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Preimplantation genetic diagnosis of thalassemia

Head of Molecular Biology Laboratory , WU HUI-MEI

Thalassemia is one of the most common inherited single-gene disorders in the world. It occurs when mutations in the gene encoding globin results in reduced production of Globins, which resulting in anemia. Hemoglobin A (HbA), the major blood oxygen carrier in adults, is a heterotetramer of α- and β-globin proteins, each bound to heme. In embryonic and fetal stages, other subtypes of globins constitute fetal hemoglobin to extract oxygen. The thalassemias are classified according to which chain of the hemoglobin molecule is affected. The production of α-globin and β-globin chain is affected in α- and β-thalassemia, respectively. The mutated gene is passed down from generation to generation. Preimplantation genetic diagnosis (PGD) can be applied to select embryos with normal globin genes to slowly eliminate the disease.

Alpha-thalassemia is associated with large deletion in α-globin gene of chromosome 16p, which results in insufficient production of α-Globin protein and hence anemia. There are fourα-globin alleles, and the severity of α-thalassemias is correlated with the number of affected α-globin alleles. Alpha-globulin is produced in small quantities during the early embryonic stage, at which time hemoglobin is composed of other globins with similar functions. As an embryo develops, it begins to produce large quantity of alpha-globins in early fetal period, approximately 6 weeks of gestation, and the function of alpha-globin cannot be replaced by other globins. A mutation in any one of the four alpha-globin genes results in a condition with no symptoms, i.e, alpha thalassemia silent carrier, and the fetus can survive. At the most severe form of alpha-thalassemia in which no alpha-globin can be produced, the fetus dies in utero. Depending on the location and number of genedeletion, the symptoms of anemia will manifest to varying degrees. In Taiwan, about 4 to 5% of the population suffers from alpha-thalassemia. Ethnic distribution of various alpha-thalassemia determinants depends on the geographic location. In Taiwan, the most prevalent type is the Southeast Asia deletion (95%), and the remaining 5% are composed of Thai type, Filipino type, etc.

Beta-thalassemia is caused by point mutations in beta-globin gene on chromosome 11. More than two hundred point mutations have been reported. Beta-globin is not produced in large quantities, but other subtypes of globin replaced its function in the fetal stage, therefore the fetus remain unaffected even is the most severe case. After birth, fetal globin synthesis declines rapidly as beta-globin production increases and substitute fetal globin by three months of age. The major difference between alpha- and beta-thalassemia is there are surrogates for insufficient beta-globin. Similar to alpha-thalassemia, there are certain geographical distribution of ethnic origins of, for examples, in Taiwan and coastal area of the mainland China, 95% beta-thalassemia results from by four types of mutations, and the remaining 5% patients are caused by dozen types of mutations.

In our IVF center, thalassemia PGD is focused on the screening of major types of mutation, and is carried out only for suspected thalassemia trait couples. The genotypes of the parents are determined by polymerase chain reaction (PCR) from blood samples. In brief, after DNA purification from WBCs, fragments of target genes were amplified by PCR with specific primer pairs. The genotype of alpha-thalassemia can be demonstrated by differentiation from gel electrophoresis, whereas restriction enzyme digestion is required prior to gel electrophoresis to determine the beta-thalassemiagenotype. The genotype of the offspring is determined by PCR from 1 or 2 blastomeres at the 8-cell stage embryo cultured at day 3 of the IVF procedure. Re-amplification is required for single cell genotyping. If there are enough embryos, only normal ones were selected for transfer; but if the available embryos were limited, the alternative is to transfer carrier embryo, and the child will have the same degree of anemia as the parents in the future.

No test is 100% accurate, so does PGD for thalassemia. We highly recommend the couples receiving PGD to have chorionic villus sampling after pregnancy to ensure the fetal genotype.

▲Figure 1. Gel electrophoresis of Southeast Asian type of alpha-thalassemia.
  1. carrier
  2. normal
▲Figure 2. Gel electrophoresis of the four most common types of beta-thalassemia.
A: -28(A→B)
B: codon17(AAG→TAG)
C: codon 41/42 (TCTT deletion)
D: IVS-2 nt 654 (C→T)
  1. normal
  2. carrier