W8L12 - Cytogenetics Flashcards
What is Cytogenetics?
The study of genetic material at the cellular level: the study of chromosomes
Where is Cytogenics used?
Prenatal investigations Constitutional studies Infertility/recurrent fetal loss Spontaneous miscarriage Leukaemia Solid tumour and lymphoma evaluation
Specimen Types in Cytogenics
Peripheral blood Amniotic fluid Chorionic villus sample Products of conception Bone marrow Tumour biopsy Skin biopsy
Chromosome Structure and Identification
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
Position of Centromere
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)
G Banding
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
Constitutional and Acquired Chromosomal Abormalities
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
Detection of Imbalances
Conventional cytogenetics
FISH - fluorescence in situ hybridisation
Quantitative fluorescence PCR
Microarray/array CGH
Detection of Imbalances - Fluorescence in situ Hybridisation
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
Repetitive Sequence Probes used with FISH
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
Unique Sequence Probes (Locus Specific Inserts)
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
Common Cytogenic Changes in Leukaemia
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.
Breast Cancer
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
Genetic Markers
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
QF-PCR Methodology
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
Difference of detection methods between aCGH and Microarrays
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
Analysis of Microarray
Depending on size and gene content of the copy number change identified, cases are generally divided into categories:
- Pathogenic
- known syndromes - Uncertain clinical significance
- incomplete/variable penetrance - Unknown clincal significance
- novel copy number changes/no previous documented cases - Likely benign
- found in the general ‘normal’ population and has no discernable phenotypic effect
Williams Syndrome
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
Advantages and Disadvantages of Array CGH and Microarray
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
Quantitative Fluorescense PCR
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
