Rare and unexpected mutations among Iranian beta-thalassemia patients and prenatal samples discovered by reverse-hybridization and DNA sequencing.

β-thalassemia is one of the most common hereditary disorders in Iran, there being an estimated two million carriers and an extensive spectrum of mutations.1-3 While peripheral diagnostic laboratories cover the most common β−globin gene mutations known to occur within their respective area, samples remaining untyped are referred to our national reference laboratory for further analysis. Between 1999-2001 we received a total of 70 DNA samples from patients (59 thalassemia trait and 4 thalassemia major subjects) and prenatal cases (4 chorionic villi and 3 amniotic fluid samples), from various geographic areas and ethnic groups within Iran. As our first approach these samples were screened for a panel of 22 relatively common β-globin mutations using an assay based on polymerase chain reaction (PCR) and reverse-hybridization to oligonucleotide arrays immobilized on test strips.4 This powerful technique covers more than 80% of known β-globin alleles in Iran in a single amplification and hybridization step, allowing even very small amounts of DNA (e.g. prenatal samples) to be rapidly and comprehensively typed.5 A total of 65 samples, which remained negative for these prevalent mutations, were subjected to DNA sequencing of the entire β-globin gene including the 5 ́-untranslated region. The combined results of both techniques revealed a total of 23 different β-globin alleles among our samples (Table 1). The most common one was IVS I-130 (G-C), which was identified in six subjects from the North of Iran, three subjects from the Southwest, as well as in one DNA of unknown geographical origin. A single homozygous individual showed a severe type of anemia and low hemoglobin (8.7 g/dL), the remaining heterozygotes had a mean Hb A2 of 5.3% (range: 4.6-6.5), and mean MCV and Hb levels of 61.7 fL (58.0-64.8) and 12.0 g/dL (10.813.2), respectively. The second most common mutation was cd 82/83 (-G), which was present in four samples each from Northern and Central Iran, and in one sample from Western Iran. Two of the samples were derived from prenatal cases; the mean Hb, MCV and Hb A2 level of the remaining seven heterozygous subjects were 12.6 g/dL (10.0-15.1), 62.7 fL (60.0-68.0) and 5.0% (3.9-6.0), respectively. The origin of both IVS I-130 (G-C) and cd 82/83 (-G), as well as a number of other mutations found among our samples, is attributed to neighbors to the North and Northwest of Iran (Turks, Kurds, Azerbaijanies).6-8 Unlike these, another group of mutations, including the third most common mutation cd 16 (-C) as well as cd 41/42 (-TTCT), is of Asian-Indian or East Asian (Chinese, Japanese) origin.8 Interestingly and in agreement with our previous observations,3 frameshift mutation cd 16 (-C) was entirely restricted to chromosomes derived from the Southeast of Iran, suggesting that it should be particularly considered for β-thalassemia testing in that part of the country. A third group of mutations we observed originates from Mediterranean populations; this group included the Portuguese type (TGG-TGA) of cd 15 mutation.8 This was found in three samples from Western and Southwestern Iran, while the Asian-Indian type (TGG-TAG) of cd 15 mutation was identified in one sample from the Southeast. Among the very rare mutations were two heterozygotes for IVS II-2,3 (+11/-2), which had been discovered earlier in an Iranian family.9 Very surprisingly, we also identified IVS I-2 (T-C) in an individual from Northern Iran, a mutation which had originally been reported to occur among American Blacks.10 A previously unknown transition from AAG (Lys) to TAG (Stop) in codon 95, was discovered in a β-thalassemia trait patient from Western Iran presenting with the following hematologic parameters: MCH: 18.7 pg, MCV: 61.1 fL, Hb A: 93.3%, Hb A2: 5.2%. In a total of 10 cases we could not detect a mutation by our two-step approach, suggesting that the thalassemia phenotype for these patients is caused by genetic defects outside the β-globin gene and 5 ́-untranslated region. These results confirm earlier reports from our group as well as other groups on the highly heterogeneous spectrum of β-thalassemia mutations in Iran.1-3 The large variety of existing alleles and their uneven distribution within the different parts of the country is mainly due to the fact that the Iranian population represents a mixture of various ethnic groups. Refining our knowledge about the distribution of common and rare mutations will further improve carrier detection and prenatal diagnosis to prevent this prevalent disorder.