• 1.
  

  a) F b) T c) F d) T e) T

The insufficient fetal DNA fractions in maternal plasma result in a reduced ability to discriminate between the diploid and aneuploid chromosomes of the fetus. It is the most common reason for a detection of false negative results in NIPT-based aneuploidy screening. The fetal DNA is mainly derived from the placenta. Pregnancies with a confined placental mosaicism are characterized by a discordant genetic make-up between the fetus and the placenta, thus often resulting in a detection of “false positive” NIPT results. In obese pregnant women, the fetal DNA fractions are usually low, increasing the chances for detection of a false negative NIPT result. Tumors are often associated with chromosomal aberrations. During pregnancy, a tumor releases its DNA into maternal plasma, which may distort the NIPT results. A fetus, which died in utero, releases its DNA into maternal plasma. If the non-viable fetus was affected by a chromosomal aberration, the NIPT result would indicate an affected pregnancy despite the presence of a viable unaffected fetus.

• 2.
  

  a) F b) F c) T d) F e) F

The method does not target any maternally inherited mutations in maternal plasma. The mutation also cannot be distinguished between the mother and the father. If the paternal mutation is detected in maternal plasma, an invasive procedure would be recommended. The lack of its detection eliminates any unnecessary invasive procedures. A female fetus would be a carrier of the mutation without clinical manifestation. In male pregnancies, the father would not transmit the mutation linked to the X chromosome to the fetus.

• 3.
  

  a) F b) T c) T d) F e) T

In practice, it is relatively more convenient to use MPS data for RHDO analysis. To achieve the same diagnostic performance using the dPCR analysis, many SNPs would need to be analyzed or, if only a single SNP is being interrogated, one would need to test a great number of molecules, increasing the demand for a large plasma volume. On the other hand, the analysis of each additional SNP would require a new dPCR design. The interrogation of maternal inheritance is based on the detection of the balance or imbalance of maternal haplotypes in maternal plasma. For a construction of the parental haplotype, the genetic information from the mother, father and the proband is necessary. The paternal inheritance is usually assessed by the presence of paternal alleles absent from the mother’s genome. However, RHDO analysis may be used for determining paternal inheritance. Informative SNPs are those that are homozygous in the father (AA) and heterozygous in the mother (AB), allowing one to assess the allelic ratio between the maternal A and B alleles. Targeted sequencing allows one to focus the sequencing on the gene locus of interest for subsequent RHDO analysis.

• 4.
  

  a) F b) T c) F d) T e) F

As the assessment of maternally inherited mutations is not possible using the exclusionary approach, it is unknown whether the fetus has inherited the mutant or the wild-type allele from the mother. All methods increased the sensitivity for the detection of fetal mutations when using conventional methods, such as real-time PCR. The gel-based method is prone to contamination, therefore is not recommended for routine use. Moreover, fetal DNA in maternal plasma is generally shorter than maternal fragments. Targeted sequencing of maternal plasma DNA permits efficient and unbiased detection of fetal alleles at genomic regions of interest, which saves the costs associated with the whole-genome sequencing. Further studies are needed to assess the cost-effectiveness of NIPT for monogenic diseases.

• 5.
  

  a) T b) T c) F d) T e) T

Complete amplification failure, contamination with foreign DNA and allele drop out are all recognised challenges. Clearly there is a time limit in fresh cycles as the timing of embryo transfer is vital. PGD is not used for whole genome amplification.

• 6.
  

  a) T b) T c) T d) T e) T

The ethnicity and presenting features are compatible with a diagnosis of hydrops fetalis with maternal mirror syndrome. Thus it is important to exclude maternal thalassemia trait and the red cell indices from a complete blood count could confirm a tentative diagnosis of thalassemia trait. The obstetric ultrasound could reveal features of hydrops fetalis, allow measurement of the amniotic fluid volume, show the placental thickness and location, and exclude undiagnosed multiple pregnancy. There is a high chance of pre-eclampsia and coagulopathy if there is indeed hydrops fetalis. Even if the blood pressure is not yet elevated, pre-eclampsia is likely to develop if hydrops fetalis is confirmed, and the blood test results can provide the baseline value for further comparison later. If fetal distress is documented in a case of hydrops fetalis due to homozygous thalassemia, perinatal death can be anticipated and a crash caesarean section not only will not help the fetus but will also put the mother at an even greater risk. Therefore vaginal delivery should be attempted following the finding of hydrops fetalis. There is a very high chance of retained placenta and need for manual removal and postpartum hemorrhage from the combined effects of uterine atony, retained placental tissue, and coagulopathy. Furthermore, there is likely to be some degree of maternal anaemia. Therefore even in the absence of tissue trauma from cesarean section or genital tract laceration, more blood loss at delivery can be anticipated and the mother is less able to withstand the effect of blood loss due to the underlying anaemia. Prophylaxis for postpartum hemorrhage, and reserving whole blood and blood products for possible transfusion and resuscitation is prudent.

• 7.
  

  a) F b) T c) T d) T e) T

The ethnicity and presenting feature suggested an increased risk of α-thalassemia trait, in which case there may be increased iron in her body. Iron supplementation should only be prescribed when iron deficiency has been confirmed. The complete blood count would provide information on her MCV and MCH as well as on her platelet and white cell count. If low MCV and MCH are found, maternal thalassemia trait must be excluded by further tests. The low MCV suggested that she could be a thalassemia trait carrier so that her partner should also be screened. If both partners are thalassemia carriers, the fetus would have increased risk of a neural tube defect. Before maternal thalassemia trait and fetal anomalies are excluded, it is prudent to prescribe folic acid supplementation. At this point in time, even though folic acid supplementation would be too late to prevent fetal neural tube defects it would improve her anaemia if she is indeed proven to have thalassemia trait.

• 8.
  

  a) T b) T c) T d) F e) F

Given her age and history of TM, it is prudent to exclude fetal anomalies and aneuploidy, and to determine the thalassemia status in her fetus unless the sperm that achieved her IVF pregnancy is known to be from a person without thalassemia trait, or that pre-implantation genetic screening or diagnosis has been performed and ruled out homozygous or severe heterozygous thalassemia syndrome in her fetus. Since the fetal safety of chelation therapy is not established, it is prudent to discontinue chelation therapy for the time being. It is likely that she has a variable degree of iron overload. Even though she may not have cardiac symptoms at this moment, her cardiac status is unknown, unless she is properly assessed. Cardiac events during pregnancy can lead to maternal mortality. Actually, her risk of thromboembolic complications is increased after splenectomy, and thromboprophylaxis should be provided. It is dangerous to assume that as she did not provide a history of infection, she does not have any of the infections that could be transmitted via transfusion. She could also have acquired these infections from other sources and infections such as HIV and hepatitis B and C could jeopardize her health and infect her baby. Screening for these infections is mandatory.

• 9.
  

  a) T b) F c) T d) T e) F

The phenotype of Hb H disease is variable, and she could develop quite severe anemia requiring transfusion before delivery. Depending on the initial hemoglobin level, her blood count should be monitored every 2-4 weeks till delivery. Unless the family physician is familiar with the management of pregnancy in subjects with Hb H disease, it is safer for an obstetrician to continue antenatal care till delivery. She has increased risk of gestational diabetes mellitus on account of both her age as well as her Hb H disease, so that an oral glucose tolerance test is prudent. Hb H disease can be associated with increased risk of preterm labor especially if there is anaemia. These symptoms could be due to preterm labor so that assessing her in hospital would be appropriate. If preterm labor is established, tocolytic treatment and corticosteroid would be indicated. There is no reason for Caesarean delivery on account of Hb H disease alone. Unless there are obstetric indications for Caesarean delivery, she should be allowed to attempt vaginal delivery especially if spontaneous labor ensues.

• 10.
  

  a) T b) T c) F d) F e) T

The maternal tissues or cells (fibroblasts and epithelial cells) can adhere to the tip of the needle during invasive procedures through the maternal abdominal wall, and they can be sucked into the syringe along with first sample withdrawal. Therefore the first 2 ml of amniotic fluid are discarded to reduce the risk of contamination of the sample with maternal cells. During invasive procedures, bleeding can occur at the puncture sites, and maternal hematopoietic cells can contaminate the amniotic fluid. Cultured amniotic cells, on the other hand, are much more likely to represent a homogeneous specimen type reflecting pure fetal origin due to an absence of coexisting contamination with cells of maternal hematopoietic origin. Maternal blood cells senesce during culture and are not expected to proliferate. Although prolonged culture may allow overgrowth of maternal fibroblasts and epithelial cells, potentially leading to detectable MCC, discarding the first draw of amniotic fluid samples reduces this possibility. CVS analysis presumes that the fetal genotype is reflected accurately in the extraembryonic tissues. Contamination of a CVS sample with cells of maternal origin may result in analysis of the maternal rather than the fetal genotype, especially when the sample size is small. In CVS samples, the culturing process increases the risk of detectable MCC given the co-localization of maternal and fetal cell lineages in the placenta. In these situations, MCC can be reduced by carefully separating the maternal decidua from chorionic villi before analysis. Highly sensitive molecular testing methods have identified the presence of MCC in 9.1% of direct or cultured fetal cell preparations, 17.8% of which had no visible evidence of maternal blood. MCC analysis for prenatal molecular testing for single-gene disorders has been recommended by several genetic societies (the American College of Medical Genetics and Genomics, the UK Clinical Molecular Genetics Society, and the Canadian College of Medical Geneticists) and the Association for Molecular Pathology. These societies recommend that labs performing prenatal molecular testing understand how MCC may affect detection of normal and abnormal results for each assay.

• 11.
  

  a) T b) F c) F d) F e) T

At mid pregnancy, ultrasound examination can readily detect the many hydropic changes found in fetuses with homozygous α-thalassemia. The cardiothoracic ratio, placental thickness, and middle cerebral artery peak systolic velocity are most used to detect fetal anaemia and hydropic signs in clinical practice. These sensitive markers are helpful for evaluation of the hemodynamic status and cardiovascular function of the affected fetuses. The major hemoglobin in the fetus is Hb F (α ₂ γ ₂ ), and later supplemented by less than 10% of Hb A (α ₂ β ₂ ) until birth. Therefore homozygous β-thalassemia causes no production of β-chains and absence of Hb A. Since Hb A only accounts for a small fraction of the total Hb, fetuses with homozygous β-thalassemia presents no anaemic or hydropic changes, and ultrasound cannot detect homozygous β-thalassemia before birth. Hydrops fetalis is a condition in the fetus characterized by an accumulation of fluid, or oedema, in at least two fetal compartments. There are two types of hydrops fetalis – immune and nonimmune. The type depends on the cause of the abnormal fluid. Immune hydrops fetalis is a complication of a severe form of Rh incompatibility. Nonimmune hydrops fetalis occurs when a disease or medical condition is beyond the body’s ability to manage fluid. There are three main causes for this type: heart or lung problems, severe anaemia (e.g. from thalassemia or infections), and genetic or developmental problems, including Turner syndrome. In some areas, α-thalassemia is the most common cause of fetal hydrops. However, even in pregnancies at-risk for α-thalassemia, there is still a chance that other causes for hydrops fetalis exist. Fetuses with Hb Bart’s disease can develop anaemia even in the first trimester. The fetal cardiac chamber becomes enlarged and cardiac output increases to compensate for the anaemia and hypoxia, resulting in an increased cardiothoracic ratio. An ultrasound findings of cardiomegaly at 12-14 weeks of gestation can be highly specific for affected fetuses in pregnancies at risk.

• 12.
  

  a) F b) T c) F d) F e) F

While fetal cardiomegaly is very sensitive in predicting an affected fetus at or after 12 weeks, it is difficult to measure the cardiothoracic ratio before 12 weeks. Placentomegaly is very sensitive in predicting an affected fetus. There is a lot of overlap in the measurements of middle cerebral artery peak systolic velocity between affected and unaffected pregnancies. There is also a lot of overlap in the measurements of nuchal translucency between affected and unaffected pregnancies. Hydrops is uncommon in an affected fetus in the first trimester.

• 13.
  

  a) T b) T c) T d) F e) F

The sensitivity of using fetal cardiomegaly, placentomegaly and middle cerebral artery peak systolic to predict an affected fetus is high in the second trimester. Although hydropic features can present in an affected fetus after 20 weeks, these features are not common. Although limb reduction can present in an affected fetus, these features are also not common.

• 14.
  

  a) T b) F c) T d) T e) T

Middle cerebral artery peak systolic velocity is not very predictive of mild anaemia which can be present in an affected fetus. Low blood viscosity in fetal anemia accounts for the increased middle cerebral artery peak systolic velocity. In a fetus affected by homozygous α ⁰ -thalassemia, the blood viscosity may not be very low as the hematocrit in affected fetuses can be around 30%. The physical properties of hemoglobin Bart’s (γ ₄ ) may differ from hemoglobin F (α2γ2) as the genes and hence protein structures are different. It is difficult to accurately measure middle cerebral artery peak systolic velocity in the first trimester because of the angle of Doppler insonation should be near zero while fetal motion is frequent at this gestation. There is a lot of overlap in the measurements of the middle cerebral artery peak systolic velocity between affected and unaffected pregnancies especially in the first trimester.

• 15.
  

  a) F b) T c) T d) T e) F

Fetal anaemia is not present in alpha thalassemia trait, and thus cardiomegaly is not a feature. Haemoglobin H disease can be associated with moderate anemia and thus cardiomegaly. Cardiomegaly is present in some congenital heart diseases with significant haemodynamic changes. Cardiomegaly can be present in fetal growth restriction. As beta genes are not required for synthesis of hemoglobin in-utero, fetal anemia and thus cardiomegaly does not occur in homozygous beta thalassemia. The latter is associated with severe anemia six months after birth with switching to beta genes.

• 16.
  

  a) F b) T c) F d) T e) T

Normal HbA ₂ levels are below 3.5% of total haemoglobin. Although it is possible that a carrier of an α- and a β-thalassaemia mutation could show normal levels of HbA2, these cases are not typical of β-thalassaemia. Microcytosis causes unusually small red blood cells, as measured by their mean corpuscular volume. This is a key indicator of anaemia. HbF levels may be increased slightly in β-thalassaemia (ranging from 0.5-4%), but an increase to greater than 10% would indicate co-inheritance of another haemoglobin variant, most likely δβ thalassaemia or HPFH. HbA ₂ levels are increased to more than 3.5% in typical β-thalassaemia carriers. Hypochromia, or abnormally low levels of haemoglobin, measured by mean corpuscular haemoglobin (MCH), is a typical feature of β-thalassaemia.

• 17.
  

  a) F b) F c) T d) F e) F

Leukaemia is a blood cancer caused by an abnormal proliferation of white blood cells. The patient’s white blood cell levels in this case are normal. Typical features of thalassaemia would include an increase in red blood cell number and a decrease in red cell distribution width (RDW). Therefore, it is not a likely cause of the patient’s anaemia in this case since a decrease in RBC count and an increase in RDW was seen. IDA is characterised by a reduced red blood cell count, with cells having an increased RDW. Blood biochemical analysis for serum iron, ferritin, unsaturated iron binding capacity (UIBC) and total iron binding capacity (TIBC) should be ordered as a common first-line procedure to detect IDA. Gastroenteritis may result in anaemia due to deficiencies in uptake of vitamins from the diet. However, it would be more appropriate to identify whether the patient is suffering from IDA first. Covert blood loss in a 17 year old is highly unlikely.

• 18.
  

  a) F b) T c) F d) T e) F

Since there is an increased level of HbH, the patient does show signs of HbH disease. However, the presence of an additional minor band suggests co-inheritance of another variant. The most likely explanation is that the patient carries HbH–Constant Spring (–/α ^cs α) thalassaemia trait. The minor band appearing at just 1–2% on capillary electrophoresis could be Hb Constant Spring. Southeast Asian ancestry also supports a possible prevalence of Hb Constant Spring. Further confirmatory testing using DNA analysis is required to confirm HbH-Constant Spring. Hb Barts disease is a result of all four α-globin alleles being absent. Affected individuals do not survive to reproductive age. HbE usually migrates together with HbA ₂ on HPLC. Therefore, it would not have been detected using this method. The absence of the HbC band from acid pH electrophoresis rules out the possibility of HbH (–/-α) with HbC trait.

• 19.
  

  a) T b) F c) F d) F e) F

α-/α- carriers (also known as α+ thalassaemia carriers) have one HBA gene from each chromosome deleted. In contrast, αα/– carriers have two deleted genes on a single chromosome. Gap-PCR uses pairs of primers designed to cover the specific mutation breakpoints. If there is a deletion, the amplicon produced following PCR will be smaller than expected if the full length amplicon were present. b), c) and e) are all methods used for detection of point mutations. They cannot be used to identify large deletions, although may be useful for detection of point mutations in the HBA genes, such as the Constant Spring mutation. HbH inclusions are only reliably present in carriers of HbH disease; they are unable to discriminate between the α-/α- and αα/– genotypes.

• 20.
  

  a) T b) F c) T d) T e) F

Iron deficiency is one of the common cause of microcytic anaemia and is correctable, and an iron profile should be tested to exclude iron deficiency. Amniotic fluid sampling or fetal blood sampling is now replaced by chorionic villi sampling as CVS can have earlier prenatal diagnosis in first trimester. Checking her husband’s blood for CBC and MCV will screen for thalassaemia carrier status on the paternal side, and a normal finding will have a very low chance that the fetus will be affected by severe thalassaemia. If the husband also has microcytosis and both couples must be screened for thalassaemia carrier status, there may be one in four chance that the fetus may have thalassaemia major if both couples carry the same type of thalassaemia trait, either alpha or beta.

• 21.
  

  a) T b) F c) F d) T e) T

Alpha thalassaemia major or Hb Bart’s hydrops fetalis syndrome will present as hydropic features during early pregnancy. Without intra-uterine transfusion, the fetus will suffer from severe anaemia from early gestation, and most of the affected fetuses result in stillbirth. If the affected baby is born prematurely and the intrauterine hypoxia is not severe, the newborn baby may survive for a short period of life. However most of these babies still die from multi-organ failure soon after birth. There are few reports of Hb Bart’s syndrome being long term survivors but most of them had received intra-uterine transfusion. Hb Bart’s hydrops fetalis syndrome happens when both couples carry the α0 mutation, i.e. deletion of both alpha 1 and 2 genes on the same chromosome. When the fetus inherits the mutated alleles from both parents, the baby will have all the 4 genes deleted and result in alpha thalassaemia major. African descendants rarely have the α0 mutation, more commonly having α+ mutation, i.e. only one alpha gene is mutated or deleted on the chromosome. Beta thalassaemia major will not be associated with hydrops fetalis syndrome because the beta globin chain is not the predominant globin during the intrauterine period. Termination of pregnancy is one of the preferred option for diagnosed Hb Bart’s hydrops fetalis syndrome because this can prevent the stillbirth or early neonatal death of the affected fetus, and also prevent the maternal complications related to the hydropic syndrome.

• 22.
  

  a) F b) F c) T d) T e) T

Hb Bart’s hydrops fetalis syndrome is due to loss or mutation of all the four alpha genes. Hb H Disease is having 3 out of 4 genes mutated or deleted, thus the fetus only inherits one or two abnormal alpha genes from the mother. The father is beta thalassaemia trait and does not carry the alpha thalassaemia mutation, thus the fetus will only have maximum of two alpha genes affected in this setting. Hb Bart’s Disease should not happen. As above, the fetus will not have the possibility of 3 alpha genes affected, thus Hb H Disease will not happen as well. The fetus will be either an alpha thalassaemia trait carrier (α ⁰ or α ⁺ ), double alpha and beta thalassaemia trait which does not have a severe thalassaemia phenotype. Thus there is no chance of having a severe thalassaemia genotype leading to transfusion dependence after birth. There is thus no need to perform prenatal diagnosis on the fetus as the clinical phenotype is normal.

• 23.
  

  a) F b) T c) F d) F e) T

Hb E carriers have a mild thalassaemia phenotype and the Hb level is usually normal. There is no significant pregnancy complication. Her husband is of Greek origin and has a higher chance of having beta thalassaemia trait. For couple with Hb E and beta thalassaemia carriage, there is one in four chance that the fetus may have Hb E-beta thalassaemia. Hb E-beta thalassaemia may have variable degree of anaemia, from moderately severe to a severe phenotype, and some may even be transfusion dependent. Antenatal diagnosis of the thalassaemia status of the fetus will provide the couples with the necessary information on the management of the pregnancy.

Hb E is common in South East Asia and rare in the Mediterranean region. Thus the chance of having homozygous Hb E disease is unlikely. HB E-beta thalassaemia is a form of non-transfusion dependent thalassaemia with moderately severe anaemia. However some patients may later develop deteriorating course and become transfusion dependent.