Chromosome analysis Flashcards
1
Q
The criteria used for the selection of metaphase spreads suitable for analysis includes all
the following EXCEPT FOR:
A
Presence of an intact cell membrane(+)
Chromosome band level (length)
The quality of G-banding
The number of overlapped chromosomes
2
Q
When reviewing a final karyotype a possible overlapped chromosome is suspected. In order to confirm the chromosome number of this cell, all the following information is needed EXCEPT FOR:
A
Patients clinical history(+) Identification number of the patient Microscope used to photograph the cell being analyzed The coordinates of the metaphase
3
Q
What is the minimum number of cells that must be counted for in situ amnio analysis?
A
Ten cells from two coverslips Ten cells from each of four cultures Fifteen cells from one culture One cell each from fifteen different colonies from at least two coverslips(+)
4
Q
Which of the following is TRUE when the banding level of chromosomes is 550-600?
A
There are two dark bands on chromosome 7q.
There are two dark bands on chromosome 11p.
There are two dark bands on chromosome 9q.
The first dark band on chromosome 10q21 is split into
two different bands.(+)
5
Q
When an abnormal cell is detected in an in situ culture all the following should be
considered to rule out pseudomosaicism EXCEPT FOR:
A
Confirm abnormality in other cells in the colony.
Confirm abnormality in another culture.
Count 20 colonies from one coverslip.(+)
Confirm abnormality in other colonies on the same
coverslip.
6
Q
All of the following will result in erroneous chromosome counts EXCEPT:
A
Nomadic intruders
Overspreading
Debris
Undertrypsinization(+)
7
Q
A Cri-du-chat patient has a chromosome abnormality of
A
t(15;2)(q11;q21)
t(5;2)(p13;p12)
t(5;10)(q13;p22)
r(5)(p15.1q35)(+)
8
Q
The peripheral blood culture of a patient suspected of having CML is 46,XY. In order to rule out the presence of the t(9;22) the following tissue would be cultured next:
A
Skin
Bone Marrow(+)
Another peripheral blood specimen
Peripheral blood from a family member
9
Q
What is it called when two different cell lines within an organism seem to come from
different sources?
A
Contamination
Mosaicism
Chimerism(+)
Pseudomosaicism
10
Q
Which of the following is the correct ISCN nomenclature for a male with fragile X
syndrome?
A
46,fra(X)(q27.3),Y
46,Y,fra(X)(q27.3)(+)
46,XY,fra(X)(q27.3)
46,X,fra(X)(q27.3)
11
Q
46,XY,der(9)t(9;10)(q32;q12). Which of the following terms best describes this written karyotype?
A
Balanced
Unbalanced(+)
Robertsonian translocation
Reciprocal
12
Q
Twenty-nine out of 30 metaphases are normal in the amniotic fluid culture of a pregnant
woman with a history of multiple miscarriages. One cell has a translocation. What does this
signify?
A
leukemia
cultural artifact(+)
Robertsonian translocation
derivative
13
Q
Deletion (15)(q11 q13) is associated with which of the following?
A
Prader-Willi Syndrome(+)
Williams Syndrome
DiGeorge Syndrome
Cri-du-Chat Syndrome
14
Q
I. Select Suitable Metaphases For Analysis
1.Chromosome morphology
A
Metaphases that are suitable for analysis:
a) Have relatively few overlaps or the overlaps do not obscure the chromosome bands.
b) Are free from cytoplasm.
c) Are banded to the degree that dark vs. light bands can be discerned.
15
Q
I. Select Suitable Metaphases For Analysis
2.Suitable banding level
A
Chromosome length is dependent on a number of variables:
a) Tissue type
b) Colcemid exposure time
c) Stage of mitosis (e.g. Prophase, Mid-, or Late-Metaphase)
Band level refers to the number of bands that occur along the length of the chromosomes in a haploid set. Typical metaphase chromosomes, which are analyzed in clinical cytogenetic laboratories, will range from a band length of 250 to 850. When chromosomes are either longer or shorter than these band levels they become difficult to analyze.
To determine the banding level of chromosomes one should follow the following criteria (Josifek et al, 1991):
a) Determine the number of bands from several chromosomes.
b) Determine the number of bands from an entire chromosome or chromosome arm.
c) Count only the dark G-bands not including the centromere bands.
Figure 1 illustrates the difference in the number of bands on the short arm of chromosome 11’s at different band levels.
16
Q
I. Select Suitable Metaphases For Analysis
- Suitable number of metaphases to analyze
a) Constitutional analysis
A
The majority of clinical cytogenetics laboratories will count 20 cells per specimen. This will rule out a mosaicism of 14% at a 95% confidence level (Hook, 1977). If one abnormal cell is detected, the number of cells analyzed should be increased to 30. This will rule out a mosaicism of 10% at a 95% confidence level. Amniocentesis coverslip cultures are the exception. In these specimens, 15 colonies from at least two coverslip cultures are counted.
Mosaicism implies that there is more than one cell line in a sample. Mosaicism is possible when an error in division or a mutation takes place during the early stages of embryonic development. Mosaicism should not be confused with Pseudomosaicism.
17
Q
I. Select Suitable Metaphases For Analysis
- Suitable number of metaphases to analyze
b) Analysis of acquired abnormalities
A
In the analysis of leukemic bone marrow or solid tumor specimens there are often normal contaminating cells found with the neoplastic cells. Again most clinical laboratories will count 20 to 30 cells. However, in this type of analysis the cytogeneticist is analyzing the chromosomes to find the malignant cell population, rather than to rule out a constitutional chromosome abnormality.
There will be instances in the analysis of acquired abnormalities when a laboratory may choose to study more than 20 to 30 cells. For example, in studying predisposition to cancer, analysis of 50 to 100 cells is required in order to pick up rare acquired rearrangements, which may be observed in the peripheral blood. In addition, for breakage studies, 50 cells often must be analyzed to determine an accurate reading that can be compared to historical controls.
18
Q
I. Select Suitable Metaphases For Analysis
-Prenatal
A
Typically, 15 colonies from a minimum of two coverslips are counted or analyzed.
In the analysis of in situ cultures it is important to analyze cells from a number of different colonies, coverslips and cultures. This will help rule out maternal cell contamination and pseudomosaicism. A suspected mosaicism in a prenatal sample may necessitate the analysis of 50 cells.
19
Q
I. Select Suitable Metaphases For Analysis
-Oncology
A
Typically, 20 cells are analyzed.
In the analysis of bone marrow cultures it is important to analyze cells grown under different culture conditions. For example, most myeloid leukemias are grown in both 24 and 48 hour cultures if sufficient sample is available. Conversely, most lymphocytic leukemias and lymphomas are grown in both 24 and 72 hour stimulated cultures whenever possible. 72 hour PHA stimulated cultures are set-up and analyzed whenever T-cell disease is suspected and 72 hour LPS stimulated cultures are set-up and analyzed whenever B-cell disease is suspected.
20
Q
II. Count and Analyze metaphase spreads in a Systematic Manner
1.Document modal number
A
The modal number is the most common chromosome count recorded from a given cytogenetic preparation.
The range is also a term referring to the number of chromosomes observed in all the cells of a population
21
Q
II. Count and Analyze metaphase spreads in a Systematic Manner
2.Document karyotype
A
It is necessary to document pertinent information about each case studied for future references. This includes the location of the metaphase and scope, the technologist’s name, the date of analysis, and the Polaroid, negative or computer filename. All this information together with the written karyotype, prepared according to the ISCN guidelines, should be recorded within the patient report (An overview of ISCN is included in pages 48-53 of this module.
Each karyotype should be labeled with the patient name, lab number, date sample was received, and the negative reference number or computer filename.
22
Q
II. Count and Analyze metaphase spreads in a Systematic Manner
3.Document slide location
A
Located on the stage of most compound microscopes are two vernier scales, one for the x-axis and another for the y-axis coordinates of the stage. These vernier scales can be read to the tenth degree and are highly accurate for the slide location of metaphase spreads.
23
Q
II. Count and Analyze metaphase spreads in a Systematic Manner
4.Document analysis of separate colonies for in situ cultures
A
Amniotic fluid cultures are often grown in situ. In this type of culture system, cells are plated onto sterile coverslips and placed in petri dishes at 37oC. These cells are then allowed to attach and form colonies. When the appropriate cell density has been achieved, the coverslips are then exposed to Colcemid, hypotonic solution, Carnoy’s fixative, banding solutions and are finally analyzed under the microscope without the removal of the cells from these coverslips.
When counting and analyzing the 15 to 30 cells of an in situ culture, one should select and document cells from as many different colonies as possible. In addition, metaphase plates should be analyzed from different cultures. If an abnormality is detected, other metaphase plates from the same colony should be examined to confirm that all cells have the same abnormality within the colony. All of these precautions, if well documented, will allow interpretation of any abnormalities detected and are the most effective measure for ruling out maternal cell contamination and pseudomosaicism.
The four main types of mosaicism:
Level I Mosaicism - Called single cell mosaicism occurs when only one colony shows metaphases with an abnormal karyotype.
Level II Mosaicism - Called Pseudomosaicism occurs when an abnormal karyotype is limited to one coverslip culture.
Confined Placental Mosaicism (CPM) - This type of mosaicism occurs when an abnormal karyotype is found only in the extraembryonic membranes of the fetus.
Level III Mosaicism - Called True mosaicism occurs when two or more colonies with the same abnormal karyotype are observed in two or more coverslip cultures.
24
Q
III.Prepare accurate karyotypes from photographic prints or computer images
1. Select good quality prints or images
A
Photographic plates, which are to be used for karyotyping, should be of sufficient quality to allow identification of each and every chromosome. Ideally, the bands should be sharp and clear showing a clear distinction between dark and light regions along the chromosomes and there should be few overlaps.
25
III.Prepare accurate karyotypes from photographic prints or computer images
2.Arrange chromosomes using approved format
Autosomes are numbered from 1 to 22 according to decreasing overall length. Chromosome number 1 is the longest chromosome of the autosomes and chromosome numbers 21 and 22 are the shortest chromosomes of the autosomes (The one exception to this rule is chromosome 22 which is larger than chromosome 21.). The sex chromosomes are designated X and Y.
The human chromosomes are also grouped into groups designated by the letters of the alphabet A - G. This grouping is based on size and centromeric position.
Location of the centromere is one of the important factors in determining the identification of a particular chromosome. It is the centromere which is the landmark used to divide the chromosome into the short arm (p) and the long arm(q). Based on the proportion of the short arm to the long arm, a chromosome is described as being one of the following:
a) Metacentric - Centromere is in the middle of the chromosome and the short and long arms of the chromosome are approximately equal in length.
b) Submetacentric - Centromere is located away from the middle of a chromosome and the p arm of the chromosome is shorter than the q arm.
c) Acrocentric - Centromere is located very close to the terminal end of the chromosome and often is characterized by a secondary constriction separating the short arm from the satellite of the chromosome.
The short arm (p) of the chromosome is always oriented in the up position in the correct karyotype format.
In addition, band designation is determined by distance from the centromere. For example, bands p11 and q11 are located closer to the centromere than bands p21 and q21.
26
III.Prepare accurate karyotypes from photographic prints or computer images
3.Provide permanent copy of final karyotype
A permanent copy of the final karyotype should be provided with each cytogenetic report. This permanent copy does not have to be the original karyotype itself, but can be a photographic copy of the original.
27
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
1.Aneuploidy (numerical abnormalities)
Polyploidy - Abnormalities of the number of haploid sets in a cell. There are 23 chromosomes per haploid set (n). Two copies of the haploid set (2n) is characteristic of normal diploid cells.
Triploidy (3n) - These cells have three copies of the haploid set. Fetuses with this constitutional abnormality are often nonviable or do not live long after birth. The written karyotype of these conceptions is either 69,XXX or 69,XXY.
Two-thirds of all triploid conceptions are the result of double fertilization of the ovum by two sperm. (Remember that normally all gametes, ovum and sperm, are haploid.)
One-third of triploid conceptions arise due to fertilization of a diploid ovum by a haploid sperm.
Tetraploidy (4n) - These cells have four copies of the haploid set. Fetuses with this constitutional abnormality are also nonviable or do not live long after birth. The written karyotype of these conceptions is either 92,XXXX or 92,XXYY.
This type of abnormal conception arises due to the failure of the first cleavage division in the zygote.
Care must be taken in analyzing fetal material, which is positive for tetraploidy mosaicism. Tetraploidy is a common artifact of tissue culture and may be found in the cultures of normal fetuses.
AneuploidyTerms - This is the gain or loss of a given chromosome.
Monosomy - One copy of a given chromosome.
Disomy - Two copies of a given chromosome. This is the normal situation.
Trisomy - 3 copies of a given chromosome.
Tetrasomy - 4 copies of a given chromosome.
Mechanisms of Aneuploidy
Nondisjunction - Nondisjunction is the failure of paired chromosomes (Meiosis I) or sister chromatids (Meiosis II or Mitosis) to separate at anaphase. If nondisjunction occurs during Meiosis I or during Meiosis II, this abnormal division can result in a constitutionally abnormal individual. If nondisjunction occurs during Mitosis, this abnormal division will result in a mosaic individual.
Anaphase lag - This mechanism of aneuploidy refers to when a chromosome lags behind as the spindle separates the chromosomes to opposite poles.
Aneuploidy is the most common karyotypic anomaly encountered in phenotypically abnormal live births. It is estimated that 20 percent of all human conceptions are aneuploid.
Constitutional autosomal monosomies are not compatible with life. Constitutional autosomal trisomies are limited to the smaller chromosomes. Clinical examples of these aneuploidies will be given on page 31 of this module.
28
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
2. Structural rearrangements
- Reciprocal Translocations
Chromosome breakage is not a rare event. Daily our cells undergo assault by external and internal clastogens. Each cell has numerous enzymes, which repair broken DNA strands. This repair goes on throughout the life of each cell. Most of the time the DNA is repaired correctly, but occasionally errors will occur. When these rearrangements occur in germ cells, they can give rise to offspring with constitutional chromosomal abnormalities. Constitutional abnormalities are present in all cells of an individual. If these rearrangements are acquired after zygote formation, they give rise to individuals with nonconstitutional chromosomal abnormalities seen in only a proportion of cells.
Chromosomal structural rearrangements can be divided into two groups.
1. Interchromosomal rearrangements
Reciprocal Translocations
These are balanced exchanges of genetic material between two or more chromosomes. In other words, no genetic material is lost during the exchange. These rearrangements can occur when two or more chromosomes simultaneously break. Whenever chromosomes break, they have "sticky ends" which can readily unite with another "sticky end."
Approximately one out of 1,000 individuals is a balanced translocation carrier. Phenotypically these individuals are normal; however they are at risk for having miscarriages or abnormal children depending on the breakpoints and chromosome involved in rearrangement. The actual risk for an abnormal outcome due to abnormal meiotic configurations is 11-12%. Twenty percent of individuals with multiple miscarriages will be balanced translocation carriers.
If alternate segregation of the chromosomes takes place, balanced gametes will be formed. These gametes will contain either the normal copies of the chromosomes or the balanced translocation.
If adjacent segregation of the chromosomes takes place, unbalanced gametes will be formed. In most instances this will lead to fetal demise. The smaller the segment of DNA involved in translocation, the more likely those abnormal fetuses will come to term.
29
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
2. Structural rearrangements
- Robertsonian translocations
These translocations occur when there is a fusion in the centromere region between two acrocentric chromosomes. Individuals with Robertsonian translocations and only one other normal homolog of each of the chromosomes involved are phenotypically normal. They have normal phenotypes because they still have only two copies of all the genes except for the short arms of the two acrocentric chromosomes involved. (The short arms contain ribosomal genes located on all acrocentrics and highly repetitive satellite DNA; the loss of these regions is well tolerated). However, these individuals will have unbalanced gamete formation and have a 2/3 theoretical risk of having abnormal conceptions if mated with a normal individual.
Approximately one out of 1,000 individuals are carriers of Robertsonian translocations. Der(13;14)(q10;q10) is the most commonly observed Robertsonian translocation followed by der(14;21)(q10;q10).
Even though the theoretical risk of a translocation Down's fetus is 1/3 in individuals who are carriers of der(14;21)(q10;q10) translocations, the actual empirical risk is 10-15% if the mother is the carrier and 0-2% if the father is the carrier.
Robertsonian translocations form a trivalent configuration. Alternate segregation produces roughly equal frequencies of balanced gametes. That is, gametes with either normal chromosomes or the Robertsonian translocation.
Adjacent segregation produces unbalanced gametes, three which are lethal (monosomy 21, monosomy 14, and trisomy 14) and one which results in translocation Down Syndrome.
30
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
2. Structural rearrangements
- Deletions
Deletions result in the loss of chromosomal material. This results in functional monosomy of the segment involved.
Terminal deletions are the result of one break with the subsequent loss of the acentric fragment during cell division due to anaphase lag.
Interstitial deletions result from two breaks within the same chromosome arm. Again the acentric fragment is lost during the following cell divisions.
31
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
2. Structural rearrangements
- Duplications
Duplications occur when a portion of chromosomal material is duplicated. This extra genetic material results in functional trisomy for this segment of DNA. Duplicated material may be inserted in the same order as the original segment or may be inverted and then inserted in the reverse order.
It is possible for duplications to result from unequal crossing over events in meiosis or from a rearrangement between two chromatids during mitosis.
32
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
2. Structural rearrangements
- Inversions
Inversions result when two breaks occur in the same chromosome and the middle segment rotates 180o before DNA repair. This results in no net loss of genetic material, but will change the gene order and will give rise to abnormal gamete formation in carriers.
Pericentric inversions occur when one break is in the short arm of the chromosome and the other break is in the long arm of the chromosome. Note that the centromere is involved with a pericentric inversion.
Most constitutional pericentric inversions do not affect the phenotype of the carrier; however, they affect the offspring of a carrier.
Each unbalanced product has a duplication and a deletion. Viability depends upon the size of the segments involved as well as what genes are involved. The risk of abnormal offspring is similar to the risk observed in an individual with a balanced reciprocal translocation.
Paracentric inversions occur when both breaks occur within the same arm of the chromosome. The centromere is not involved in this inversion.
In meiosis an inverted chromosome must form a loop in the region of the inversion in order to pair with its normal homolog. Unbalanced products are either acentric or dicentric. Neither dicentric or acentric chromosomes are stable, so the risk of unbalanced offspring is low. Individuals who have paracentric inversions as constitutional abnormalities may have reduced fertility as a consequence of this unbalanced gamete formation.
33
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
2. Structural rearrangements
- Isochromosomes
An isochromosome is formed when there is a misdivision of the centromere. Normally, the centromere divides vertically during cell division. Occasionally, the centromere will divide horizontally and the result will be one daughter cell with two sister chromatids of the short arm and the other daughter cell with two sister chromatids of the long arm.
During the S-phase following this mitotic error, the centromere as well as both copies of the short or long arms will duplicate. One arm of an isochromosome is therefore a mirror image of the other arm.
A cell with an isochromosome is trisomic for one chromosome arm and monosomic for the other arm.
34
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
2. Structural rearrangements
- Ring chromosomes
Ring chromosomes are formed when there are two terminal breaks occurring on the same chromosome. If these breaks form a union, a continuous DNA molecule is formed with one centromere. These structures, like dicentrics, are highly unstable because they either break or lag behind during anaphase.
35
Syndromes associated with sex chromosome abnormalities
Extra copies of the sex chromosomes are better tolerated than extra copies of the autosomal chromosomes. However, the clinical phenotype becomes progressively worse as the number of additional sex chromosomes increases. (For example, 47,XXX, individuals have a more normal phenotype than 48,XXXX individuals, and these 48,XXXX individuals have a more normal phenotype than 49,XXXXX individuals).
36
Syndromes associated with autosomal chromosome abnormalities
47,XX,+13 Patau
47,XX,+18 Edward
47,XX,+21 Down
Most autosomal trisomies are nonviable and result in spontaneous abortions.
Viable autosomal trisomies include +13, +18 and +21. Many of these conceptions will die in utero and those that make it to term will have clinically abnormal phenotypes.
Nonviability of the fetus and severity of the clinical phenotype increases as the size of the chromosome increases and as the proportion of euchromatin to heterochromatin increases. (13, 18, & 21 are relatively small chromosomes with a large proportion of dark heterochromatic G-bands).
37
Syndromes associated with autosomal deletions and duplications
```
Wolf Hirschhorn del(4)(p16)
Cri-du-Chat del(5)(p15)
Retinoblastoma del(13)(q14)
Cat's Eye dup(22)(pter-q11) tetrasomy of 22p
```
38
Syndromes associated with microdeletions
Microdeletions are often due to meiotic recombination errors. These deletions can only be detected with high resolution chromosome banding or molecular cytogenetic techniques (FISH) when the deletion is submicroscopic.
Some of these microdeletions have a complex clinical phenotype because they occur in chromosomal regions containing a number of important structural genes. Therefore, the clinical phenotype is related to both the size of the deletion as well as those genes actually deleted. Sometimes these syndromes are referred to as contiguous gene syndromes.
```
Prader-Willi
Angelman
Langer-Giedion
Miller-Dieker
DiGeorge
Aniridia-Wilms' Tumor
```
39
Syndromes associated with autosomal chromosome abnormalities
Uniparental disomy (UPD) - This term is used to describe events, which lead to a zygote inheriting two identical chromosomes from the same parent without inheriting the matching chromosome from the other parent. It is believed that the majority of UPD reflect maternal age-related nondisjunction, which leads to trisomy or monosomy of a particular chromosome. These lethal conditions may be "rescued" by a second event (duplication in cases of monosomy or deletion in cases of trisomy). This duplication of an identical set of genes leads to loss of heterozygosity. This increases the risk of recessive disorders and can cause maternal/paternal gene dosage imbalances, which may be responsible for abnormal fetal development.
Imprinting - Hypermethylation of clinically relevant genes can result in a phenotype similar to that observed in patients with microdeletions and uniparental disomy. When DNA is hypermethylated gene expression is blocked and the gene is effectively silenced. Differential methylation of DNA can therefore cause genetic disorders. Evidence also exists for antisense RNA silencers, which can prevent gene expression by binding to complementary DNA.
40
Acquired abnormalities
These abnormalities are nonconstitutional. They are present in only a small percentage of cells in the human body. Clone is a term used to define a subpopulation of cells with a related chromosome profile.
Acquired chromosomal abnormalities originate from external or internal environmental forces which cause the chromosomes to break (leads to structural rearrangements) which adversely affect the mitotic apparatus (leads to numerical abnormalities).
Acquired Chromosome Abnormalities
Clastogens are agents which cause chromosome breaks. Commonly known clastogens include:
```
Viruses
Radiation (g or UV)
Pesticides
Chemotherapeutic agents
Toxins
Others
Tumorigenesis is the process whereby normal cells become tumor cells through the acquisition of a number of distinct aberrant traits which allow neoplastic growth, proliferation and metastasis to distant sites.
```
Tumors must acquire continual growth stimulus.
Tumors must successfully evade the immune system.
Tumors must acquire vasculature to supply nourishment and for the removal of metabolic waste.
Tumors must be able to invade adjacent tissue and invade the bloodstream for metastasis to distant sites.
Characteristics of Genes Which When Mutated Enhance Cancer Susceptibility
Genes which:
affect metabolism of potentially carcinogenic compounds.
repair genetic damage.
affect growth regulation.
influence the ability of the immune system to detect and destroy potential tumor cells.
Oncogenes - Genes which when mutated become activated and impart malignant traits to cells through the acceleration of cellular growth (proto-oncogenes term which refers to the normal form of these cellular genes)
Tumor Suppressor Genes - Genes which when mutated become inactivated and impart malignant traits to cells because they fail to restrict cellular growth.
41
Leukemias
This cancer of the hematopoietic system arises from clonal expansion of lymphoid or myeloid cells which replace the normal bone marrow. (Potter and Watmore, and Pui, et. al., 1990)
Acute Leukemias - Clonal expansion of immature blasts associated with an abrupt onset of clinical symptoms and a rapidly progressing clinical disease. Acute leukemias are classified as such when greater than 30% blasts are found in the peripheral blood or in the bone marrow.
Chronic Leukemias - Clonal expansion of mature-appearing cells with a slow onset of clinical symptoms and a slowly progressing clinical disease.
42
MDS
-5,-7,+8, del(5q), del(7q)
refractory anemia
refractory anemia with ringed sideroblasts
refractory anemia with excess of blasts
refractory anemia with excess of blasts in transformation
CMML
43
AML
M0-AML with minimal myeloid differentiation
M1- AML without differentiation
M2-AML with differentiation t(8;21)(q22;q22)
M3- acute promyelocytic leukemia t(15;17)(q22;q11-12)
M4- acute myelomonocytic leukemia inv(16)(p13q22)
M5-acute monocytic leukemia t(9;11)(p22;q23); other 11q23 rearrangements
M6-acute erythroleukemia del(5q), del(7q), +8
M7-acute megakaryocytic leukemia
44
ALL
Acute lymphoblastic leukemia
- pre B t(1;19)(q23;p13)
- B-cell t(9;22)(q34;q11); t(4;11)(q21;q23); t(8;14)(q24;q32); t(2;8)(p12;q24); t(8;22)(q24;q11)
- T-cell translocations involving 14q11, 7p15, 7q34
45
CLL
Chronic lymphocytic leukemia B-cell +12, 14q32 translocations
46
Myeloproliferative disorders:
CML=chronic myelogenous leukemia t(9;22), i(17q), +8, +Ph, +19
Polycythemia vera del(20)
Essential thrombocythemia unknown
Myelofibrosis unknown
47
Lymphomas
During maturation, B-lymphocytes undergo immunoglobulin gene rearrangement and T lymphocytes undergo T cell receptor gene rearrangement. This cutting and rejoining of DNA provides the variability and specificity of antibodies in B-lymphocytes and the T cell receptor in T lymphocytes. This process is prone to recombination errors, which result in DNA translocations. If this rearrangement results in turning on an oncogene, that cell will divide (proliferate) out of control and result in a lymphoma (NOTE: This is also true of the T and B-cell leukemias). C-myc, located on 8q24, is a common oncogene, which is translocated to these regions.
The breakpoints involved in consistent chromosome translocations observed in the cytogenetic study of lymphomas correspond to the sites of the B cell immunoglobulin and T cell receptor genes.
48
B cell immunoglobulin genes
14q32 Immunoglobulin heavy chain gene
2p12 Immunoglobulin light chain gene, kappa
22q11 Immunoglobulin light chain gene, lambda
49
T cell receptor genes
14q11 T cell receptor gene, alpha and delta chains
7q34 T cell receptor gene, beta chain
7p15 T cell receptor gene, gamma chain
50
Hodgkin's lymphoma
currently no specifically identified chromosome abnormalities
51
Burkitt's lymphoma
t(8;14)(q24;q32)
t(2;8)(p12;q24)
t(8;22)(q24;q11)
52
Follicular lymphoma
t(14;18)(q32;q21)
53
Anaplastic large cell lymphoma
t(2;5)(p23;q35)
54
Mantle cell lymphoma
t(11;14)(q13;q32)
55
T-cell lymphoma
rearrangements of 14q11, 7p15, 7q34
56
Cytogenetic Analysis of Solid Tumors (CAST)
Tumor Heterogeneity
1. Cancer is a genetic disease.
2. Most cancers are monoclonal in origin.
3. Tumor cells are genetically very unstable.
4. This instability may be inherited or acquired.
5. Malignant tumors contain multiple clones and subclones.
6. Tumor metastasis is a clonal and controlled process.
7. Some cancers are resistant in nature to treatment.
8. Gene amplification is found in some resistant tumors.
Terminology
1. oma - tumor (benign or malignant)
2. carcinoma - malignant epithelial tumor
3. sarcoma - malignant mesenchymal tumor
4. fibro - fibrous
5. adeno - gland
6. lipo - fat
7. leio - smooth muscle
8. rhabdo - striated muscle
9. osteo - bone
10. chondro - cartilage
57
Mosaicism/Chimerism
Mosaicism arises due to mutational events, which occur after conception. These postzygotic changes can then expand in a subset of an individual's cells during embryogenesis. Depending on the abnormality and the distribution of abnormal cells, the effect on the phenotype can range from benign to severe. page39
Chimerism - Chimeric individuals have two or more cell lines within their body. This is quite different from mosaicism and results from either:
a. The fusion of fraternal twin zygotes.
b. Double fertilization of an egg and polar body.
c. Exchange of hemopoietic stem cells by dizygotic twins.
58
Culture artifacts
a. de novo translocations - Occasionally chromosome translocations and deletions will occur in cells when grown artificially in the laboratory. These spurious translocations are often nonclonal and are attributed to culture conditions or normal random mutations.
b. Confined placental mosaicism - This refers to nondisjunction events, which occur in the outer cell membranes. The following describe possible placental, fetal combinations, which can occur in pregnancies where mosaicism for trisomy 21 is present.
1. Normal fetus and mosaic trisomy 21 villus
2. Mosaic trisomy 21 fetus and mosaic trisomy 21 villus
3. Mosaic trisomy 21 fetus and normal villus
c. Maternal cell contamination - Chorionic villus sampling and amniocentesis procedures may result in maternal cells being removed along with fetal cells. During cell culture, if the maternal cells also form colonies it becomes difficult to discern the fetal cells from the maternal cells. Experienced cytogeneticists can make use of the sex chromosomes to determine maternal cell contamination in cases where the fetus is male, but must use chromosome polymorphisms to determine maternal cell contamination in cases where the fetus is female.
Amniocentesis
Maternal cells can enter the needle inserted through the abdomen of the mother during the collection of the amniotic fluid that surrounds the fetus. To prevent maternal cell contamination, which arises from this procedure, the first 1-2cc of amniotic fluid collected should be discarded. Maternal cell contamination has also been associated with bloody taps.
Chorionic villus sampling
During the CVS procedure a biopsy is taken either transabdominally or transcervically with the assistance of ultrasound. The biopsy contains the fetal villus and the surrounding maternal decidua. Before culturing, the maternal decidual tissue is dissected away from the villi using a dissecting microscope. Following dissection, the villi are digested with a protease to remove any remaining maternal cells and the cytotrophoblast layer.
The fetal villi are morphologically distinct from the maternal decidua in that they have multiple branches and buds whereas the maternal decidua does not. Even with this morphological distinction and the precautions taken to ensure 100% fetal cells are cultured, it is impossible to remove all the maternal cells in all instances.
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Pseudomosaicism
"Pseudo", meaning false refers to any clonal or nonclonal change that can be attributed to cultural artifacts and do not reflect the true karyotype of the fetus.
A chromosome abnormality observed in only one cell culture but not in another cell culture established from the same individual.
A chromosome abnormality observed in only one colony of an in situ culture.
A chromosome abnormality observed in only one cell from a specimen, culture, or colony.
A chromosome abnormality not confirmed in abortus material or in the blood culture of the fetus or newborn.
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COMMON MULTI-CELL PSEUDOMOSAICISMS
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2 2%
7 10.8%
X 8.8%
17 6.1%
20 6.1%
9 5.4%
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Normal variants
Human chromosomes have variable regions, which can have differing amounts of DNA. Individuals with size variants of these regions are phenotypically normal. Normal variants are also referred to as polymorphisms.
These chromosomal regions include the C-band regions of all chromosomes and the satellites of the acrocentric chromosomes.
These regions contain highly repetitive DNA sequences that are heterochromatic, late replicating, for the most part genetically inert, and contain few functional genes. These characteristics allow duplications or deletions of these regions to be well tolerated and clinically associated with normal human development.
True abnormalities involve rearrangements of the euchromatic portions of chromosomes. These chromosomal regions contain many unique sequences of DNA and are therefore gene rich. Deletions and duplications in these regions have a greater chance of having an adverse effect on normal human development.
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Distinguish chromosome heteromorphisms for tracking purposes and as case specific identifiers.
Chromosome hetermorphisms of chromosomes 1, 9, 16, Y and the short arms of the acrocentric chromosomes are useful in tracking particular chromosome variants that are present in various members of a family. They can also provide useful clues in the determination of maternal cell contamination as well as sample mix-ups.
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Identify variants
C-band variants - satellite and heterochromatic areas
Size heteromorphisms are common in the C-band regions of human chromosomes. The most commonly observed heteromorphisms involve chromosomes 1, 9, 16, and Y. These chromosomes contain the largest amounts of C-band material in the majority of individuals. Size heteromorphisms are also commonly observed among the satellites of acrocentric chromosomes. Satellites can range in size from nonexistent to large, and can be present in a duplicated form termed "double decker" satellites.
Inversions within the C-band material located near the centromere are also commonly observed. Inversions of chromosome 9 are fairly common within the population. Also inversions of chromosomes 1 and 16 are observed at a lower frequency. These inversions are associated with a normal phenotype; however, individuals with these inversions have an increased risk for producing unbalanced gametes. This risk increases with the size of the inversion (risk of crossing-over within inversion loop during meiosis increases).
C-band variants - satellite and heterochromatic areas (cont.)
Fragile sites - There have been over 100 fragile sites reported in the literature. These fragile sites may be expressed spontaneously in rare individuals, but for the most part these fragile sites are induced through a variety of culture conditions. (e.g. Fragile X needs folate deficient media to be expressed).
Fluorescent intensity - Certain heterochromatic regions will stain very brightly with fluorescent dyes such as quinacrine dihydrochloride and DAPI. These regions include the centromere region of chromosome 3 , 4 and the acrocentric chromosomes as well as the q12 band of chromosome Y.
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Use established format for recording final result
The following information should be included in the final cytogenetic report:
a. Patient data, including name, address, age, date of birth, sex, social security number, referring physician, clinical indication for cytogenetic testing, and family history.
b. Specimen data, including tissues studied, the date sample was received, pertinent information relating to quality of specimen and factors occurring during transportation or set-up procedures which may have influenced the final report.
c. Laboratory data, including the number of cultures initiated, the number of cells analyzed, karyotypes, banding techniques used, and quality of preparation.
d. Cytogenetic data
Results stated in the current ISCN format.
Interpretation of results for the benefit of the physician. This interpretation should be in layman terms for the non-cytogeneticist. Interpretation should also include the correlation of the cytogenetic results to the clinical features of the patient.
Recommendations for:
a. Analysis of other tissue.
b. Family studies.
c. Other necessary testing.
d. Genetic counseling.
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Notify designated authority with final result
The cytogenetic report, after approval by a CAP certified pathologist, is then sent to the referring physician for signature. A copy of the signed report is placed in the patient's file, given to the patient's physician and sent to medical records.
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Awareness of possible miscommunication in oral reporting
Oral reports are necessary if a sample is received on stat status. Stat indicates that the results of the testing are vital to the patient's welfare and clinical outcome.
Whenever there is oral communication between two different individuals with two different perspectives, there is the chance of miscommunication. When results are communicated over the telephone, miscommunication is even more commonplace. To ensure the proper documentation of an oral report, the reporting individual should ask the individual accepting the report to repeat the results and the interpretation back to them (feedback). This alone will cut down on the number of miscommunications.
Two critical elements often interfere with the communication process.
Perception is our interpretation of reality. This is a primary source of miscommunication because people's perceptions are often different.
Noise is anything that interferes with the transfer of meaning.
Physical noise - For example others talking nearby, construction, etc...
Nonverbal noise - Someone waiting to talk to you, another phone call, etc…
Oral reporting of the cytogenetic report should always be followed by a written report as soon as possible.
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Use ISCN nomenclature
Order of the written karyotype.
51,X,t(X;18)(p11;q11),+1,+del(1)(q13),+inv(1)(p13q13),+8,+10,+21,-22[20]
The number of chromosomes in the cell is indicated first followed by the sex chromosomes. Sex chromosome abnormalities precede autosomal abnormalities and the autosomal abnormalities are listed in order from 1 to 22. If more than one abnormality of a particular chromosome number is observed, the abnormalities are placed in alphabetical order. The number in brackets at the end of the written karyotype indicates the number of cells analyzed with this karyotype.
Numerical Abnormalities
1. Sex chromosome aneuploidy
Constitutional
47, XXY
Extra copies of the sex chromosomes are indicated without separation with a comma or with a plus sign; the same is true for missing copies of the sex chromosomes.
45,X
Acquired
47,XY,+X or 45,X,-X
Nonconstitutional gains or losses of the sex chromosomes are indicated with plus and minus signs.
2. Autosomal chromosome aneuploidy
47,XX,+21 or 45,XX,-21
Extra copies of autosomes are separated from the sex chromosomes with a comma. The same is true for missing copies of autosomes. This format is used for both constitutional and acquired karyotypes.
Structural rearrangements
Interchromosomal rearrangements
a. Balanced translocations
Translocations involving two chromosomes
46,XX,t(8;14)(q24;q12)
This indicates that the distal segment of chromosome 8q24 is translocated to the distal segment of chromosome 14q12 and the distal segment of chromosome 14q12 has rejoined at 8q24.
Translocations involving three chromosomes
46,XX,t(8;22;14)(q24;q11;q12)
This indicates that the distal segment of chromosome 8q24 has been translocated to chromosome 22 at the band q11 and the distal segment of chromosome 22q11 has moved to 14q12. The segment distal to band 14q12 in turn has translocated to chromosome 8 at band q24. Note that this is an exception to the rule that chromosomes should be placed in ascending order.
b. Unbalanced rearrangements
Robertsonian translocations
45,XX,der(14;21)(q10;q10)
Robertsonian translocation as discussed earlier in this section is the result of centric translocations between the acrocentric chromosomes with the subsequent loss of the short arms. Band q10 indicates that the breaks occurred in the centromeres of chromosomes 14 and 21.
Dicentric chromosomes
45,XY,dic(2;4)(p12;p14)
This written karyotype indicates that a translocation has occurred between chromosomes 2 and 4 resulting in a dicentric chromosome with loss of the segment distal to 2p12 and distal to 4p14. Note that the missing normal chromosomes 2 and 4 are not indicated with minus signs because the dicentric chromosome replaces them.
Insertions
1. Direct insertions imply that the inserted segment maintains its original orientation after insertion.
Within a chromosome
46,XY,ins(5)(q14q21q31)
The segment between bands 5q21 and 5q31 has been inserted at band 5q14.
Between two chromosomes
46,XY,ins(5;1)(q14;q22q32)
The segment between bands 1q22 and 1q32 has been inserted at band 5q14. Note that the chromosomal band where the insertion occurs is always written first irrespective of numerical order.
Inverted insertions imply that the inserted segment has reversed its original orientation after insertion.
Within a chromosome
46,XY,ins(5)(q14q31q21)
The segment between bands 5q21 and 5q31 has been inverted and inserted at band 5q14. The band 5q21 is now more distal to the centromere than 5q31 which is now more proximal to the centromere.
Between two chromosomes
46,XY,ins(5;1)(q14;q32q22)
The segment between bands 1q32 and 1q22 has been inserted at band 5q14 in reverse order relative to the centromere. (1q32 is now more proximal to the centromere and 1q22 is more distal.)
Intrachromosomal rearrangements - A common feature of intrachromosomal rearrangements is that the written karyotype does not have a semicolon between the rearranged bands.
Deletions
Terminal Deletions
46,XX,del(7)(p11)
This indicates that the segment of chromosome 7 distal to 7p11 has been deleted from this cell.
Interstitial Deletions
46,XX,del(7)(p11p13)
This karyotype indicates that the segment of chromosome 7 between bands 7p11 and 7p13 has been deleted from this cell.
Inversions
Pericentric - These inversions result from a break occurring in both the short and long arms of a chromosome and the broken piece rotating 180o before restitution of the broken ends. This inversion involves the centromere. Note that the p arm is indicated first.
46,XY,inv(3)(p21q14)
Paracentric - These inversions result from two breaks occurring in one arm of a chromosome and the broken piece rotating 180o before restitution of the broken ends. This inversion does not involve the centromere. Note that the band most proximal to the centromere is indicated first.
46,XY,inv(3)(p14p21)
Duplications
Direct - A direct duplication indicates that the duplicated segment retains its original orientation.
46,XX,dup(2)(q22q25)
Inverted - An inverted duplication indicates that the duplicated segment is inverted relative to its original orientation.
46,XX,dup(2)(q25q22)
Isochromosomes - Isochromosomes arise due to misdivision of the centromere during mitosis. The resulting chromosome will have a mirror image of one of its whole arms. The break point is assigned based on the arm involved.
46,XX,i(17)(q10)
This written karyotype indicates that this cell is monosomic for the short arm of chromosome 17 and trisomic for the long arm of chromosome 17.
Ring chromosomes
46,XX,r(4)(p14q31)
Fragile Sites
Fragile sites are genetically unstable regions of chromosomes that are prone to break when exposed to various mutagens or exposed to a folic acid deficient environment (Yunis, 1989). Fragile X is an example of this phenomenon. This inheritable syndrome is the main cause of mental retardation in males and is the only inherited disorder associated with a chromosome fragile site. At the molecular level there is an expansion of CGG trinucleotide repeats in the FMR-1 gene located in band (X)(q27.3). Five to fifty percent of cells in affected individuals will express this fragile site when grown in folic deficient media (Thompson, 1991).
Female
46,X,fra(X)(q27.3)
Males
46,Y,fra(X)(q27.3)
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Use ISCN nomenclature
In Situ Hybridization
With the advent of DNA probe methodologies, the sensitivity of chromosomal tests has dramatically increased. Hence, the following new ISH ISCN nomenclature was proposed in 1995.
ish= metaphase FISH
nuc ish= interphase FISH
fib ish= FISH performed on DNA fibers
rev ish= FISH using probes made using reverse transcriptase
PAGE53!!!!!pics
Metaphase FISH
Centromeric probes - A metaphase cell from an individual with a 46,XY karyotype showing two signals after hybridization with chromosome 8 centromeric probe.
46,XY.ish 8cen(D8Z2x2)
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The period (.) separates the cytogenetic results from the FISH results.
D8Z2 is the terminology that describes where the probe lies on the physical genome map.
X2 indicates that two signals are observed in this cell.
Painting probes - A metaphase cell from an individual with a 46,XY karyotype showing two positive chromosome signals after hybridization with a chromosome 9 painting probe.
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46,XY.ish 9(wcp 9x2)
wcp = whole chromosome painting probe
Regional (locus) specific - Interphase cells from an individual with a 46,XY,t(9;22)(q34;q11.2) karyotype. Cells show three signals after hybridization with the abl and bcr cosmid probes, one red, one green, and one hybrid red/white/green signal.
Interphase FISH
Centromeric probes - Interphase cells from an individual with a 46,XY karyotype following bone marrow transplantation. Cells show two signals after hybridization with chromosome X and Y probes, one red and one green.
nuc ish Xcen(DXZ1x1), Yq12(DYZ1x1)
Regional (Locus) Specific - Interphase cells from an individual with a 46,XY,t(9;22)(q34;q11.2) karyotype. Cells show three signals after hybridization with the abl and bcr cosmid probes, one red, one green, and one hybrid red/white/green signal.
nuc ish 9q34(ABLx2), 22q11.2(BCRx2)(ABL con BCRx1)
con = connected
Regional (Locus) Specific ES (extra signal) probes - Interphase cells from an individual with a 46,XY,t(9;22)(q34;q11.2) karyotype. Cells show four signals after hybridization with a bcr/ab; ES probe, two red, one green, and one hybrid red/white/green signal.
nuc ish 9q34(ASSx2,ABLx2), 22q11.2(BCRx2)(ABL con BCRx1)
OR
nuc ish 9q34(ABLx3), 22q11.2(BCRx2)(ABL con BCRx1)
Regional (Locus) Specific D (dual fusion) FISH probes - Interphase cells from an individual with a 46,XY,t(11;14)(q13;q32) karyotype. Cells show four signals after hybridization with a lgH/CCND1 (Cyclin-D) probe, one red, one green, and two hybrid red/white/green signals.
nuc ish 11q13(CCND1x3),14q32(IgHx3)(CCND1 con IgHx2)
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Evaluate the need for additional studies
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Evaluate the need for additional studies
Repeat culture
Clotted blood or bone marrow
Amnio or CVS culture which shows no cell attachment within 3-5 days.
Contaminated culture
No growth
Analysis of other tissues
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Peripheral blood lymphocytes
Leukemias or lymphomas with circulating immature blasts
To rule out constitutional abnormality
Family studies when a balanced translocation carrier is expected.
To confirm abnormal prenatal findings in newborns.
Abortus tissue - This tissue is cultured and studied cytogenetically in order to confirm abnormal prenatal cytogenetic findings.
Request of family studies
Unbalanced rearrangements detected in prenatal samples or phenotypically abnormal newborns necessitate the need for genetic counseling and follow-up family studies. These unbalanced rearrangements may have arisen due to the presence of balanced translocations in the parents. Therefore, it is important to identify those families containing balanced translocations who are consequently at risk for chromosomally abnormal fetuses.
For example Robertsonian translocation carriers produce abnormal gametes in a ratio of 2:1 to normal gamete production.
Request of family studies page55-60
Autosomal dominant patterns of inheritance
The phenotype appears in every generation with each affected individual having an affected parent.
Any child of an affected parent has a 50% risk of being affected.
Phenotypically normal individuals have only phenotypically normal children.
Males and females are equally likely to transmit the phenotype to children of either sex. Distinctive from sex-linked dominant traits in that males can transmit the phenotype and can have unaffected daughters.
Autosomal recessive patterns of inheritance
The abnormal phenotype is typically observed among siblings and is not observed in parents, offspring, or other relatives.
The risk for each sibling of the proband having the abnormal phenotype is one-fourth.
Can occur in children of a consanguineous mating (mating between related individuals).
Males and females are equally likely to be affected.
X-linked dominant patterns of inheritance
The abnormal phenotype is seen equally among males and females when the mother transmits the gene responsible for the condition.
The abnormal phenotype is never transmitted directly from father to son.
All daughters of an affected male inherit the phenotype.
X-linked recessive patterns of inheritance
The abnormal phenotype is observed more frequently in males than females.
There is no male to male transmission of the phenotype.
All daughters of an affected male are usually phenotypically normal but have a 50% risk of having phenotypically abnormal male children.
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Biochemical or molecular analysis
Alpha fetoprotein (AFP) and the Triple screen are common prenatal biochemical tests used for the detection of neural tube and cytogenetic defects.
Alpha fetoprotein (AFP)
Alpha fetoprotein is a plasma protein made by the fetal liver. It has similar chemical and physical characteristics to those of albumin. AFP can be detected during pregnancy, both in the amniotic fluid (AFAFP) and in the blood of the mother (MSAFP). If a fetus has a neural tube defect (NTD) there will be leakage of fetal plasma into the amniotic fluid. This leakage will significantly elevate AFAFP and MSAFP levels.
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Triple Screen Used to study the levels of the following products produced by the fetal/placental unit in the amniotic fluid or maternal serum.
MSAFP (maternal serum alpha fetoprotein
uE3 (estriol)
hcG (human chorionic gonadotropin)
With this test, trisomy 18 and trisomy 21 can be predicted. In both cases estriol and AFP levels will be low; however, hcG will be low in trisomy 18 cases and elevated in trisomy 21 cases.
Note: Other factors may result in abnormal triple screen tests even though the fetus is normal. For example, obese women and pregnancies with more than one fetus will result in abnormal product levels in the maternal serum or amniotic fluid.
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All of the following criteria are desirable when choosing a metaphase for karyotyping except:
A. Long chromosomes
B. Highly contrasted chromosomes(+)
C. Few overlapping chromosomes
D. Clearly banded chromosomes
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All of the following will result in erroneous chromosome count except:
A. Nomadic intruders
B. Overspreading
C. Debris
D. Undertrypsinization(+)
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The criteria used for the selection of metaphase spreads suitable for analysis includes all of the following except:
A. Presence of an intact cell membrane(+)
B. Chromosome band level (length)
C. The quality of G-banding
D. The number of overlapped chromosomes
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Which of the following is true when the banding level is 550-600?
A. There are 2 dark bands on 7q
B. There are 2 dark bands on 11p
C. There are two dark bands on 9q
D. The first dark band on 10q is split into 2 different bands(+)
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The acceptable minimal band level for most specimens is
A. 300
B. 400(+)
C. 500
D. 600
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The minimal band level to detect PWS syndrome is:
A. 350
B. 450
C. 550(+)
D. 850
