Introduction

α-Thalassaemias result from deletions or mutations involving α-globin genes (HBA1 and HBA2) on chromosome 16, causing either the absence or reduced production of α-globin chains [1]. Additionally, non-deletional mutations of the α-globin genes can lead to the production of α-globin variants, which may have further adverse effects on erythroid differentiation and cell metabolism. Clinically significant α-thalassaemias can be broadly categorized into two main conditions: HbH disease and haemoglobin Bart’s hydrops foetalis.

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The prevention and control of α-thalassaemias aims to identify couples at risk of conceiving a foetus with the most severe form of α-thalassaemia, Hb Bart’s hydrops foetalis, or other clinically severe forms of HbH disease. Identifying (more...)

This chapter will cover the prenatal screening for a couple at risk of having a foetus with Hb Bart’s hydrops foetalis and foetal diagnostic methods. Additionally, screening process for severe forms of HbH disease (HbH hydrops foetalis) and homozygous non-deletional forms of α-thalassaemia (e.g., homozygous Hb Constant Spring (CS) will be reviewed.

Haemoglobin Bart’s hydrops foetalis

Haemoglobin Bart’s hydrops foetalis or α-thalassaemia major, results from homozygosity or compound heterozygosity of in-cis deletions of duplicated α-globin genes (α⁰-thalassaemia). The deletion of all four α-globin genes (--/--) results in an absent α-globin production. Fetuses with Hb Bart’s hydrops foetalis from a⁰-thalassaemia deletions that spare the ζ-globin gene (HBZ), such as the Southeast Asian a⁰-thalassaemia deletion (--SEA), typically presents with features of hydrops in the second or third trimester. Their haemoglobin mainly consist of Hb Bart’s (γ₄) and Hb Portland I (ζ₂γ₂) [2–8]. Other haemoglobins that may be present in small amount include Hb Portland II (ζ₂β₂), Hb epsilon4 (e₄), Hb Gower I and HbH (β₄) [2, 6, 7]. Hb Bart’s has an extremely high oxygen affinity and is non-functional for delivering oxygen to foetal tissue. Hb Portland I is a functional Hb that delivers oxygen in the earlier gestation to the foetuses. Foetuses with Hb Bart’s hydrops foetalis present with severe anaemia, enlarged liver and spleen, developmental abnormalities, and heart failure. If left untreated, hydrops foetalis will ensue and will ultimately leads to foetal demise in utero, stillbirth, or early neonatal death. It also affects the mother, potentially causing preeclampsia or eclampsia (mirror syndrome), and increasing maternal mortality [9]. Foetuses who are homozygous for larger α⁰-thalassaemia deletions involving the ζ-globin gene (HBZ), such as the --FIL or --THAI α⁰-thalassaemia deletions are unable to produce any functional haemoglobin and result in early pregnancy loss [9, 10]. Additionally, foetuses who are compound heterozygous for --THAI and --SEA α⁰-thalassaemia deletions have been reported with earlier onset of hydrops foetalis and few long-term survivors [11].

While it is rare, some affected pregnancies may result in childbirth and long-term survival without any foetal therapy. However, with the availability of intensive perinatal care and intrauterine transfusion, there has been increasing number of surviving patients with Hb Bart’s hydrops foetalis [12, 13]. An international registry reported outcomes of 69 patients with Hb Bart’s hydrops foetalis who survived into childhood and adulthood [12]. All patients require lifelong red cell transfusion and iron chelation. About two-thirds of the patients have congenital abnormalities, most frequently urogenital or limb abnormalities [12]. The congenital abnormalities are compatible with a vascular disruption defect caused by vascular occlusion or hypoxia associated with Hb Bart’s hydrops foetalis [14]. Intrauterine transfusion contributes to better outcome during the perinatal period. However, growth retardation and neurodevelopmental delay remain the major problems in 40–50% and 20% of the patients, respectively [12]. Haematopoietic stem cell transplantation resulted in transfusion independency in 14 out of 18 patients who underwent the treatment but did not improve the growth retardation or neurodevelopmental delay in some patients. Despite intrauterine transfusion, there are associated maternal complications, such as amniotic fluid abnormalities, preeclampsia, and preterm birth [12]. A report of foetuses with Hb Bart’s hydrops foetalis from Canada showed that all 12 Foetuses who did not receive intrauterine transfusion died within the first week of life [13]. Nine out of 13 foetuses who received intrauterine transfusion survived, were transfusion-dependent and experienced earlier iron overload, and had more frequent endocrinopathies and short stature. Neurocognitive outcome was not significantly affected in 5 patients who were assessed. MRI of the brain showed silent ischemic infarcts in 3 patients [13]. Given this information, clinical characteristics, intrauterine management, long-term complications, especially growth retardation, neurodevelopmental delay, and congenital abnormalities, should be discussed in detail with parents during counselling for management [12, 13].

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Due to the lethal nature of Hb Bart’s hydrops foetalis and its adverse effects on foetal and maternal health, genetic counselling and prenatal screening for couples at risk of having a foetus with Hb Bart’s hydrops foetalis should be offered (more...)

Prenatal screening for carriers of α⁰-thalassaemia

Both individuals in a couple at risk of foetal Hb Bart’s hydrops foetalis must carry α⁰-thalassaemia allele. They are either α⁰-thalassaemia carriers or individuals with HbH disease. Carriers of α⁰-thalassaemia have microcytic and hypochromic red blood cells. The commonly used cut-off mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) for screening of α⁰-thalassaemia is typically <78-80 fL and <27 pg, respectively [15–17]. HbH disease can be detected by hypochromic, microcytic anaemia in blood count and presence of HbH and/or Hb Bart’s during Hb analysis. Haemoglobin analysis can also reveal co-inheritance of β-thalassaemias or other Hb variants but it is not able to identify α⁰-thalassaemia or some unstable globin variants.

When both individuals in a couple meet the MCV or MCH criteria, they undergo DNA-based testing for α⁰-thalassaemia. The test for α⁰-thalassaemia is required regardless of β-thalassaemia status or iron deficiency as coinheritance of α-thalassaemia with β-thalassaemia, other β-haemoglobinopathies, and iron deficiency is common [18]. The exception is in the African population where α⁺-thalassaemia is common but α⁰-thalassaemia is rare [16, 19-21]. Genetic testing for α⁰-thalassaemia should be performed when both partners have low MCV or MCH and β-thalassaemia carrier state has been ruled out through Hb analysis [16].

DNA-based method for identification of α-thalassaemia

Approximately 5% of the world’s population carries α-thalassaemia mutations [22]. Common α⁰-thalassaemias and their prevalent regions are summarized in Table 1. Ethnicity information is crucial for selecting the types of deletions to be tested.

Table 1

Common α⁰-thalassaemias and the prevalent regions

Gap-polymerase chain reaction (gap-PCR) technique is employed for detection of common types of α⁰-thalassaemia. The technique is particularly useful for identifying large gene deletions. PCR primers are designed to flank known deletion breakpoints that are specific for each deletion. In cases of α⁰-thalassaemia deletions with unknown breakpoints, multiplex ligation-dependent probe amplification (MLPA) of α-globin gene cluster is utilized to determine the region of deletion.

Couples at risk of foetal Hb Bart’s hydrops foetalis

Pregnant women are strongly encouraged to seek early antenatal care. During the initial antenatal care visit, couples should receive counselling regarding the risk of foetal thalassaemia diseases, and prenatal testing to identify those at risk should be conducted at this stage [23]. For pregnant women at risk of foetal Hb Bart’s hydrops foetalis, the first ultrasound examination should be scheduled for 12-13 weeks of gestation. Couples facing this risk should receive counselling on the clinical presentation of foetuses with Hb Bart’s hydrops foetalis, foetal diagnostic methods, and management options.

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There are two foetal diagnostic approaches available: The conventional approach: This method involves obstetric procedures to obtain foetal DNA or blood for molecular and/or Hb analysis. It is invasive and carries a slightly higher risk of procedure-related (more...)

Conventional foetal diagnosis of Hb Bart’s hydrops foetalis: analysis of foetal DNA or blood

The conventional foetal diagnostic approach entails obstetric procedures to obtain foetal DNA or blood for molecular and/or Hb analysis. the details of each obstetric procedure are as shown in Table 2.

Table 2

Obstetric procedures for prenatal diagnosis

DNA-based diagnosis of the foetus is performed using gap-PCR to detect the α⁰-thalassaemia deletions that may have been inherited from the parents. In cases where the specifics of α⁰-thalassaemia deletion is unknown, the foetal cord blood should be utilized for Hb analysis. In foetuses with Hb Bart’s hydrops foetalis, the predominant haemoglobins are Hb Bart’s (75-90%) and Hb Portland (10-15%) [2–8]. The possibility of maternal DNA or blood contamination should be assessed using standard methods, as described elsewhere [27].

Noninvasive foetal diagnosis of Hb Bart’s hydrops foetalis: ultrasound

Ultrasound can be used to evaluate signs of foetal anaemia and hydrops in an affected foetus in cases that the couple opts against invasive prenatal diagnosis, or when the risk identification is incomplete, such as a single mother who is an a⁰-thalassaemia carrier. If signs of foetal anaemia or hydrops are present, DNA-based foetal diagnosis needs to be done to confirm the final diagnosis.

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The ultrasound approach for the known couple at risk of having a foetus with Hb Bart’s hydrops foetalis is as follows [24]: The mother should undergo serial foetal ultrasound scans starting from 12–14 weeks of pregnancy. The ultrasound (more...)

The ultrasound algorithm for prenatal diagnosis of Hb Bart’s hydrops foetalis is shown in Figure 1. The ultrasound algorithm is based on the evidence that [24, 29]: (1) Ultrasound is highly effective in early detection of Hb Bart hydrops foetalis in pre-hydropic phase. (2) All of the affected foetuses can be detected before 24 weeks of gestation. (3) The most sensitive parameters in predicting foetal anaemia in Hb Bart’s hydrops foetalis appear to be the cardiac diameter to thoracic diameter ratio (CTR), followed by middle cerebral artery peak systolic velocity (MCA-PSV), and placental thickness, respectively. (4) On serial ultrasound examination, either increased CTR or increased MCA-PSV are highly suggestive of hydrops and confirmation of diagnosis through obtaining foetal blood is strongly recommended. (5) Several other ultrasound markers are helpful in increasing specificity, such as hepatosplenomegaly.

Figure 1

Ultrasound algorithm for prenatal diagnosis of Hb Bart’s hydrops foetalis among at-risk pregnancies. The ultrasound algorithm is adapted from reference [24]. (with permission) *Positive sonomarker [28]: Abnormally increased cardiac diameter-to-thoracic (more...)

Other consideration: noninvasive prenatal testing of Hb Bart’s hydrops foetalis by analysis of cell-free foetal DNA

Noninvasive prenatal testing (NIPT), which involves the analysis of cell-free foetal DNA (cff-DNA), has found widespread use in prenatal diagnosis of foetal aneuploidies and autosomal dominant diseases [30]. NIPT using cff-DNA has also been employed for prenatal diagnosis of Hb Bart’s hydrops foetalis [31–38]. Various highly sensitive molecular detection techniques have been researched for this purpose, including real-time quantitative PCR, digital PCR, next-generation sequencing, and mass spectrometry methods. The sensitivity and specificity of the tests are approaching 100% with the advanced techniques. With the advancement of these techniques, the sensitivity and specificity of the tests are approaching nearly 100%. As these methods continue to improve, NIPT for Hb Bart’s hydrops foetalis using cff-DNA should be considered as a screening test and used in conjunction with other established methods for foetal diagnosis.

HbH hydrops foetalis

HbH disease arises from mutations of three out of four α-globin genes, typically involving a compound heterozygosity of α⁰-thalassaemia on one chromosome and either a deletion or a point mutation of one α-globin gene on the other. Deletional HbH disease (genotype --/-α) is generally associated with mild and non-transfusion-dependent haemolytic anaemia. In contrast, non-deletional HbH disease (genotype --/α^Tα or --/αα^T) result in a more severe phenotype [39, 40]. While the majority of these patients remain non-transfusion dependent, on demand transfusion is common and some will require regular transfusions.

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HbH hydrops foetalis represents a subtype of HbH disease characterized by severe foetal haemolytic anaemia, which can lead to hydrops foetalis [41]. HbH hydrops foetalis typically results from a compound heterozygosity involving α⁰-thalassaemia (more...)

Given the rarity of HbH hydrops foetalis and the limited available data, the monitoring for foetal anaemia in pregnancies with HbH hydrops foetalis is based on guidelines established for Hb Bart’s hydrops foetalis. It is crucial to obtain a definitive molecular diagnosis and identify potential couples at risk for appropriate management.

Hydrops foetalis due to homozygous Hb Constant Spring mutation or other rare non-deletional α-thalassaemia mutations

Homozygous HbCS (α^CSα/α^CSα) is associated with mild anaemia in older children and adults, although the condition is an emerging cause of severe foetal anaemia and hydrops [44–48]. Foetuses with hydrops foetalis associated with homozygous HbCS and other rare non-deletional α⁺-thalassaemia mutations (e.g. Hb Taybi and Hb Pakse) present from mid-gestation with varying degree of anaemia and/or hydropic signs. Haemoglobin analysis in foetuses with homozygous HbCS shows presence of HbF, HbA, Hb Bart’s and HbCS (Hb F/A/Bart’s/CS pattern) with Hb Bart’s of 7–17%, lower than those seen in HbH hydrops foetalis [8, 44, 45]. Intrauterine transfusion helps to reverse the anaemia and hydropic changes. After intrauterine transfusion, patients with homozygous HbCS usually have mild anaemia at birth and are non-transfusion dependent thereafter. The finding of anaemia in foetal period that improves after birth suggests an adverse interaction between the CS variant and the γ-globin, although this has not been documented. Compound heterozygosity of HbCS and Hb Pakse can result in foetal anaemia with comparable findings as homozygous HbCS and should be managed similarly [8].

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As intrauterine transfusion can improve the perinatal outcome in foetuses with hydrops foetalis associated with homozygous HbCS, the ultrasound monitoring for foetal anaemia will be beneficial. Based on limited evidence, the ultrasound algorithm for prenatal (more...)

Summary and recommendations

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