Current prenatal testing strategies
Tissues for prenatal genetic testing
Amniotic Fluid
Fetal skin
Epithelial surfaces of fetal:
Respiratory system
Geniturinary tract
GI system
Umbilical cord
Amnion
-~
mls
Fetal blood samples
Fetal lymphocytes
Chorionic villi
Cytotrophoblast
Extraembryonic mesoderm
10mg
Desirable Criteria for Prenatal Diagnosis
Accurate
Low/no risk to pregancy
Performed early in gestation
Result rapidly available
Inexpensive
Reasons for prenatal genetic testing
FASP
High risk: biochemical/combined screening
Ultrasound scan abnormalities
Chromosomal imbalance?
Familial genetic disorder
Known pathogenic variant
Historical tecnhiques
Chromosomal karyotyping
Copy number changes
Aneuploidy
Triploidy
Deletions/Dups
Resolution of 5-10Mb
Chromsomal rearrangements
Insertion
Balanced Translocation (mostly non disease causing)
Requires cell culture and metaphase chromosome analysis
10-14 days
Testing strategy
Biochemical/combined screening indicates risk of aneuploidy
Rapid testing
Structural abnormalities on US scan
Rapid testing
Follow up tests/further analysis
Follow up test/further analysis
Rapid testing: FISH
Interphase fluorescence in situ hybridisation
Uncultured amniocytes
Result in <3 days
Direct CVB karyotypes can be achieved on uncultured, actively dividing cytotrophoblasts
Current techniques
Rapid testing: QF-PCR
DNA extraction can be from
Amniotic fluid (~1ml)
Chorionic villus cell digest (pre-culture)
Fetal blood (~6ul)
Cell cultures set up for:
Back up (precious sample)
Follow up tests
Quick
Cheap
Several markers per chromsome
A semi-quantitative PCR of 24-26 cycles
Microsatellite markers
Short tandem repeats are fluorescently tagged and sized
PCR of repeat analysed on a fluorescent sequencer
Polyacrylamide gel separates them
Detects allele size
Detects amount of fluorescence
Can distinguish paternal and maternal alleles
STRs
STRs are scattered throught the genome (>10,000)
Homozygote = both alleles same length
Heterozygote = alleles differ and can be resolved from one another
Markers in the region of interest are chosen with the highest heterozygosity in the population to make it easier it distinguish them to make the test more informative
Multiplex primers
Design a combination of fluorescently tagged prinmers for Chr 13, 18, 21, X and Y or buy a kit
Use different fluorophores to maximise information from a single PCR reaction = stops alleles of similar sizes on different chromosomes from being indiscriminable
Need to know the limits of allele sizes (number of repeats)
Need to run a size marker (DNA ladder) to calculate fragment sizes
Analysis
Normal = 2 alleles of different lengths of a ratio of 1:1
Trisomy = 3 alleles of different lengths at a 1:1:1 ratio or 2 alleles of different lengths at a ratio of 2:1 (1:2)
Homozygosity = single peak = could be 2 alleles or 3 alleles all of the same length = uninformative
If all single peak markers = depends on heterozygosity of your markers and affected by consanguinity
use different markers/primer multiplex
Technical complications:
Preferential Amplification
Stutter bands
MCC
Primer-binding site polymorphism
Somatic microsatellite mutation
Submicroscopic duplication
Smaller allele is preferentially amplified which gives a skewed peak ratio
Often if a >20bp size difference
Polymerase slippage during PCR
Different by single repeat size each time
3 alleles, peak height for shared should equal two others
Generally affects all markers
Mosaicism
Can be difficult to distinguish between this and MCC
Only one chromoome would be affected
15% or greater detected
Polymorphism on the template DNA strand where a PCR primer anneals
Primer binding less efficient
Can resolve by lowering annealing temperature
A mutation occurring after conception where repeat size has expanded
Only one marker will have been affected and usually mosaic
Of a larger section of DNA containing MS marker region
Clinical Considerations
The markers only give indicative information about that region
The markers may not contain critical regions
Indicative but not a definitive diagnosis
What makes an abnormal QF-PCR result
Triallelic markers
Biallelic markers (2:1 [1.8-2.4:1]/1:2[0.45-0.65:1])
Need at least two informative markers
Problems for single abnormal markers
Microarray
Comparative Genomic Hybridisation
DNA extraction from:
Amniotic fluid
Supernatant
Cell cultures
Chorionic Villus
bulk
Cell cultures
Fetal blood
Cell cultures will be set up for:
Back up
Follow up tests
Technical complications
Triploidy not detectable
Test/Reference starting DNA concentration
Normalisation
Mosaicism
Threshold for detection
MCC
DNA requirements (~1ug)
CVB sample size
Gestation at amniocentesis
Cell culture
Microarray Vs. Karyo
Increased resolution over karyotyping
Additional clinically relevant findings
-6% cases with structural anomalies
1.7% cases with positive results
Minimum recommended resolution 400kb
UK evaluation and Recommendations
Indications for microarray testing
One or more structural anomalies on US
Isolated NT >3.5mm when CRL measures from 45-84mm
Fetuses with a sex chromosome aneu that is unlikely to explain the US
Does not include soft markers
Microarrays have potential for findings
Secondary to current clinical presentation
VOUS
Reporting recommendations
Any variant that will potentially inform the management of the pregnancy, or of the family, in the clinical context in which CMA was done or in the future
Be clear if finding is an unsolicited clinically actionable one and recommend CGU
NOT to be reported
VOUS not linked to potential phenotype based on:
Genes involved
Penetrance
Unsolicited findings with no available intervention
15q13.1q13.3 dup
15q11 BP1-BP2 dup/del
Xp22.31 (STS) dup
16p13 dup
Heterozygous del of recessive genes that cannot be linked to the presenting phenotype
Targeted 'mutation' testing
Most pathogenic vars SNVs/indels
PCR: Amplify DNA of interest
Can use Sanger
Doesn't require large samples
Sequencing
Primer extension in the presence of ddNTP terminators
Others
MLPA (multiplez ligation dependent probe amplification
Dosage assay: Dels/Dups
Targeted testing for familial pathogenic variants
Must be definitive genetic diagnosis
Clearly causative
Can detect the familial variant prior to accepting referral
Considerations
Amplification of low starting quality DNA
Risk dependent on sampling technique/Starting material/sample quality