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13.1 When was amniocentesis invented? How is it a less risky procedure now than when it was first used?
Amniocentesis was first developed in 1877, but became used in widespread practice only in the second half of the 20th century. Since the 1970s, ultrasound has been used to guide the sampling needle, thus reducing risks of the foetus suffering mechanical damage from the sampling technique.
13.2 Describe the similarities and differences between transabdominal and transcervical chorionic villus sampling.
Transabdominal samples are retrieved by passing a fine needle through the abdominal wall of the mother, through the uterine wall, and through the amniotic sac. Transcervical samples are retrieved by passing the needle through the vagina and cervix. In both cases, fine-needle biopsy is used to take a sample of the placental tissue. Transcervical sampling has a slightly higher complication rate owing to infection than transabdominal sampling.
13.3 What are the main risks of PUBS? Given these, when is the technique used?
The main risk of PUBS is excessive bleeding at the site of venepuncture, leading to pathological foetal blood loss. Given the higher rate of procedure-associated complication of PUBS compared with other sampling methods, it is used only as a second-line technique, where other techniques have failed, or in complex cases where details of foetal blood chemistry results are needed. The procedure carries a higher risk earlier in pregnancy, and for this reason is generally performed at a gestational age of 18 weeks or more – preferably when the foetus has a chance of life outside the uterus, should an emergency delivery be required.
13.4 What are the normal physiological functions of AFP, oestriol, hCG, inhibin A and PAPP-A? In an abnormal pregnancy, how would levels of these proteins in maternal serum likely differ from the normal ranges?
AFP is the principal foetal serum protein, with responsibility for maintaining osmotic balance between blood and tissue and transporting a range of macromolecules. In aneuploid pregnancies, maternal serum AFP levels are lower than expected and, where there are foetal structure abnormalities, such as neural tube defects, AFP maternal serum levels are higher than expected.
Oestriol is a hormone produced by the placenta and is necessary for the proper growth and development of a wide range of tissues. Maternal serum oestriol levels are lower than expected in aneuploid pregnancies and those with developmental delay.
hCG is produced by the placenta upon implantation of the embryo. It stimulates the production of progesterone, which is essential for maintaining the placenta. Down syndrome pregnancies typically have two-fold raised maternal serum levels of hCG compared with expected levels.
Inhibin A is a hormone produced by the ovaries and placenta, and is necessary for the proper growth and development of the foetus. Higher than expected levels of inhibin A in maternal serum correlate with Down syndrome and other aneuploidies.
PAPP-A plays a role in the insulin-like growth factor pathway, and is thus involved in a wide range of developmental functions. Down syndrome pregnancies tend to have maternal serum levels of PAPP-A that are lower than expected.
13.5 Which tests could be performed on foetal cells that have been recovered from maternal blood?
Assuming sufficient numbers of cells were recovered, cytogenetic tests (such as karyotyping and FISH), PCR-based tests and sequencing, could all be performed on foetal cells recovered from maternal blood.
13.6 How can cffDNA be distinguished from cfDNA?
cffDNA is typically around 200 bp in length, whereas cfDNA of maternal origin is typically present in longer fragments, upon to 23 kbp in size. Several analysis techniques exploit this difference in fragment length – for example, size-selection of DNA fragments following gel electrophoresis. Alternatively, cffDNA may be distinguished by the presence of foetal genome markers that are not present in the maternal genome - for example, if the foetus is male, the presence of genes from the Y-chromosome can be used to reconfirm the sampling of cffDNA in maternal blood.
13.7 Outline the principles of the bacterial growth inhibition assay, isoelectric focusing and immunoassays.
In the bacterial growth inhibition assay, an agar plate is seeded with Bacillus subtilis. The bacteria are unable to grow in the absence of extra phenylalanine. A piece of filter paper soaked with a neo-natal blood sample is placed on the plate. Bacterial growth near the blood sample indicate excessive levels of phenylalanine in the infant’s blood.
Isoelectric focussing separates proteins by charge. A protein mixture is placed on an immobilised pH gradient, and an electric current is passed through the medium. The proteins will move to the location in which they have no net charge.
Immunoassays use antibodies to specifically recognise a molecular target. Presence of target is reported using a chromogenic (colour-generating) or luminescent (light-generating) enzyme that is linked to the primary antibody, or to a secondary antibody that binds to the primary antibody. The read-out is proportional to the amount of target present; thus the methods are quantitative.
13.8 How can mutations in the Fibroblast Growth Factor Receptor (FGFR) family of genes cause a genetic disorder?
The normal role of the FGF signalling pathway is to down-regulate apoptosis and promote growth and differentiation of osteoblasts. However, when this pathway is over-stimulated by a mutant, over-active receptor, ossification may occur too early in development and to too great a degree. Apert Sydrome and Achondroplasia are two genetic disorders that exemplify this point, involving excessive early ossification of the cranium and long bones, respectively.
13.9 Why does Down syndrome have a wide range of associated phenotypes, across many physiological systems?
Down syndrome is genetically complex, as all of the genes on chromosome 21 are present in three copies, rather than the usual two copies. This wide range of potentially-overexpressed genes can lead to abnormalities in many different systems of body.
13.10 What do cri du chat syndrome and Smith-Magenis syndrome have in common?
Cri du chat syndrome and Smith-Magenis syndrome are both caused by specific chromosomal deletions. They also both lead to (different) characteristic facial features, intellectual disability and behavioural problems.
13.11 Why do males suffer from sex-linked diseases more commonly than females?
Most sex-linked diseases are X-linked recessive; the X chromosome contains many more genes than the Y chromosome, and loss-of-function mutations are more common than gain-of-function mutations. As males have only one copy of the X chromosome, any recessive alleles inherited from the mother will show in the phenotype, whereas in females, a dominant, functional gene on the second X chromosome may mask the effect of the recessive allele, and thus give a normal phenotype.
13.12 What is the neural tube? What may happen if it fails to close?
The neural tube is a structure in the early embryo, from which the brain and spinal cord subsequently form. If it does not properly close, spina bifida, or - in the most severe cases – anencephaly, may result.
13.13 Which molecules accumulate in: MSUD; Gaucher disease; and MCADD?
The abnormally-accumulating molecules are as follows: MSUD – amino acids with branched chain R-groups (leucine, isoleucine, and valine); Gaucher disease – a sphingolipid (glucocerebroside); MCADD - fatty acid chains that are between 6 and 12 carbon molecules in length.
13.14 What is the genetic and functional basis of sickle cell anaemia?
The condition is caused by a single missense point mutation in the gene for -globin, which results in the hydrophilic amino acid glutamic acid being substituted by the hydrophobic amino acid valine. The existence of a valine residue in an outward-facing position on the molecule causes the globin chains to aggregate in long chains. The aggregates are poor at carrying oxygen, and also distort the shape of the red blood cells, giving the typical 'sickle cell' appearance. Sickle-shaped cells are poor at passing through fine capillaries, leading to haemolysis and vessel blockage.
13.15 Why do the symptoms of CAH differ between XX and XY newborns?
CAH may result in an overproduction of the hormone testosterone. In males, the presence of testosterone is normal, so development of sexual organs is also normal. However, in females, the presence of excess testosterone can cause virilisation – the development of male-like structures – and thus XX karyotype infants with CAH may be born with ambiguous or male-like genitalia.
13.16 Why is the percentage of Down syndrome foetuses electively aborted after pre-natal diagnosis likely to be different from the overall percentage of Down syndrome foetuses that do not result in a live birth?
Down syndrome is associated with a high rate of spontaneous abortion. Therefore, among those foetuses that either do not undergo pre-natal testing, or do undergo pre-natal testing but the mother elects to continue the pregnancy, a sizeable proportion will die naturally, prior to birth.
13.17 Which technology offers the potential for pregnant women to opt for sex-selective abortion from the seventh week of pregnancy?
The analysis of cffDNA from a maternal blood sample can be used to determine foetal sex, from the 7th week of gestational age onwards.
13.18 Which technology may increase the rate of pre-natal Down syndrome diagnosis, if it were to be made generally available at no extra cost to the pregnant woman?
The analysis of cffDNA from a maternal blood sample – typically by next-generation sequencing and subsequent calculation of ratios between reads of different foetal chromosomal origins – can indicate the risk that the foetus has Down syndrome at a very high level of sensitivity and specificity – and thus, arguably without the need for invasive testing – at an early stage of the pregnancy. It is thought that making this technology generally available at no extra cost may increase the number of elective abortions of Down syndrome foetuses, as abortion is perceived by some to be an easier choice to make at an earlier gestational age.