W8L12 - Cytogenetics

Question 1

What is Cytogenetics?

Answer 1

The study of genetic material at the cellular level: the study of chromosomes

Question 2

Where is Cytogenics used?

Answer 2

Prenatal investigations
Constitutional studies
Infertility/recurrent fetal loss
Spontaneous miscarriage
Leukaemia
Solid tumour and lymphoma evaluation

Question 3

Specimen Types in Cytogenics

Answer 3

Peripheral blood
Amniotic fluid
Chorionic villus sample
Products of conception
Bone marrow
Tumour biopsy
Skin biopsy

Question 4

Chromosome Structure and Identification

Answer 4

Chromosomes are arranged in a karyogram based on centromere position, band pattern and length of chromosome
The smaller arm of the chromosome is the “p” arm and the long arm is the “q” arm
Ordered from largest to smallest

Question 5

Position of Centromere

Answer 5

Metacentric 
- centromere near the middle
- p and q arms similar size
Sub-metacentric
- centromere closer to one end
- smaller p arm and larger q arm
Acrocentric 
- centromere at top
- very small p arms and often have satellites (NOR region) on the end
Telocentric
- have the centromere at the end with no p arm (not present in humans)

Question 6

G Banding

Answer 6

Preferred method of staining for chromosomes
Treat slides with protease such as trypsin
Banding patterns obtained are thought to reflect both structural and functional composition

Question 7

Constitutional and Acquired Chromosomal Abormalities

Answer 7

Constitutional
- present from early fetal development
- either at the zygote stage or shortly afterwards in embryogenesis e.g. down syndrome
Acquired
- occur during the life of a person e.g. leukaemia or solid tumour growth

Question 8

Detection of Imbalances

Answer 8

Conventional cytogenetics
FISH - fluorescence in situ hybridisation
Quantitative fluorescence PCR
Microarray/array CGH

Question 9

Detection of Imbalances - Fluorescence in situ Hybridisation

Answer 9

Method using DNA probes to bind to specific sequences on chromosomes in cytogenetic preps and then detecting these probes by labelling them with fluroescent dyes
Often used for finding specific features in metaphase and interphase cells
- identification of numerical abnormalities
- investigation of structural aberrations

Question 10

Repetitive Sequence Probes used with FISH

Answer 10

Centromeric or Alpha Satellite DNA probes
- centromeres of each chromosome can be distinguished with individual probe
- chromosomes 13&21 and 14&22 dont work as their centromeric regions are too similar and will cross hybridise
- used for identifying aneuploidy, mosaicism, markers
Whole chromosome paints (WCP)
- designed to mark the entire chromosome of interest
- useful for identifying marker chromosomes and complex rearrangements e.g. insertions
- small rearrangements of <2-3Mb will not be detected

Question 11

Unique Sequence Probes (Locus Specific Inserts)

Answer 11

Target regions that are not repeated in the genome and may code for a gene
Used for:
- microdeletions/deletions
- oncogenes such as n-myc, c-myc
- sub-telomeric region specific for each chromosome end
Can be probes that break apart and fusion probes that are used in leukemic investigations

Question 12

Common Cytogenic Changes in Leukaemia

Answer 12

Most well known is the Philadelphia chromosome in CML
This results from a translocation between chromosomes 9 and 22 creating the BCR/ABL fusion gene
Most common changes in Myelodyplastic syndromes include del(5q), monosomy 7, trisomy 8 and del(20q)
Abnormalities specific to AML include t(8;21)(q22;q22) etc.

Question 13

Breast Cancer

Answer 13

Mutations of the BRCA1 and BRCA2 gene associated with an increased risk for breast and ovarian cancer
Can be seen with:
- gains of 1q, 6q, 7, 8, 18 and 20
- loss of 2 and 5
- deletions of 1p, 3p, 6q, 11q, 17p
- abnormalities of 18p
- activation and overexpression of HER2/neu gene on chromosome 17

Question 14

Genetic Markers

Answer 14

Short segments of DNA located throughout the genome that have a repeated sequence
Polymorphic
Diploid organisms have two copies of any particular genetic marker
PCR amplification of genetic markers using fluorescently tagged primers

Question 15

QF-PCR Methodology

Answer 15

Amniotic fluid/CVS sample
Fetal DNA extracted out of sample
Multiplex PCR using 25 pairs of fluorescent primers
PCR products size separated using capillary gel electrophoresis using a genetic analyser
Markers differentiated by size and fluorescent tag
Peak pattern and area for each allele are analysed
Sequence copy number determined from the relative quantitation of the markers

Question 16

Difference of detection methods between aCGH and Microarrays

Answer 16

aCGH
- detection of copy number changes using normal reference DNA and patient DNA hybridised to an array allowing for comparison between the two
Microarray
- allows detection of copy number changes as well as SNP information
- comparison to computer reference instead of labelled reference DNA

Question 17

Analysis of Microarray

Answer 17

Depending on size and gene content of the copy number change identified, cases are generally divided into categories:

1. Pathogenic
   - known syndromes
2. Uncertain clinical significance
   - incomplete/variable penetrance
3. Unknown clincal significance
   - novel copy number changes/no previous documented cases
4. Likely benign
   - found in the general ‘normal’ population and has no discernable phenotypic effect

Question 18

Williams Syndrome

Answer 18

Caused by a deletion of genetic material from the chromosomal region 7q11.23
The deleted region includes more than 20 genes (ELN being the most critical)
Symptoms:
- distinctive ‘elfin’ face
- low nasal bridge
- unusually cheerful with demeanor and ease with strangers
- mental retardation

Question 19

Advantages and Disadvantages of Array CGH and Microarray

Answer 19

Advantages
- commercial arrays available 
- SNP arrays also available for genotyping and trio analysis for parental studies
- multiple loci (thousands/millions loci tested)
Disadvantages
- multiple loci (ambiguous results)
- low-throughput
- consumable costs far greater than FISH
- balanced rearrangements not detected

Question 20

Quantitative Fluorescense PCR

Answer 20

Rapid prenatal diagnosis of chromosome copy number changes
- chromosomes 13, 18, 21, X and Y
- detects roughly 85-90 % clinically significant chromosomal abnormalities detected at birth
Aimed at pregnant women with increased risk of chromosome abnormality
Undergo invasive sampling of amniotic fluid or chorionic villus sampling