David H.K. Chui, MD, Department of Pathology and Molecular Medicine, Room 2N31, McMaster University Medical Centre, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada. E-mail: email@example.com
Haemoglobin H (Hb H) disease is caused by deletion or inactivation of three α-globin genes, leaving only one intact and active α-globin gene. People with Hb H disease usually have moderate anaemia, but are generally thought to be asymptomatic. Some Hb H disease patients require transfusions, and there are reports of fetuses with Hb H disease who have severe anaemia in utero resulting in fatal hydrops foetalis syndrome. We now report a case of Hb H hydrops foetalis syndrome, caused by the inheritance of a hitherto novel α-globin gene point mutation (codon 35 TCC→CCC or Serine→Proline) and an α-thalassaemia deletion of the Filipino type removing all ζ-α-globin genes on the other chromosome 16. The infant was delivered prematurely because of pericardial effusion and fetal distress, and was found to have severe anaemia and congenital anomalies. A review of the relevant literature on this syndrome is presented, and serves to underscore the phenotypic variations of Hb H disease and the need for surveillance for this condition among newborns and genetic counselling in communities with a high proportion of at-risk populations.
The human α-globin gene cluster is made up of one embryonic ζ-globin gene and two α-globin genes, arranged in the order of 5′-ζ2 –α2 –α1–3′ on chromosome 16pter-p13.3 (Higgs, 2001). Each individual usually has a total of four α-globin genes. Both α2- and α1-globin genes code for identical α-globin peptides, but α2-globin gene accounts for significantly more α-globin chain production than α1-globin gene (Liebhaber et al, 1986).
α-Thalassaemia is a common hereditary condition caused by mutations affecting one or more of the four α-globin genes, leading to decreased or absent α-globin chain production (Higgs, 2001; Higgs & Bowden, 2001). When three α-globin genes are deleted or defective, only one intact α-globin gene remains. The excess β-globin chains form β4 tetramers (Hb H). These affected individuals are said to have haemoglobin H (Hb H) disease. They have moderate anaemia but, generally, are thought to be well.
Fetuses that have deletion of all four α-globin genes (homozygous α°-thalassaemia) have 80–90% Hb Bart's (γ4 tetramers). These fetuses almost always succumb in utero during the second or third trimester of gestation, or die within hours after birth (Chui & Waye, 1998; Higgs & Bowden, 2001). This is known as Hb Bart's hydrops foetalis syndrome, which is by far the most common cause of hydrops in South-east Asia.
There are sporadic reports of fetuses with Hb H disease that developed the hydrops foetalis syndrome (Chui & Waye, 1998; Higgs & Bowden, 2001). We now report another newborn with this syndrome who had inherited a novel α-globin gene mutation, plus a complete deletion of the ζ-α-globin gene cluster on the other chromosome 16. A summary of relevant literature on this serious hereditary condition is also presented.
Both parents were of Filipino ancestry, lived in California and were well. The mother had two previous pregnancies, one of which ended at 6 weeks with spontaneous miscarriage of unknown cause. During the third pregnancy, she developed gestational diabetes that was controlled by diet alone. Several weeks before delivery, ultrasonography revealed pericardial effusion in the fetus. Subsequently fetal distress was noted, and the newborn was delivered by Caesarean section at 34·5 weeks of gestation.
At birth, the Apgar scores were 5 and 7 at 1 and 5 min. The neonate weighed 1·84 kg, and was severely anaemic (Hb 8·2 g/dl; haematocrit (Hct) 0·328; mean cell volume (MCV) 106 fl), and thrombocytopenic (platelet count 16 × 109/l). He was found to have 54% Hb Bart's (γ4) detected as a fast-moving haemoglobin by high-performance liquid chromatography (HPLC). Hb Bart's has high-oxygen affinity and is incapable of oxygen delivery to tissues. Furthermore, levels above 25% at birth are indicative of Hb H disease (Lorey et al, 2001).
The newborn was jaundiced (total/direct bilirubin 116/63 µmol/l), and had hepato-splenomegaly with both organs enlarged to the level of their respective iliac crests. He also had ambiguous genitalia with fourth degree hypospadias, bifid scrotum with some rugae present and bilateral inguinal testes. Karyotyping later showed normal 46 XY chromosomes. Clinical problems encountered during the first month of life included pulmonary hypertension requiring ventilatory support for 4 d, global hypertrophic cardiomyopathy shown by echocardiogram and suspected but not proven sepsis.
The infant was transfused at birth, and five more times subsequently during the first 4 months of life. Thereafter, the haemoglobin level stabilized at 8·6–8·9 g/dl without transfusion. At 13 months of age, the infant weighed 8·9 kg (10th percentile), with a head circumference of 47·5 cm (50th percentile). His Hb was 8·9 g/dl; Hct. 0·295; MCV 62 fl; reticulocyte count 0·05; leucocyte count 15·7 × 109/l; and platelet count 374 × 109/l. The spleen was palpable 1·5 cm below the left costal margin. There was little change in the appearance of the ambiguous genitalia (Fig 1). The child's motor and social developmental milestones were in keeping with an estimated chronological age of 40 weeks or 10 months of age.
Materials and methods
Haematological studies Complete blood counts were performed using a Coulter-S blood cell counter. Hb isoelectric focusing (IEF) at pH 6–8, Hb A2 quantification using HPLC (BioRad Laboratories, Hercules, CA, USA) and Hb H inclusion bodies determination were carried out (Bain et al, 1998). Assays to detect unstable haemoglobin by heat instability and by isopropanol precipitation were performed (Halchuk et al, 1992; Dacie & Lewis, 1995a pp.268–270). Newly synthesized globin chains were labelled in vitro with [3H]-leucine, and later isolated by carboxymethylcellulose (CMC)-urea column chromatography (Clegg, 1983). Screening for glucose-6-phosphate dehydrogenase (G6PD) activity was also undertaken (Dacie & Lewis, 1995b pp. 227–228).
Molecular analyses Genomic DNA was extracted from peripheral blood leucocytes. The α-globin genotype was determined using Southern blot analysis (Waye et al, 1993). The diagnosis of (––FIL) α-thalassaemia deletion was by a gap-polymerase chain reaction (PCR) method using primers designed specifically to detect this deletion (Fischel-Ghodsian et al, 1988; Eng et al, 2000). To detect point mutation, regions of the α2-globin gene were amplified using PCR, and the mutation was identified by direct nucleotide sequencing of the PCR product using a BigDye terminator cycle sequencing kit (Applied Biosystems, CA, USA) and an ABI 310 automated sequencer.
The haematological data and α-globin genotypes of both parents are summarized in Table I. The mother was a carrier of the Filipino type (––FIL) α-thalassaemia deletion removing all ζ-α-globin genes in cis. The father was heterozygous for a novel mutation involving the α2-globin gene (codon 35 TCC→CCC or Serine→Proline) as shown in Fig 2. No variant α-globin chain/haemoglobin was detected using several techniques (Table I) including in vitro labelling of newly synthesized globin chains with [3H]-leucine for either 30 min or 2 h followed by globin chain separation using CMC-urea column chromatography (data not shown). The newborn had inherited both parental mutations, and his α-globin genotype was (––FIL/αcodon 35 TCC→CCC or Ser→Proα).
Table I. Summary of haematological findings and α-globin genotypes of both parents.
RBC, red blood cell.
Reticulocyte count (× 109/l)
Peripheral blood morphology
Mild hypochromia and microcytosis
Moderate hypochromia and microcytosis
Hb H inclusion bodies
Occasional RBC seen with Hb H inclusions
None detected by IEF and anion HPLC
None detected by IEF and anion HPLC
Unstable Hb testing
α:β Globin synthetic ratio
Free erythrocyte protoporphyrin (normal 0·3–0·9)
(αcodon 35 TCC→CCC or Ser→Proα/αα)
The proband had only one normal α-globin gene. He developed pericardial effusion and fetal distress in utero, and was found to have severe anaemia and congenital anomalies at birth. The correct and early diagnosis of Hb H disease in this infant was facilitated by the universal newborn screening programme for Hb H disease in the State of California (Lorey et al, 2001).
The nucleotide change from TCC to CCC in codon 35 of the α2-globin gene results in a serine to proline substitution at the end of the B helix of the α-globin chain (Bunn & Forget, 1986; Huisman, 1993). Because proline residue is incapable of participating in an α-helix formation, except in the first three positions of the helix, this substitution is likely to introduce a profound conformational change of the α-globin chain (Huisman, 1993). There are at least 51 known α- and β-globin chain variants, all caused by the introduction of a proline residue [Hardison et al, 1998 (http://globin.cse.psu.edu)]. More than half of these variant haemoglobins are unstable, and many result in Heinz body haemolytic anaemia, such as Hb Bibba (α2codon 136 CTG→CCG or Leu→Pro). Several of these highly unstable variant haemoglobins such as Hb Quong Sze (α2codon 125 CTG→CCG or Leu→Pro) result in a thalassaemic phenotype (Liebhaber & Kan, 1983). In addition, the serine residue in codon 35 of the α-globin chain serves as part of the α1β1 contact site (Bunn & Forget, 1986; Huisman, 1993). For example, the substitution of tyrosine in this site (Hb Shinagawa) results in an unstable haemoglobin [Hardison et al, 1998 (http://globin.cse.psu.edu)].
The father of the proband was a carrier of the novel codon 35 α-globin gene mutation. He had borderline anaemia and mild microcytosis, consistent with the diagnosis of α-thalassaemia trait. No abnormal haemoglobin was detected, and the results of haemoglobin stability assays were negative (Table I). Furthermore, no variant α-globin chain was found in in vitro-radioactive labelling of newly synthesized globin chains. Taken together, these findings point to hyper-instability of the putative mutant globin/haemoglobin.
The substitution of proline for serine does not lead to a net charge change of the variant globin, and a separation of the putative variant α-globin chain from the normal using CMC-urea column chromatography is therefore not certain. On the other hand, should a haemoglobin tetramer containing variant α-globin chains of distorted higher-order structure be formed, its isoelectric point (pI) would probably differ from that of Hb A. Two known variant haemoglobins containing α-globin chains with Ser→Pro in codon 131 (Hb Questembert) and codon 138 (Hb Atteboro) can be resolved from Hb A using IEF and cellulose acetate electrophoresis [Hardison et al, 1998 (http://globin.cse.psu.edu)]. Additional studies using reversed phase HPLC and mass spectrometry techniques might be informative.
Patients with Hb H disease are generally thought to be asymptomatic. However, some patients require regular transfusions (Kattamis et al, 1988; Galanello et al, 1992; Hall et al, 1993; Kanavakis et al, 1996; Chen et al, 2000; Higgs & Bowden, 2001; Waye et al, 2001). The natural history of this hereditary disorder, particularly during infancy and childhood, has not been well documented. A recent report has suggested that there are more morbidities than previously appreciated (Chen et al, 2000). For example, in 13% of children with Hb H disease, their growth rate was below the third percentile. Almost half of the patients had been transfused, though not regularly. The majority of adult patients (85%) had iron overload, leading to hepatic cirrhosis or cardiac dysfunction in some. Hypersplenism and cholelithiasis were other complications.
Table II. Summary of published cases of Hb H hydrops foetalis syndrome.
Gestational age at delivery
Clinical manifestations at birth
Subsequent clinical course
Hb analysis at birth
Oron-Karni et al (2000) reported brief descriptions of four pregnancies in one couple. One pregnancy terminated in hydrops fetalis, three others in newborns with Hb H disease, one of whom had severe anaemia at birth that required transfusions. Their α-globin genotypes were [-α3·7 (codon 125 CTG→CAG or Leu→Gln)/-α3·7 (codon 125 CTG→CAG or Leu→Gln)].
* (αα) TH Undetermined non-deletional α-thalassaemia mutation of either α-globin gene.
Patients with Hb H disease caused by a combination of two α-globin gene deletions plus a non-deletional α-globin gene mutation such as αConstant Spring (codon 142 TAA→CAA or Ter→Gln) are more anaemic than those with deletional mutations removing three α-globin genes (Kattamis et al, 1988; Galanello et al, 1992; Kanavakis et al, 1996; Styles et al, 1997; Higgs & Bowden, 2001). Nevertheless, fetuses with genotypes of either (– –SEA/αConstant Springα) or (– –THAI/αConstant Springα) do not develop the severe hydrops foetalis syndrome (Higgs & Bowden, 2001; Lorey et al, 2001). It is conceivable that the incorporation of variant α-globin chains caused by the novel codon 35 mutation found in this family leads to extremely unstable haemoglobin molecules, and therefore results in severe anaemia in utero. Parallel findings of β-globin gene mutations that result in highly unstable haemoglobins were recently reviewed (Thein, 1999).
This report serves to emphasize the phenotypic diversity of Hb H disease, which can range from being relatively well in adulthood to fetal death in utero. This variability is illustrated by the fact that (– –SEA/αcodon 59 Gly→Aspα) can cause fatal Hb H hydrops foetalis syndrome, whereas two patients with (- (α)20·5/ααcodon 59 Gly→Asp) survived to adulthood, albeit with severe anaemia (Curuk et al, 1993; Chan et al, 1997). Non-deletional mutation involving the α2-globin gene leads to a more severe phenotype than the same mutation found in the α1-globin gene. Another cause for fatal Hb H hydrops fetalis syndrome is (– –TOT/αdeletion of GAG or Glu in codon 30α), whereas individuals with (– –SEA/αdeletion of GAG or Glu in codon 30α) can survive to adulthood (Chan et al, 1997; Chen et al, 2000). The deletion of embryonic ζ-globin gene in the (– –TOT) α-thalassaemia deletion might cause more severe anaemia in affected fetuses in utero.
Genitalia abnormalities are also part of the clinical manifestations found in the α-thalassaemia/mental retardation syndromes (Gibbons & Higgs, 2001). In the ATR−16 syndrome caused by large deletions involving the 16p telomere, these congenital defects are probably caused by deletion of ‘dosage-sensitive’ genes that are important in genitalia development. Individuals found to have monosomy involving 350 kb of the 16p telomere, including the region removed in the (– –FIL) α-thalassaemia deletion, did not have significant phenotypic anomalies other than α-thalassaemia trait (Horsley et al, 2001). Thus the candidate genes, if present, are located more proximal to the 350 kb already examined. The ATR–X syndrome is caused by mutations of the ATRX gene on chromosome Xq13. The ATRX protein is involved in chromatin-mediated transcriptional regulation and, presumably, might modulate expression of genes essential for normal developmental processes including those located near the α-globin gene cluster and elsewhere. In both ATR syndromes, the fetuses do not have anaemia to the same severe degree as those fetuses with Hb Bart's or Hb H hydrops foetalis syndromes. Thus, the causes for their genital anomalies are likely to be different in these two groups of fetuses.
It is now increasingly evident that there are genetic modifiers that can modulate clinical manifestations of many hereditary disorders such as haemoglobinopathy (Chui and Dover, 2001). Taken together, the present and other reports reaffirm the importance of surveillance of Hb H disease among newborns, particularly in communities with a high proportion of populations at risk (Lorey et al, 2001). These public health efforts will help determine the disease prevalence and monitor phenotypic variations. Furthermore, the affected infants can be provided with appropriate care, such as prompt treatment of infections, avoidance of oxidant drugs and compounds, and transfusions when indicated. Definitive genotyping is necessary to ensure that proper genetic counselling regarding reproductive risks, especially for the Hb H hydrops foetalis syndrome, is given to appropriate family members.
Note added in proof
At 20 months of age, the infant did not require transfusion. Splenomegaly at 3 cm below the left costal margin was present. His Hb was 9·2 g/dl, reticulocyte count 0·06, and platelet count 350 × 109/l. He was to undergo urological surgery sometime during the following months.