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The Hong Kong Association of Blood Transfusion and Haematology
A Young Boy with Chronic Anaemia

Dr. Raymond Chu
Department of Clinical Pathology, Pamela Youde Nethersole Eastern Hospital

Case Presentation

A 5-year-old boy was admitted into hospital for vomiting and diarrhoea for one day. He has been seen by private practitioner previously because of anaemia and was told to have thalassaemia. He has not been transfused previously.

Physical examination showed pallor and jaundice. Thalassaemic facies with frontal boxing and depressed nasal bridge were noted. He has hepatosplenomegaly with liver 3 cm and spleen 5 cm below costal margin. Growth parameters showed that his weight was 1 kg below the 3rd percentile while his height and head circumference were at the 10th percentile.

On admission, his haemoglobin level was 7.2 g/dl, which dropped to 5.6 g/dl after rehydration. There was reticulocytosis (retic 5.2%) with increased total bilirubin level (48 umol/L). Liver and renal function tests were otherwise normal. His iron status was normal. Haptoglobin level was markedly reduced with negative results for direct antiglobulin test and G6PD deficiency screening. 270 ml blood was given and his haemoglobin was 9.9 g/dl after transfusion. Haemoglobin study showed a haemoglobin variant band and a family study was performed. Results are as follows:

  Father Mother Patient
Sex M F M
Age (yrs) 35 37 5
Hb (g/L) 13.6 12.9 5.6
RBC (x 1012/L) 6.68 5.11 2.73
Hct 0.423 0.394 0.173
MCV (fL) 63.3 77.0 63.4
MCH (pg) 20.3 25.3 20.6
HbA % 55 68.4 13.7
HbF % 0.9 8.6 69
HbA2 % 3.9 3.3 3.1
Hb Variant %      
(estimated by densitometry of the alkaline gel) Neg 17.7 9.1
Hb H bodies Neg Neg Neg

Paragon gel electrophoresis at alkaline and acid pH of the whole family:

Fig. 1
(Lane 2 = Patient, Lane 3 = Father, Lane 4 = Mother)
Fig. 2

Globin chain electrophoresis:

Fig. 3
(Lane 4 = Patient, Lane 5 = Father, Lane 6 = Mother)

HPLC tracings of the family:

Patient
Fig. 4
Fig. 5
Father
Mother
Fig. 6

Discussion

This patient presented with features of thalassaemia intermedia. He has markedly elevated Hb F (69%), normal Hb A2 level and a Hb variant band in the alkaline gel electrophoresis, which moves between Hb S and Hb A2. Globin chain electrophoresis suggested that the Hb variant is an alpha chain variant. His HPLC tracing showed three abnormal peaks, one between Hb F and Hb A (Fig. 4, Black Arrow), the other two smaller bands in the D-window (Fig. 4, Red Arrow).

The father's findings are consistent with beta thalassemia trait.

Interestingly the mother showed a Hb variant, which moves in a different position in alkaline gel electrophoresis when compared with his son's variant band. It moves between Hb S and Hb F, which is faster than the child's variant band. In her HPLC tracing, the bands are at the D/S windows (Fig. 6, Red Arrow), which are at similar positions as her son's two minor bands (Fig. 4, Red Arrow). Her Hb A2 level of 3.3% is within normal limits while her Hb F is elevated to 8.6%. She has mildly decreased MCV at 77 fl and blood film showed mild microcytosis, compatible with thalassaemic trait picture. Her globin chain electrophoresis also suggested that the Hb variant is an alpha chain variant. However, alpha chain haemoglobinopathy alone cannot account for the elevated Hb F. Futhermore, it cannot account for the microcytosis unless it belongs to those a chain variants that can give rise to the phenotype of a+ thalassaemia, e.g. Hb Constant Spring, Hb Q, etc. However, its position in alkaline gel electrophoresis and HPLC tracing are different from those known a chain variants that can give rise to the phenotype of a+ thalassaemia. At this stage, it can reasonably be deduced that she probably has another globin gene abnormality, likely to be a normal A2 b thalassaemia, This could then explain the microcytosis with elevated Hb F level . This could also account for her child's clinical and laboratory findings (thalassaemia intermedia picture with markedly elevated Hb F level). If the child has the b-thalassemia gene from his father together with the alpha haemoglobinopathy gene from his mother only, this still could not account for his findings. Therefore, it is highly likely that this boy has more than two globin gene abnormalities and the third abnormality is likely to be a normal A2 b thalassaemia inherited from his mother.

Globin genotyping gives the answer to this diagnostic problem. b-globin genotyping showed that the father is heterozygous for codon 17 (A->T) b0-mutation while the mother is heterozygous for nt-29 (A->G) b+-mutation. The child was shown to be compound heterozygous for codon 17 and nt-29 mutations (b0 / b+ configuration), which explains the thalassaemia intermedia presentation with markedly elevated Hb F level. a-globin genotyping showed that both the mother and child have a point mutation in the a1 gene (116 GAG -> GCG; Glu a Ala), which confirms the alpha haemoglobinopathy to be Hb Ube-4. This haemoglobin variant has been described in a Japanese and a Korean family. [1] [2]. In heterozygote, there is no abnormal haematology apart from the presence of such haemoglobin at 10-15% levels as this haemoglobin shows normal functional and stability studies. The different electrophoretic mobilities of the Hb variant in alkaline gel can be explained by the binding of the a-globin variant with the abundant g globin chain in the child (avariantg) while the a-globin variant binds with the b-globin chain in the mother (avariantb). There is still some avariantb in the child as evidenced by the presence of minor peaks in the D-window of the HPLC tracing (Fig. 4, Red Arrow) at similar positions to the mother's bands (Fig. 6, Red Arrow).

References

  1. Ohba Y, Miyaji T, Matsuoka M, Morito M, Iuchi I. Characterization of Hb Ube-4: alpha 116(GH4) Glu yield Ala. Hemoglobin 2:181-6, 1978.
  2. Iuchi I, Hidaka K, Ueda S, Shibata S, Hitomi F, Takesue A. Hb Ube-4 (alpha 116 Glu leads to Ala): second independent instance found in a Korean family of Japan. Hemoglobin 2:561-3, 1978.

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