Cytogenetic basis of inheritance and techniques Flashcards
What is conventional cytogenetics?
-Metaphase chromosome analysis= G banding
What is molecular cytogenetics?
- Cytogenetic analysis at all stages of cell cycle (DNA/in situ)
- FISH, Microarray CGH, MLPA, Next generation sequencing, QF-PCR, qPCR
How do cytogenetic abnormalities produce an abnormal phenotype?
- Dosage effect
- Disruption of a gene
- Effect due to the parental origin
- Position effect
- Unmasking of recessive disorder
Describe Trisomy 21
- 1/700
- 75% spont abort
- Eyes: upwards slanting, brush field spots
- Ears: abnormally shaped/low set
- Tongue: protruding
- brachycephalic, short neck
Describe Trisomy 21 in adults
- Fertility: fem-normal males-infertile
- Average life 55-68
- Medical problems: alzheimer’s, hypothyroid, obesity, diabetes, hearing loss, seizures, inc risk of cancer
Describe Edwards Syndrome
- Trisomy 18
- 1/6000 livebirths 95% spont abort
- Head: microcephaly, low set ears, micrognathia, cleft lip/palate
- Hands&feet: clenched hands, overlapping fingers, rockerbottom feet
- Low birth weight
- Short sternum
- Severe mental retardation
What organ malformations occur in trisomy 18?
- Umbilical/inguinal hernia
- Congenital heart disease (90%)
- Congenital kidney abnormalities
- Eye abnormalities (cataracts, micropthalmia)
Describe Patau Syndrome
- Trisomy 13
- 1/12000 livebirths (95% spont abort)
- Small at birth
- Mental retardation severe
- Microcephaly/sloping forehead
- Defects of brain-holoprosencephaly
Describe the trisomy 13 phenotype
- Abnormal genitalia
- Heart defects
- Polydactyly & fingers flexed
- Ears abnormal & low
- Cleft lip/palate
- Eyes-retina dysplasia
What are features of Turner’s syndrome?
- Loss of ovarian function
- No puberty
- Infertility
- Webbed neck
- Swelling of hands/feet
- Skeletal abnormalities (short)
- IQ normal
- Coarctation of aorta
Describe Klinefelter’s syndrome
- Most undiagnosed
- Identified through infertility/hypogonadism
- Infertility
- May lack secondary sexual characteristics
- Testicular dysgenesis
- 30-50% gynaecomastia
- Normal growth in infants then accelerates
- Adults long legs &arms
- IQ normal
What types of duplications are there?
- Direct gain= 2 copies of segment on another chromosome is exactly the same way
- Indirect gain= 2 copies of segment of a chromosome one the same way the other inverted
What is a ring chromosome?
-Breakage occurs then ends of chromosome join to form circularisation
How is the whole genome tested and how is a targeted part of the genome tested?
Whole= G-banding, microarray, next generation sequencing Part= FISH, MLPA, qPCR/QF-PCR
What are the stages of G-banding?
1) Cell culture
2) Mitotic arrest
3) Hypotonic
4) Fixation
5) Trypsin & Leishman’s stain
6) Banding-AT & GC rich regions
What molecular cytogenetic techniques are there?
-FISH
-MLPA
-Array CGH
-Next generation sequencing
-qPCR
QF-PCR
What is the process of FISH?
1) Labelling (fluorochrome)
2) Denaturation
3) Target and hybridisation
4) Visualisation 24hr by post-hybridisation washing
5) UV light
What types of probes can be used?
- Unique sequence
- Centromeric
- Paints
What are the applications of FISH?
- Copy number imbalance
- Aneuploidy
- Confirmation/clarification G-banding or array CGH
- Identifying specific abnormalities in cancer
What is copy number variation?
A DNA segment with a variable copy number (compared with a reference genome.-the number of copies of a particular gene varies for individuals)
What are the consequences of a copy number?
-Disease susceptibility or resistance (autism or cancer)
What happens in microarray CGH?
- Genomic imbalances at high resolution so inc detection rates
- Hybridise sample & control DNA to a microarray chip with 1000s of DNA spots
What is required for microarray CGH?
- 3ml blood in EDTA
- Control DNA from same sex
- 1-2ml lithium heparin blood for cell culture
What patients are used for array CGH?
- Dysmorphic infants
- Moderate-severe learning & developmental disabilities
- > 40 genomic disorders
What are the advantages of array CGH?
- Early diagnosis
- Greater accuracy of location/size of imbalance
- Information on relevant genes
- High resolution
- 1st line test reduces need for other tests
What are disadvantages of array CGH?
- Dosage changes only not balanced rearrangements/mutations
- Low level mosaicism not detected
- Non-pathogenic & uncertain pathogenic changes detected
- Needs good quality DNA
What is the process of next generation sequencing?
1) sequencing libraries generated by fragmenting genomic DNA & adaptor ligation (ligated DNA selected)
2) Cluster generation (attach DNA to flow cell & amplify)
3) Sequencing (extent strand)
What is QF-PCR
- PCR amplification of short tandem repeats using fluorescent primers
- Product visualised & quantified as peak areas using automated DNA sequencer
How is prenatal aneuploidy detected?
- DNA extraction from amniotic fluid/ chorionic villi
- PCR amplification (primers from chromosomes 13,18,21,X,Y)
- Aneuploidy = >2 markers with abnormal dosage
What does relative quantification compare?
Difference in concentration between patient sample & normal control assessed by 2 different primer sets. Expressed as a ratio relative to 1 deletion= 0.5 and duplication=1.5
What are the applications of cytogenetics?
- Diagnosis
- Family studies
- Assessing reproductive risks
- Cancer management
What is a Trivalent?
Forms a chain at anaphase- meiosis 1
What types of prenatal diagnoses are available?
- Amniocentesis
- Chorionic villus biopsy
- NIPT
What is NIPT?
- Non invasive prenatal testing
- Maternal blood sample
- Extract circulating free metal DNA
- Assess aneuploidy of 13,18,21
- Risk for aneuploidy-invasive test to confirm
- Reduces no. of invasive tests
What are indicators for prenatal diagnosis?
- inc maternal age
- Serum screen risk
- Abnormal ultrasound scan
- FH/previous chromosome abnormality
