosome number and structure in relation to human disease. Cytogenetic abnormalities account for 1 in 154 live births and are collectively more common than all mendelian single gene disorders together. The incidence in mothers over 35 increases to about 1 in 50. A high percentage of pregnancies where the fetus has chromosomal abnormalities will result in abortion.

Question 2

List the 6 major clinical indications for cytogenetic analysis

Answer 2

1) Problems with early childhood growth and development–failure to thrive, abnormal physical appearance and/or internal structure abnormalities, mental retardation, ambiguous genitalia. 2) Stillbirths and neonatal deaths that have the appearance of a cytogenetic abnormality 3) A history of infertility or recurrent miscarriages 4) A known or suspected chromosome abnormality in a first degree relative (parent or sibling) 5) Maternal age over 35 6) Cancer (chromosomal analysis of cancerous tissue)

Question 3

Identify 6 common cell types and their sources that are used for cytogenetic analysis

Answer 3

6 common types of dividing nucleated (must be nucleated or contain DNA to analyze it) cells or tissues used for cytogenetic analysis are: 1) White blood cells (T lymphocytes) from peripheral blood 2) Amniotic cells (amniocentesis) 3) Cells from the chronic villi- extra embryonic fetal tissue that form the surface of the chorionic sac. (Chorionic Villus Sampling, CVS) 4) Fibroblasts (skin biopsy) 5) Cancer cells (tumor biopsy) 6) Bone marrow cells (bone marrow biopsy for hematological malignancies)

Question 4

List the 7 types of techniques that can be used to detect abnormalities of chromosome number or structure. Also, understand the resolution of various chromosomal and genomic diagnostic approaches and when each could be used

Answer 4

1) Standard Karyotyping: looking at the haploid genome or the whole chromosome. 2) Routine Banding: Looking at a chromosome band 3) High-resolution banding: Looking at a chromosome band 4) Comparative genome hybridization: Looking at submicroscopic regions of the genome here (50-250,000bp) 5) FISH Analysis: Looking at submicroscopic regions of the genome here (50-250,000bp) 6) Chromosomal Microarray: Looking at submicroscopic regions of the genome here (50-250,000bp) 7) Whole-Genome Sequencing: Looking at the nucleotide level

Question 5

Metacentric

Answer 5

Centromere is near the middle of the chromosome. The p and q arms are about equal length

Question 6

Submetacentric

Answer 6

Centromere is offset a bit with the q arm slightly larger than the p arm

Question 7

Acrocentric

Answer 7

Centromere is way off to one end off the chromosome, with the p arm being very small and q very large. The p-arm is called a satellite.

Question 8

p-arm

Answer 8

It is the petite arm or the smaller arm

Question 9

q-arm

Answer 9

It is the larger arm.

Question 10

Ideogram

Answer 10

It is when all the stained chromosomes are all lined up in numerical order with the sex chromosomes as the last 2. Each chromosome has a specific staining pattern with different dyes. The chromosomal DNA will take up the dyes and this can be used to classify chromosomes because they each have a unique staining pattern.

Question 11

Explain the numbering system used to identify specific chromosome bands.

Answer 11

The numbering system itself is based on the Giemsa staining patterns. Starting at the centromere you have the p-arm in one direction and the q-arm in another direction. The numbering goes outward in both directions from here. For chromosome 5 for example you would say “5-p-2-3” which indicates chromosome 5, the p-arm, region 2, band 3. This is based on what phase the chromosome is in too because during metaphase when the chromosome is more condensed you would see less bands but during prometaphase it would be less condensed so you would see more bands. Also, different areas of the chromosome will stain differently with different dyes because it depends on the the characteristics of the DNA.

Question 12

Describe the advantages of the chromosome band numbering system when describing cytogenetic abnormalities and for identifying genes that may have been affected.

Answer 12

The numbering gives a universal way of looking for specific chromosomal abnormalities. For example, if there is a gene deletion, one can compare two stained chromosome and may be able to see the deletion of the region of the chromosome. This is a good way to diagnose disease as well.

Question 13

Aneuploidy

Answer 13

It is abnormalities of chromosome number and they are the most common type of human chromosome disorders. They are mainly monosomies or trisomies. Aneuploidy usually results from meiotic nondisjunction. Partial aneuploidies arise from insertions (duplications) or deletions of portions of the chromosomes.

Question 14

Describe and compare the outcomes of nondisjunction occurring during either the first or second meiotic divisions (meiosis I and meiosis II) during gametogenesis and indicate how the two may be distinguished.

Answer 14

Chromosomal nondisjunction is the failure of chromosomes to detach from one another during one of the two meiotic divisions. This would lead to gametes or germ cells being diploid instead of being haploid like they should be. If nondisjunction occurred in meiosis I then the germ cells would have two different chromosomes. If it occurred during meiosis II then the germ cell would have 2 of the SAME chromosomes.

Question 15

Q-banding

Answer 15

Chromosomes are stained with quinacrine.

Question 16

R-Banding

Answer 16

It is the reverse of Giemsa staining results. What is a dark band in giemsa is a light band here.

Question 17

Fluorescent R-banding

Answer 17

untreated chromosomes are stained with acridine orange and examined by fluorescent microscopy

Question 18

High Resolution Banding (Prometaphase banding)

Answer 18

G or R banding of chromosomes obtained at an early stage of mitosis when they are less condensed.

Question 19

Monosomy

Answer 19

It is when only one chromosome is received from the parents. Monosomes almost always result in death except monosomy X or Turners syndrome in females. ONLY ONE IS VIABLE (MONOSOMY X)

Question 20

Trisomy

Answer 20

When three chromosomes are inherited. Only 3 autosomal trisomies are capable of producing a viable offspring: 1) Trisomy 13 (Patau syndrome)- frequency of 1’22,700 live births 2) Trisomy 18 (Edward Syndrome)- frequency of 1/7,500 3) Trisomy 21 (Down Syndrome)- frequency of 1/580 There are also 3 trisomies that occur in sex chromosomes to produce viable offspring: 1) Trisomy X in females- 1/900 2) Klinefelter Syndrome (47, XXY or 48, XXXY) 1/1000 3) 47, XYY- 1/1000

Question 21

Compare cytogenetic and clinical consequences of monosomy due to nondisjunction with partial monosomy due to a partial deletion.

Answer 21

If there is a monosomy that resulted from nondisjunction, then there is a complete loss of that chromosome, giving a complete loss of all of the genetic material on that chromosome. However, if there is partial monosomy due to a deletion, then the rest of the chromosome is still present. If the deletion did not occur in a coding region one may not see as serious consequences as would be seen with complete monosomy

Question 22

Describe how common chromosomal abnormalities such as insertions, deletions, isochromosomes, dicentric chromosomes, and ring chromosomes may come about.

Answer 22

Duplications and deletions can occur from unequal crossing over between misaligned sister chromatids or homologous chromosomes that contain long repeated stretches of highly similar DNA sequences. Deletions tend to produce a more serious phenotype than insertions or duplications. Generation of an isochromosome is caused usually by the loss of a p or q arm of a chromosome containing the two sister chromatids. When the p arm is lost for example, the two q arms will now be on one chromosome. This will then be replicated and treated as one chromosome because the centromere was retained. Now there will be a partial trisomy and a partial monosomy. Ring chromosomes are formed from double stranded breaks on each end of the chromosome that results in the chromosome then joining by the two ends. Dicentric chromosomes (those with 2 chromosomes) can form via translocations and PARACENTRIC INVERSIONS. These are inviable usually.

Question 23

Identify and explain features of normal and abnormal chromosomes that determine whether or not they will be stable

Answer 23

If a chromosome is acentric (lacking a centromere) or dicentric (having two centromeres) they are usually inviable and unstable. Stable chromosomes still retain one centromere because it is crucial for the kinetochore to bind to. A dicentric chromosome will also be attached to twice and thus is subject to destruction because it is likely to be pulled apart to opposite poles during division. Also, they cannot have drastic deletions that will lead to the organism being inviable. Also, ring chromosomes tend to be unstable through mitosis.

Question 24

Explain what is meant by the terms balanced and unbalanced in reference to chromosomal rearrangements and compare the resulting phenotype (normal or abnormal) of an individual that expresses one or the other.

Answer 24

Unbalanced rearrangements are likely to produce an abnormal phenotype because duplications of a part of a chromosome leads to partial trisomy and deletion of part of a chromosome leads to partial monosomy. Unbalanced simply means that there is a net loss or gain of genetic material and these are seen with insertions and deletions, unequal crossing over, isochromosome formation and the formation of a ring chromosome. Balanced rearrangements are unlikely to produce a phenotypic effect because all of the chromosomal material is present even though it is packaged differently. There is no net gain or loss of genetic info! An exception is when it disrupts an essential gene. Examples of these are inversions, reciprocal translocations and robertsonian translocations

Question 25

What are the 4 types of unbalanced rearrangements

Answer 25

1) Deletions and insertions: Deleting a part of a chromosome and also inserting a duplicated portion of a chromosome. 2) Unequal crossover: results in duplications and deletions because of misaligned chromosomes that contain large repeats. Now one chromosome will have a larger chunk or smaller chunk of the chromosome and when the cell divides it will result in that cell having unbalanced genetic material 3) Generation of Isochromosomes: when an arm of a chromosome is deleted with retention of a centromere. Results in partial monosomy and partial trisomy 4) Ring Chromosomes: They form from double stranded breaks and thus lost that material

Question 26

What are the 3 types of Balanced Rearrangements

Answer 26

1) Inversions: the chromosome section simply flips when there are two breaks, there is no net loss of genetic material. They can be paracentric (break on the same chromosome arm and does not include centromere) or pericentric (breaks on each arm and thus inverts the centromere as well). Paracentric results in 4 gamates, two of which are balanced, 2 of which are inviable due to acentric and dicentric. Pericentric inversions result in normal, balanced or unbalanced gametes. 2) Reciprocal translocations: Carriers are balanced and usually unaffected as long as it is not in an essential gene region. However, these carriers are associated with high risk for unbalanced gametes and abnormal progeny. These are rearrangements between two different chromosomes and there is no net loss or gain of genetic material in the parental cell so it is balanced. It is usually a 2:2 segregation but they can also give 3:1 segregation as well when forming germ cells so there are 12 possible germ cells that could be produced, only 1 is normal and 1 is balanced. The rest are unbalanced. 3) Robertsonian Translocations: These are a type of reciprocal translocation that only involve the 5 human acrocentric chromosomes (13, 14, 15, 21, and 22). The satellite portion that codes for ribosomes is lost. then, the two q-arms join between the two chromosomes and thus no genetic material is lost and it is balanced. This results in one normal chromosome 14 for example, one chromosome that is a combination of 14/21 and another that is a normal chromosome 21. When it splits it can result in trisomy, monosomy, normal or a balanced carrier.

Question 27

Compare the cytogenetic features of reciprocal and Robertsonian translocations and explain why the loss of the p-arms of acrocentric chromosomes in a Robertsonian translocation fails to produce a discernibly abnormal phenotype

Answer 27

In reciprocal translocation, two non-homologous chromosomes are crossing over and exchanging genetic material within the chromosome. In robertsonian the crossing over or recombination takes place via the satellite regions, causing the retention of the centromere and loss of ribosomal coding regions. Robertsonian is balanced because it is only losing ribosomal coding regions and there is enough of this on the other acrocentric chromosomes to make up for this. It is not losing coding regions.

Question 28

Draw a diagram describing how a carrier of a balanced translocation can give rise to unbalanced offspring

Answer 28

Question 29

For a balanced carrier of a reciprocal translocation, identify which modes of chromosomal segregation (alternate, adjacent, 2:2 or 3:1) are likely to result in normal offspring and which are likely to result in unbalanced carriers

Answer 29

2:2 alternate segregtion results in a normal and a balanced carrier. 2:2 adjacent results in all unbalanced and 3:1 results in all unbalanced.

Question 30

Microdeletion and duplication syndromes

Answer 30

These can result from unequal crossing over between misalligned sister chromatids or homologous chromosomes containing highly similar DNA sequence repeats. Very small deletions can be difficult to detect by classic cytogenetic techniques. The severity of the resulting syndrome often depends on the # of genes affected. Because a group of neighboring genes is involved, these disorders have also been called Contiguous Gene Syndromes.

Question 31

Prader-Willi / Angelman Syndrome

Answer 31

It is a deletion in the 15q11-q13 region. PWS is a deletion of this region in dad which is supposed to be expressed because it is imprinted by mom where AS is a deletion in moms that is supposed to be expressed because it is silenced or imprinted in dads.

Question 32

Compare the genetic and phenotypic consequences of chromosome microdeletions and duplications

Answer 32

The duplications will cause a double amount of certain genes on a genotypic level. Phenotypically speaking these are not usually severe unless it disrupts a coding region. Deletions are losing DNA and phenotypically are more severe

Question 33

Identify common diseases caused by microdeletions from descriptions of the resulting phenotypes

Answer 33

Prader-Willi Syndrome: obesity, excessive eating habits, small hands and feet, short stature, hypogonadism, and mental retardation

Angelman Syndrome: Unusual facial features, short stature, severe mental retardation, spasicity, and seizures

Question 34

Describe and compare FISH and array comparative genome hybridization (array CGH) for the detection of chromosomal abnormalities

Answer 34

1) Fluorescent In Situ Hybridization (FISH) can be used to detect gross chromosomal abnormalities (spectral karyotyping) as well as small deletions or duplications that can not be detected by high resolution chromosome banding techniques. It is an easier way to look at chromosomes. It can be used to look at a whole chromosome to detect trisomies and monosomies or to detect deletions and duplications.
2) Array Comparative Genome Hybridization (Array CGH): You are labeling with fluorophores still like FISH but you take DNA from a normal patient and label it red and then you label your patient’s DNA green and co-hybrize these DNA samples to known DNA sequences on a microarray. If there are equal numbers of these regions it will be yellow from the combination of the red and the green. If it is green then there is a net gain in your patient in that region (duplication). If it is red then there is a net loss in your patient in that region (deletion). This can show where a disease has come from then.

Whole genome sequencing is also becoming increasingly practical. The point is that multiple different techniques can be used to receive the same information.

Question 35

Explain the relationship between genetic imprinting and parent-of-origin effects in microdeletion disorders

Answer 35

If the parent that is supposed to express that gene has a deletion, then the offspring will not receive that gene because the other parent of opposite sex will have it imprinted or silenced.

Question 36

Explain uniparental disomy and its relationship to PWS and AS

Answer 36

Uniparental disomy is when one parent happens to pass on both of their chromosomes instead of just one and the other parent then passes on none of those chromosomes. If the parent that passed on both of those chromosomes happened to be imprinted it will act as “deletion” and thus will get PWS or AS dpending on which sex is passing it on.