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We report a rare case of haemoglobin (Hb) Q-H disease in a Chinese newborn, with detailed description of the haemoglobin analysis results. To our best knowledge, this is the first report in the literature which describes cation exchange high performance liquid chromatography (HPLC) and capillary electrophoresis results of a newborn with Hb Q-H disease.
The patient was a newborn male, born of non-consanguineous marriage. His father and mother were both of Chinese origins. Both parents were asymptomatic. The mother was found to be heterozygous Hb Q-Thailand during haemoglobin pattern study on antenatal screening, which was subsequently confirmed by Sanger sequencing of the HBA1 gene [NM_000558.5(HBA1):c.223G>C (p.Asp75His)]. Multiplex gap-polymerase chain reaction (gap-PCR) of α-globin genes showed heterozygous -α4.2 deletion. Deletions for --SEA and -α3.7 were not detected. The father was an α-thalassaemia trait. Molecular analysis of the α-globin genes using multiplex gap-PCR showed heterozygous --SEA deletion. Gene deletions of the -α3.7 and -α4.2 type were negative. Haematological parameters and haemoglobin analysis results by cation exchange HPLC are shown in Table 1.
Table 1Red cell indices and haemaglobin study results of the proband and his parents
The patient was born full term (38 weeks of gestation) with a birth weight of 3.31 kg. He presented with hypochromic microcytic anaemia shortly after birth. His red cell indices after birth are shown in Table 1. Peripheral blood smear showed prominent anisopoikilocytosis with hypochromic microcytic red cells, some target cells, elliptocytes and teardrop cells. There was moderate polychromasia. Reticulocytes accounted for 7.6% of the erythrocytes.
Haemoglobin analysis was carried out by HPLC on the Variant-II Hemoglobin Testing System (Bio-Rad Laboratories, USA) according to the procedures provided by the manufacturer. Examination of the chromatogram showed absence of Hb A, Hb F and Hb A2, with presence of Hb Bart's and two variant peaks. The variant peaks had retention times of 3.64 min and 4.56 min (Fig. 1A), and they were most likely to represent αQ2γ2 and αQ2β2 variants, respectively.
Fig. 1(A) Cation exchange high performance liquid chromatography analysis of the proband. (B) Capillary electrophoresis result of the proband before 1:1 mixing with normal adult blood. (C) Capillary electrophoresis result of the proband after 1:1 mixing with normal adult blood.
On capillary electrophoresis by the Capillarys 2 Flex Piercing system (Sebia, France), the two variants migrated to Z(S) (αQ2γ2) and Z(F) (αQ2β2), respectively (Fig. 1B,C). The percentages of Hb Bart's, αQ2γ2 and αQ2β2 were 30.4%, 37.7% and 31.3%, respectively. Alkaline cellulose acetate gel electrophoresis revealed two variants which migrated to the Hb S/D/G position (αQ2γ2) and a position slightly anodal to Hb S/D/G position (αQ2β2), respectively (Fig. 2A); while on acid agarose gel electrophoresis, the two variants migrated to a position between Hb A and Hb F (αQ2γ2) and a position slightly anodal to Hb S (αQ2β2), respectively (Fig. 2B). Sanger sequencing of the HBA1 gene identified a missense mutation NM_000558.5: c.223G > C (p.Asp75His) leading to Hb Q-Thailand. Gene deletions of the --SEA and -α4.2 were detected by multiplex gap-PCR. --THAI, --MED, --FIL, -α3.7 and -(α)20.5 type deletions were negative. The results were compatible with compound heterozygous --SEA deletion and Hb Q-Thailand.
Fig. 2(A) Alkaline cellulose acetate gel electrophoresis result of the proband. (B) Acid agarose gel electrophoresis result of the proband.
A follow-up assessment at the age of 7 months showed mild pallor, no clinical jaundice, satisfactory growth and no hepatosplenomegaly. The patient was on oral folate supplementation and did not require transfusion.
Hb Q-Thailand is an α-globin chain variant caused by a point mutation (GAC → CAC; Asp → His) in codon 74 of HBA1 gene on chromosome 16, and it is associated with a leftward single α-globin gene deletion (-α4.2).
The α-globin gene adjacent to the gene for Hb Q—α 74 Asp → His is deleted, but not that adjacent to the gene for Hb G—α 30 Glu → Gln; three fourths of the α-globin gene are deleted in Hb Q-α-thalassemia.
Hb Q-H disease is caused by the co-inheritance of Hb Q-Thailand and α0-thalassemia (mainly --SEA deletion). It is a rare disease which is mostly identified in patients of Chinese origins. Hb Q-H disease is associated with the absence of Hb A, presence of Hb Bart's, with Hb Q-Thailand being the predominant fraction of haemoglobin in adults.
Although Hb A is absent in patients with Hb Q-H disease, clinical features and blood indices of these patients are similar to that of deletional Hb H disease.
This is also supported by the clinical features of the newborn. He did not show hydrops or growth retardation in antenatal follow-up and did not require transfusion up to last follow-up.
To our best knowledge, haemoglobin analyses of newborns with Hb Q-H disease have rarely been reported. The results of cation exchange HPLC and capillary electrophoresis of these patients have not been reported in the literature previously.
Our case demonstrates the haemoglobin analysis results in a newborn with Hb Q-H disease. There was absence of HbF and HbA, and presence of Hb Bart's with two major haemoglobin variants, namely αQ2γ2 and αQ2β2. The percentage of αQ2γ2 variant was slightly higher than that of the αQ2β2 variant. The αQ2γ2 variant had a retention time of about 3.6 min on HPLC by the Variant-II Hemoglobin Testing System. It migrated to Z(S) on capillary electrophoresis by the Capillarys 2 Flex Piercing system. The αQ2γ2 variant migrated to Hb S/D/G position on alkaline cellulose acetate gel electrophoresis and a position between Hb A and Hb F on acid agarose gel electrophoresis. This knowledge is useful for haematologists and laboratory scientists for rapid diagnosis of Hb Q-H disease in newborns, especially if molecular tests are not readily available in the laboratory.
Conflicts of interest and sources of funding
The authors state that there are no conflicts of interest to disclose.
References
Lie-Injo L.E.
Dozy A.M.
Kan Y.W.
et al.
The α-globin gene adjacent to the gene for Hb Q—α 74 Asp → His is deleted, but not that adjacent to the gene for Hb G—α 30 Glu → Gln; three fourths of the α-globin gene are deleted in Hb Q-α-thalassemia.
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