MCD – Genetics 4 - Prenatal diagnosis of genetic diseases
Anil Chopra
1. Describe the use of non-invasive tests - maternal serum screening and ultrasound.
Indications for Prenatal Testing:
• High Risk of Aneuploidy
» High risk on Down Syndrome screening
» Previous aneuploid foetus
» Maternal request e.g. Age
• Known Genetic Disorder
» Achondroplasia
» Cystic Fibrosis
» Haemoglobinopathies
» X Linked disorder
» Parental Balanced Translocation
• Structural Anomaly detected in Foetus on Routine Ultrasound Screening
Maternal Serum Screening
 Maternal serum screening – α-fetoprotein – at 16 weeks.
 High in pregnancies with a neural tube defect.
 Low in Down’s syndrome pregnancies.
 However, there is a large overlap between levels in normal and affected
pregnancies.
Ultrasound
 Can detect neural tube defects and any gross abnormality.
 Down’s syndrome – nuchal thickness and translucency detected on ultrasound.
Down’s Syndrome Testing
 α-fetoprotein detects 40% of Down’s babies.
 triple test (at 16 weeks) also measures unconjugated estriol and human chorionic
gonadotropin. Identifies about 70% of cases but with a fairly high false positive
rate.
 This is combined with ultrasound (18 weeks) detecting nuchal fold.
 Invasive tests then follow on suspected cases or on older mothers.
In the screening of Downs Syndrome:
- May see Nuchal translucency (be able to see through the tissue just behind the
skull) from ultrasound nuchal scan.
- Irregular serum markers.
- Absent nasal bones on ultrasound. (Limited as some ethnic groups contain no
nasal bones).
If they see any of these they do a number of different tests:
• Triple test
14-21 weeks: AFP, unconjugated oestriol (uE3), and hCG together with
maternal age.
• Nuchal Translucency Scan (NT scan)
11-136 weeks: measurement of the fold of skin on the back of the foetal neck
(Nuchal Translucency) together with the maternal age.
•
Quadruple test
14-21 weeks: AFP, uE3, free β-hCG (or total hCG) and inhibin-A together
with maternal age.
• Combined test
10-136 weeks: NT measurement with free β-hCG, PAPP-A and maternal age.
• Integrated test - this is the most accurate and reliable.
Integration of NT measurement and PAPP-A in the first trimester with serum
AFP, β-hCG, uE3 and inhibin A in the second
2. Describe the use of invasive tests – amniocentesis and chorionic villus sampling
The invasive tests used will show karyotype, allow for DNA analysis and biochemical
analysis. They include:
Amniocentesis
Process of Amniocentesis
• Needle applied to abdomen of mother and amniotic fluid drawn out.
• Amniocentesis is performed more than 15 weeks into pregnancy.
• It is an aseptic technique - gloves, no touch.
• There is continuous Ultrasound guidance.
• The placenta is avoided.
• Uses a 22G needle with a stylet.
• The first 2ml is discarded and only the final 15-20ml is aspirated.
Complications of Amniocentesis
• Has 1% procedure related miscarriage.
• There is the complication of Rh (rhesus) sensitivity and so all Rh
negative women get Anti D within 72h.
• 1.3% procedure-related liquor leakage.
• Infection, <0.1%, if suspected do repeat amnio, if GS + suggest emptying uterus
Analysis of Amniotic Fluid
• Foetal cells concentrated in centrifuge (skin, pulmonary, urogenital, extraembryonic membrane cells).
• Cells cultured in multiple cultures (14 days) (Culture failure rate 0.5% (1:200))
• Maternal contamination is rare.
• Human error
• If mosaicism is seen in only one cell then it is said to be a culture artefact (problem
arising from culture). Only if it is in more than one cell can this be significant.
Chorionic Villus Sampling CVS
The process of chorionic villus sampling.
• Parts of the chorionic villus are taken in by needle, cultured and
tested on.
• CVS is performed from 11 weeks onwards. (an advantage over
amniocentesis)
• Transabdominal or transcervical – ultrasound guided.
• Short term culture - gives count in 48 hours
• Ideal for DNA analysis
• Tertiary referral unit
• Risk of miscarriage 0.5-2%.
The risks of chorionic villus sampling are similar to those of amniocentesis apart
from:
• 2% increased risk from limb defects.
Analysis of Chorionic villi
• Culture cells for 14 days - Primarily fibroblasts which are derived from inner cell
mass and therefore more representative of foetus.
• 1:200 Mosaicism - Usually confined placental mosaicism (<10% confirmed in
foetus)
Foetal Blood Sampling
• A transplacental needle is inserted into where the umbilical cord meets the
placenta or a transamniotic into the intrahepatic vein.
• Transabdominal ultrasound scanner guided.
• Done from 18 weeks onward.
• Asceptic conditions
• CVS and amniocentesis are preferred for karyotype
• Primary use if for assessing foetal anaemia.
3. Describe the use of karyotype analysis and FISH for detection of chromosomal
abnormalities.
FISH Fluorescent in situ hybridisation
 Fluorescent DNA probes are used and hybridised with the normal DNA.
 They will bind to those sequences that are complementary to themselves and so show up
any similar DNA fragments.
4. Describe the use of PCR for mutation detection with
examples
 PCR involves the use of DNA primer to amplify a specific small
region of the genome.
 DNA in this region can then be analysed for mutations.
 Advantages of PCR:
- very little DNA needed – 1 cell
- very fast – 1 day
- can be automated
 Disadvantages of PCR:
- easy to get contamination
- can only amplify 2kb at a time
- need to know precise region you are interested in
Detection of CFTR by PCR
 Allele specific PCR – also known as amplifican refractory mutation system or
ARMS test.
 Uses different set of primers to recognise normal and mutant sequence.
 For each set up two reactions:
- PCR with primer A and the common primer – amplifies normal DNA
- PCR with primer C and the common primer – amplifies mutant DNA
 Just A indicates homozygous normal.
 A and C indicated heterozygous (carrier)


Just C indicates homozygous mutant (CF phenotype)
As there are over 1000 different mutations it is hard to test for all of them –
generally 5 tested for which picks up 80% of cases.
Detection of Deletions in DMD
 Using PCR, amplify up to 10 exons at once.
 As long as the products are all different sizes you can detect whether all the exons
are present.
5. Outline factors to consider for counselling of genetic disease.
 Non-invasive screening – generally carried out on all pregnancies and can detect
major abnormalities, neural tube disorders and Down’s syndrome
 Invasive screening – only carried out if there is a high risk detected by screening
or due to family history of genetic disease.
 No population screening programs for genetic disease in place in this country due
to:
- invasion of privacy – genetic counselling always given prior to testing
- stigma attached to carriers – insurance
- racism
- cost
 Screening for β-thalasseamia major implemented in Sardinia where it is very
common.
Preimplantation Genetic Diagnosis
 Superovulate mother.
 Collect eggs.
 Fertilise with sperm from father.
 Culture to 8 cell stage (3 days).
 Take one cell and assay by PCR – select embryos to implant in mother.
 Children born almost always normal – no side effects.
 Can be used to create a saviour sibling with matching MHC for blood transplant
from umbilical cord to cure β-thalassaemia.
 Use of PGD carefully regulated in Britain by Human Fertilisation and
Embryology Authority (HFEA).
 Ten clinics hold HFEA licenses for PGD – conditions include:
- β thalassaemia
- DMD
- Cystic Fibrosis
- Huntington’s Disease
- Haemophilia
- Familial adenomatous polyposis
 In addition one centre has a license for PGD plus tissue typing (to create a saviour
sibling). The benefit must primarily be for the unborn child – cannot be tissue
typed if not already at risk of disease.