HISTORY OF GENTETIC TESTING
has been more than 150 years since scientists discovered cells,
chromosomes, and genes. In 1859, the genetical theory of
natural selection had been proposed by Charles Darwin5. Six
years later, Gregor Mendel identified
inheritance pattern from his
pea plants, resulting in Mendelian principles7. Then the world
of genetics has been explored continuously until
one hundred years later, Jerome Lejeune and his
colleague found that an additional chromosome, trisomy 21, is the
cause of Down Syndrome in 1959, after the discovery
of karyotyping techniques.
the advancement in understanding the genetic mechanism, early
diagnostic methods for different genetic diseases have been
developed. The establishment of amniotic fluid cell
culture for detecting fetal chromosomal
disorders, in 1966, and the change in the law
and people’s perception of abortion in pregnant women
at high risk of fetal chromosomal abnormality would have been the beginning of
the history of prenatal screening and diagnosis8. In the 1980s, the aim
of prenatal diagnosis was mainly to detect trisomy 21 (Down
syndrome), however, another form of chromosome abnormalities and
rare diseases could also be identified accidentally with the
the 20th century, there were two well-known procedures,
used generally for prenatal diagnosis; amniocentesis and chorionic
villus sampling (CVS)9. Although both
could prenatally detect chromosome abnormalities,
they were also considered as invasive procedures which
carry the risk of fetal loss.
previously stated, amniocentesis had been introduced
and developed as a diagnostic procedure for high-risk patients
in the 1970s. It is normally performed at the gestational age of 16-18
weeks or 20 weeks in some literatures9-12. Some institutions
offered this procedure at an early stage of pregnancy (<15 weeks), but found that it carries a higher risk of miscarriage9. The reason is that this procedure can be performed by inserting a needle transabdominally to aspirate fluid from the amniotic sac (Figure 1). Therefore, waiting until 16 weeks of gestation is essential that the fluid surrounded the fetus would be adequate enough to prevent fetal deformities or pregnancy losses. However, this procedure still carries the risk of miscarriage about 1% (range 0.75-3.30%)12. Moving on now to consider the advantages of amniocentesis. This test can prenatally detect genetic or chromosomal abnormalities by analysing cultured fetal cells acquired from amniotic fluid. The detection of affected pregnancy leads to selective abortion or establishing predelivery guidance, healthcare support, and family planning in order to take the best care of the affected child. Another significant aspect of amniocentesis is that current prenatal cytogenic tests have been developed far more than traditional karyotyping in the past13.
The integral molecular tests, including chromosomal microarray
analysis (CMA), interphase fluorescent in situ hybridization (I-FISH),
quantitative fluorescence polymerase chain reaction (QF-PCR),
and comparative genomic hybridization (CGH), provide an opportunity to identify the
wider range of chromosomal abnormality, for example, aneuploidy, translocation,
mosaicism, and pseudo-mosaicism.
the advantages, there are some disadvantages of
performing the mid-trimester amniocentesis. Firstly, it would lead to maternal and fetal complications9.
Bacterial invasion occurring through puncture sites, skin or even bowel,
possibly cause maternal infection, septic shock or death in a very severe case.
Moreover, the needle insertion would result in various degrees of fetal injury from
a small skin dimple to severe internal organ injuries. Secondly, it might
increase the risk of preterm labor as well as other third-trimester
complications, including chorioamnionitis, oligohydramnios, and preterm
premature rupture of the newborn, as a consequence of unnoticeable amniotic
fluid leakage. Thirdly, errors could occur by maternal cell
contamination, laboratory error, and typographic mistakes13.
Previously, the error rates of amniocentesis were reported about 0.1-0.6% in
the 1970s14. These were significantly reduced to 0.01-0.02% in the
1990s15, 16, possibly stemming from more developed laboratories and
more experienced operator. Nonetheless, following the introduction of prenatal
cytogenetic diagnosis, some chromosome abnormalities, such as low-level chromosome
mosaicism, uniparental disomy, subtle structural abnormality, and microdeletion
syndrome, can be undetectable. Moreover, the relatively late diagnosis during
mid-trimester pregnancy would lead to tight mother-child bonding and ethical
issues regarding the termination of an abnormal fetus. Consequently, decision
making after the diagnosis might become more problematic. Although there is an
improvement in ultrasonographic technology which could support early amniocentesis
in the first-trimester, its benefits must be balanced against the higher risk of pregnancy losses and other complications such as amniotic
Figure 1 Transabdominal amniotic fluid
aspiration in amniocentesis12
In the late 1980s, chorionic villus sampling (CVS) had
been established and become more popular than amniocentesis17.
The reason why physicians shifted their practices towards CVS is that it can be
done in the first trimester (10-14 weeks gestational age). In addition, it is
also safe, effective and reliable for the diagnosis of chromosomal, molecular
and biochemical disorders, comparable to mid-trimester amniocentesis. Chorionic
villus, which is floating freely within the intervillous space (chorion frondosum in Figure 2), will be obtained by inserting a needle through the
maternal abdomen or introducing a catheter into the cervical canal.
Confirmation of the sampling tissue is necessary to
ensure that there is the presence of required placental tissue and the amount is adequate enough to
reduce maternal cell contamination (10 mg of well-formed villus tissue)17.
CVS samples will be analysed by tissue culture together with direct analysis,
which the latter method could provide results within two days. Despite that
advanced cytogenetic analysis applying through CVS sample is quite similar to
amniocentesis, the advantages of this method are still noticeable. First, it
expands the opportunities to detect genetic disorders, including mosaicism, at
an early stage of pregnancy, especially in the families at risk for single gene
disorders17. Additionally, CVS may require a much shorter time
period for the prenatal cytogenetic results in the setting with technical
feasibility. Concerning the perinatal and maternal morbidity, there is the less
reported rate of premature birth, premature rupture of membrane and maternal morbidity
in this procedure, as compared to amniocentesis. Hence, this procedure has a
significant advantage in term of leading to less maternal mental distress.
2 Chorionic villus can be retrieved from the chorion frondosum during appropriate
gestational age, before amnion is fused with chorion17.
Nevertheless, there are some possible drawbacks of CVS which
need to be discussed with the families. The most concerned one is that it may be
related to limb reduction defects in fetuses, which was reported with an event
rate of 1-7.3:10000 cases in the previous studies9, 17-22.
One pathogenesis which could explain this complication is that excessive
placental tissue sampling, usually found in the less experienced centres, can
result in massive vascular disruption of the placenta. Subsequently, profuse
fetal hemorrhage, hypovolemia, and hypoxia may cause the thin-walled peripheral
vascular rupture, tissue necrosis and subsequently limb reduction9.
Another concern is about maternal infection which is
frequently detected in the patients who underwent the transcervical CVS. The
explanation of bacterial invasion in this method is the same as in amniocentesis
which was mentioned earlier in this essay. However, the occurrence of post-CVS
chorioamnionitis is very low, causing miscarriage in only 0.3%17. Unlike
the transcervical CVS and amniocentesis, the transabdominal-CVS may result in
less number of patients with chorioamnionitis or amniotic fluid leakage because
the needle does not break the amniotic membrane9. Interestingly,
another study41 reported the rate of maternal bacteremia post
transabdominal CVS is higher than post transabdominal CVS.
Moreover, the most important issues which need to be pointed
out are about the false-positive and false-negative results of this test and
the pregnancy loss rate. Even though direct visualization of the chorionic
villi can provide a very fast outcome, it also comes up with occasionally
inaccurate results, depending on different laboratories. Regarding the rate of
miscarriage, some studies reported that there was probably 5-10% higher rate of
spontaneous abortion post-CVS comparing to that for amniocentesis, however, the
rate is different among different centres, based on operators’ level of
experience10. Hence, this information should be discussed with the
family before both physicians and families decide to choose a suitable
procedure for prenatal diagnosis.
Non-invasive screening tests
The previous section has shown that both amniocentesis
and CVS are highly invasive, hence, a number of non-invasive screening test
have been developed. Primarily, the aim of screening tests is
mainly for the early detection of Down syndrome and other types of aneuploidy. Thus,
multiple maternal serum markers such as maternal serum human chorionic
gonadotrophin (hCG), the free-? subunit of hCG, pregnancy-associated plasma
protein (PAPP)-A, unconjugated estriol (uE3), inhibin A and Alpha-fetoprotein
(AFP), and ultrasound nuchal translucency (NT) were initiated and integrated in
the prenatal screening protocol. Different serum markers have their own screening
performance, which is dependent on the different time period of gestational age25.
The best marker during the first and second trimester are PAPP-A and free ?-hCG,
respectively. Whilst, AFP and uE3 are the weakest ones. Despite its
own performance in screening Down syndrome, its cutoff levels are diverse among
different institutions. Therefore, a statistical model was applied to develop a
suitable protocol for the use of combined markers.
The “Combined” test, including NT, PAPP-A and
either hCG or free ?-hCG, is proposed to use for the first-trimester screening.
Whilst, the “Quad” test, using hCG or free ?-hCG, AFP, uE3 and inhibin A, is the
best test for the second-trimester. However, the more complex the protocol is,
the better performance will be acquired. Hence, “Contingent” method is applied
by utilising both first- and second-trimester tests to detect 1% of the highest
risk pregnancy during the first-trimester in order to offer a prenatal
diagnostic test. Blood test for Quad markers will be taken from the other
15-20% of women who have the lower risk of pregnancy in order to re-evaluate
their risk during mid- trimester25.
Subsequently, the advanced ultrasonography is added
to the screening protocol both in first- and second-trimester to improve the
screening yield. The additional ultrasonographic findings in the early
pregnancy included absent fetal nasal bone, increased frontal-maxillary facial
angle, tricuspid regurgitation detected by Doppler ultrasound, and the absent
or reversed flow of ductus venosus. The markers for the mid-pregnancy are femur
or humerus length, and the “facial profile” markers including nuchal skin-fold,
pre-nasal thickness, and nasal bone length25. Nonetheless, the
operator-dependent nature of this test is an arguable problem, the performance
may increase only in the sufficient skill hands