Heterozygous β‐thalassemia with complete absence of hemoglobin A2 in a Chinese adult

Sir, β-thalassemia is caused by the reduced (β+) or absent (β0) synthesis of the β-globin chains of the hemoglobin (Hb) tetramer, which is made up of two α-globin and two β-globin chains (α2β2). Carriers of β-thalassemia are clinically asymptomatic. The characteristic hematological features are microcytosis, hypochromia, and increased HbA2 (α2δ2). In a thalassemia endemic region, if both partners of a couple are defined carriers for β-thalassemia, each of their offspring has a 25% risk of being affected by homozygous β-thalassemia, which is a severe transfusion-dependent anemia. Therefore, carrier screening is extremely useful by allowing couples at risk to make informed decision on their reproductive choices. Through genetic counseling and the option of prenatal testing, such a couple can opt to bring to term only those pregnancies in which the fetus is unaffected. Elevation of HbA2 is the most important feature in the detection of heterozygous β-thalassemia, but there are sometimes β-thalassemia heterozygotes with normal HbA2, compromising the screening effectiveness 1-5. β-thalassemia with the absence of HbA2 is very rare. We herein report such a case in a Chinese adult during our prenatal thalassemia screening program. The work was approved by the ethics committee of Guangzhou Women & Children Medical Center. Informed consent was obtained from the patients who were studied. A woman was screened positive for thalassemia by determinant of MCV at her first trimester. Hb analysis was followed to determine the type of thalassemia. She showed a classical β-thalassemia trait (MCV 68.2 fl, MCH 20.7 pg, Hb 12.4 g/dL, Hb A2 4.1%). Reverse dot-blot (RDB) method was used to detect the 17 types of Chinese β-thalassemia mutations, and a heterozygous codon 41-42 (-CTTT) (HBB:c.126_129delCTTT) mutation was found. Gap-PCR was also used to detect the three common Chinese α-gene deletion mutations (−α3.7, −α4.2, and −SEA), but failed to identify any of the three defects. The husband was called in to do a screening test for thalassemia. The 26-year-old man was healthy and had no history of anemia during his development. Hematological analysis showed reduced red cell indices but with normal Hb level (MCV 70.2 fl, MCH 22.7 pg, Hb 13.7 g/dL). Unexpectedly, he showed a complete absence of Hb A2 peak on Sebia capillary electrophoresis system CAPILLARYS 2 (Figure 1a). This finding led us to suspect the possible absence of δ-globin chain production. Considering the presence of β-thalassemia trait in his wife, the man was also tested for β-thalassemia mutation although he had no increased HbA2. The RDB method failed to identify any of the 17 mutations. However, a further analysis by DNA sequencing discovered the −90 C>T (HBB:c.−140C>T) mutation of the β-gene (Figure 2a). The couple was referred to genetic counseling and notified of having a one in four chance of giving birth to a baby with severe β-thalassemia syndrome. They opted for prenatal diagnosis. Invasive testing by chorionic villus sampling at 10 weeks gestation revealed that the fetus inherited both mutant alleles (β−90/βCDs41–42) from parents. The pregnancy was terminated by the couple's request. To explore the underlying mechanisms of the absence of Hb A2 in the husband, blood samples were collected from his parents. The father presented normocytic normochromic hematological characteristics (MCV 90.2 fl, MCH 30.1 pg, Hb 14.4 g/dL) with a reduced Hb A2 value (1.3%) which was the phenotype of δ-thalassemia (Figure 1b). The mother had a predominantly microcytic hypochromic blood picture (MCV 69.3 fl, MCH 20.2 pg, Hb 13.0 g/dL) with a normal Hb A2 value (3.0%) (Figure 1c). DNA sequencing confirmed the same −90 C>T mutation in the mother. These findings suggest that the mother might also have δ-thalassemia, thus masking the diagnosis of β-thalassemia. Both of them might pass on the δ-thalassemia to their son who had no production of δ-globin. To confirm these hypotheses, the investigation of δ-gene was conducted by DNA sequencing. As expected, a heterozygous T-C substitution was detected at position −77 (HBD:c.−127T>C) of the δ-gene in both the father and mother (Figure 2b), and the same −77 mutation as a homozygous state was detected in the son (Figure 2c) 6. The expression level of the δ-gene is very low throughout life. The δ-thalassemia itself has no clinical implication. However, δ-gene mutations may have serious consequences for the diagnosis of β-thalassemia trait in special ethnic groups like Chinese 7, 8. β-thalassemia heterozygotes usually have microcytosis combined with elevated levels of Hb A2. In rare instances, carriers of β-thalassemia have normal levels or even no Hb A2 as evidence in this family. Understanding and identification of these underlying causes in unusual cases are very important. Failure to do so may cause potential pitfalls in genetic counseling and prenatal diagnosis. In a previous study, we found a 0.4% carrier rate of δ-gene mutations in our population 9. The −77 was the most common mutation in the Chinese, which is located within the highly conserved core sequence (GATA-1 motif) of the δ-gene, and impairs expression by affecting the GATA-1 binding 10. Expression defects of the δ-gene reduce or even abolish the Hb A2 production, and therefore, carriers of β-thalassemia could be overlooked. If one is unaware of this coexistence, it may lead to misdiagnosis in the screening schedules for β-thalassemia. A subject with low MCV but apparently nonincreased Hb A2 level cannot just be assumed to have iron deficiency or α-thalassemia, especially when the other partner has already been identified to have β-thalassemia trait. A detailed molecular analysis for both α- and β-thalassemia is mandatory. None. None.