Hemoglobin, 35(4):323–330, (2011)Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.3109/03630269.2011.571527

ORIGINAL ARTICLE

THE CARRIER FREQUENCY OF α-GLOBIN GENE TRIPLICATION IN AN

IRANIAN POPULATION WITH NORMAL OR BORDERLINE

HEMATOLOGICAL PARAMETERS

Seyedeh Fatemeh Moosavi,1 Azam Amirian,1 Behnaz Zarbakhsh,1

Alireza Kordafshari,1 Hasan Mirzahoseini,2 Sirous Zeinali,1

and Morteza Karimipoor1

1Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran2Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran

� The −α3.7 rightward deletion is the most frequent α-globin mutation worldwide, whilefrequencies of the αααanti 3.7 triplication are only sporadically known. Carriers of the αααanti 3.7

triplication show no clinical symptoms or significant hematological changes, but co-inheritance withβ-thalassemia (β-thal) has been reported to worsen the clinical and hematological features of thepatient as well as the trait. We have screened the α-globin gene rearrangements of 280 individualswith normal hematological indices and 117 persons with borderline hematological parameters.We used multiplex polymerase chain reaction (m-PCR) and multiplex ligation-dependentprobe amplification (MLPA) technology to detect triplications and quadruplications. Only theαααanti 3.7 triplication was observed. The carrier frequency in the first group was 2.14% andin the second group 1.7%. No phenotype aggravation was noticed in two carriers of β-thal and theαααanti 3.7 triplication, while a mild β-thalassemia intermedia (β-TI) was observed in a β-thalcarrier with six α-globin genes. Due to the high consanguinity in the country, homozygosity forthe αααanti 3.7 triplication and for other rearrangements can be expected. Therefore, an accuratedetermination of the frequencies and a routine control for these mutations is essential for a correctgenotype-phenotype prediction during genetic counseling for β-thal.

Keywords α-Thalassemia (α-thal), αααanti 3.7 Triplication, Multiplex polymerase chainreaction (m-PCR), multiplex ligation-dependent probe amplification (MLPA)

Received 12 January 2011; Accepted 12 February 2011.Address correspondence to Morteza Karimipoor, M.D., Ph.D., Molecular Medicine Department,

Pasteur Institute of Iran, Farvardin Str., Tehran, Iran; Tel.:/Fax: +98-21-66480780; E-mail: morteza-karimi@yahoo.com

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324 S.F. Moosavi et al.

INTRODUCTION

Due to the high frequency of β-thalassemia (β-thal) and otherhemoglobinopathies in Iran, a national program for prevention of β-thalmajor (β-TM) has been established since 1997 by carrier screening ofcouples at the premarital stage (1). Due to the occurrence of α-thalassemia(α-thal), co-inheritance is not infrequent in our multiethnic population,and complex combinations of globin gene defects are regularly observedin many couples making an appropriate genetic counseling difficult. Theaverage frequency of β-thal alleles in the country fluctuate around 4-5% withpeaks in specific regions, while frequencies of 30% have been reported forα-thal carriers in some southern regions (2).

Although α-globin gene triplications are not associated with abnormalhematological parameters, it is known that the inheritance of α-globingene triplication exacerbates the severity of the phenotype in β-TM andβ-thal intermedia (β-TI). Conversely, although different β-TI cases havebeen reported due to co-inheritance of α-globin gene triplication andheterozygous β-thal alleles (3–6), so far, it is unclear to what extentthe additional α/β-globin chain imbalance caused by α triplications mayaggravate the mild phenotype of the β-thal carrier.

The genetic mechanisms that generate α gene triplications are called anunequal crossover and is caused by misalignment of homologous sequenceson the α2 and α1 genes during recombination that generates two products,one is the triplicated (αααanti 3.7) and the other is the hybrid (–α3.7) gene(7–10). The last, due to malaria selection, is the most frequent α-globinmutation in Iran (11) and similarly, the frequency of β-thal gene is close to10% in some northern and southern regions (12). Different α triplicationfrequencies, reported in some populations, give rise to many questions.Why so low (0.36%) in American Blacks who are 16% carriers of the −α3.7

deletion? Why less than 0.04% in Sardinians and 5% in Greek Cypriots(9). On the other hand, comparable data in Portugal (2%) (13), 1% ina multi ethnic population in The Netherlands (14) and around 1% inIndia (15), seems to converge. So far, the frequency of the αααanti 3.7 globingene triplication and cluster duplications has not been studied in Iran, andtherefore we have determined the carrier frequency of this anomaly in twoIranian populations with normal and borderline hematological parameters.

SUBJECTS AND METHODS

Two groups of individuals from Tehran, Iran, were included in our study:1) 280 unrelated individuals with normal hematological indices (MCV >80fL, MCH > 27pg, Hb A2 <3.4% and Hb F <3%); 2) 117 persons withborderline hematological parameters (MCV <80 fL, MCH <27 pg, and

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The Frequency of α Triplications in Iran 325

normal Hb A2 and Hb F). The last were referred to our laboratory forfurther molecular analysis from primary health care centers. One patientwith a mild β-TI phenotype and heterozygous for a β-thal allele was alsoincluded as well as four individuals from a family of a β-thal carrier fromgroup 2. Written informed consent was obtained from all participants.

Peripheral blood was collected in EDTA. Hematological parameterswere measured on a Sysmex KX-21 cell counter (Kobe, Japan). HbA2 and Hb F levels were measured by acetate cellulose (pH 8.6) andcolumn chromatography (ION Exchange BioSystems S. A., Barcelona,Spain). Genomic DNA was extracted from peripheral blood using thesalting out method (16). Multiplex polymerase chain reaction (m-PCR)assay as developed by Wang et al . (17) was used to detect the αααanti 3.7

triplication and ααααanti 3.7 quadruplication. The designed primers (AT3.7F and AT 3.7R) were specific to amplify the junction region in thesenew rearrangements. Other primers (Lis1-2.5F and Lis1-2.5R) were usedas internal controls (17). The PCR was carried out in 25 μL reactionscontaining 1.5 mM MgCl2, 200 μM dNTP mix, 1 × AMS (ammoniumsulfate) PCR buffer, 10% dimethyl sulfoxide (DMSO), 7 pmol Lis1-2.5F andLis1-2.5R primers, 5.5 pmol AT 3.7F and AT 3.7R primers, 1 U SmarTaqDNA polymerase (CinnaGen, Tehran, Iran) and 200-500 ng genomic DNA.Amplification was performed with an initial 5 min. denaturation at 94◦C,followed by 35 cycles at 94◦C for 1 min., 64◦C annealing for 2 min., and72◦C extension for 3 min., and a final 7 min. extension at 72◦C in a GradientMasterCycler (Eppendorf, Hamburg, Germany). After amplification, 10 μLof PCR product was run on 1% agarose gel in 0.5 × TBE (Tris-Borate-EDTA)buffer and then visualized on UV transilluminator after staining withethidium bromide.

The multiplex ligation-dependent probe amplification (MLPA) (18)technique was performed to confirm results of multiplex PCR and alsoto discriminate between heterozygote and homozygote triplication. TheMLPA was performed using SALSA MLPA P140-B2 HBA kit accordingto manufacturer recommendations (MRC Holland, Amsterdam, TheNetherlands). Briefly, genomic DNA was denatured and hybridized to theSALSA MLPA probes specific to the α-globin gene cluster. After ligationof the probes, the PCR reaction was performed by primers specific for theprobes. Then the PCR products were analyzed on an ABI 3130XL GeneticAnalyzer. The MLPA data were analyzed by Gene Marker® software (version1.6) (SoftGenetics, PA, USA).

RESULTS

We were able to identify eight cases of α gene rearrangements (Figure 1),six out of the 280 individuals of group 1 and two out of the 117 cases

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326 S.F. Moosavi et al.

in group 2. The MLPA results showed that all the positive cases had oneadditional α-globin gene, confirming that they were all heterozygous for theαααanti 3.7 triplication (data not shown). Therefore, the carrier frequencyof the αααanti 3.7 triplication in the individuals with normal hematologicalindices was 2.14% and 1.7% in the group with microcytosis. Thehematological and molecular data of seven of them are shown in Table 1.

We have also analyzed the family members of a proband from groupone (subject 1). The husband of the index case was a carrier of IVS-I-5(G>C) [HBB: c.92+5] and his α-globin gene clusters were normal (FamilyA, Table 2 and Figure 2). All the children were heterozygous for the IVS-I-5mutation, while two had also co-inherited the αααanti 3.7 mutation.

In addition, one mild β-TI patient was also studied. Multiplex PCRrevealed the anti 3.7 triplication/quadruplication. DNA analysis indicatedthat this patient was heterozygous for the codons 8/9 (+G) β-thal mutation.The MLPA test confirmed that this case is either homozygous for the anti3.7 triplication or heterozygous for the anti3.7 quadruplication.

DISCUSSION

The frequency data we have found (± 2%) are in agreement withfigures reported from other authors. We also found carriers in people withnormal CBC parameters and the borderline CBC parameters from the twoindividuals in the second group cannot be attributed to the α triplicationgenotype.

It has also been reported that the co-inheritance of extra α-genes mayaffect the clinical and hematological picture of β-thal trait, ranging from

FIGURE 1 Detection of α-globin gene triplication by multiplex-PCR. Lanes 1-4 correspond to the PCRproducts of the normal samples, lane 5: α triplication, and lane 6: no template control (NTC). Lane M,1 kb DNA ladder.

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328 S.F. Moosavi et al.

TABLE 2 Hematological and Molecular Data of Members of Family A

Parameters I-1 I-2 II-1 II-2 II-3

Hb (g/dL) 15.4 13.9 12.2 10.9 13.3PCV (L/L) 0.46 0.46 0.41 0.37 0.51RBC (1012/L) 5.40 7.30 6.80 6.00 7.40MCV (fL) 85.0 62.9 60.8 61.6 68.2MCH (pg) 28.3 19.2 18.0 18.2 17.9MCHC (g/dL) 33.3 30.5 29.7 29.5 26.2Hb A2 (%) 2.2 3.6 4.1 4.3 4.5Hb F (%) 0.3 0.7 0.3 0.1 0.5α Genotype αααanti 3.7/αα αα/αα αα/αα αααanti 3.7/αα αααanti 3.7/αα

β Genotype βA/βA βIVS-I-5/βA βIVS-I-5/βA βIVS-I-5/βA βIVS-I-5/βA

FIGURE 2 The pedigree of Family A.

an asymptomatic presentation to a mild to moderate β-TI (8–9,14,19).Kanavakis et al. (20) reported individuals with interaction of these twodeterminants that were indistinguishable from β-thal carriers. Conversely,Sampietro et al. (21) reported one case with this interaction that gave rise toa picture of mild β-TI, while a range of variable phenotypes was reported byCamaschella and colleagues (22). The latter reported that the combinationof these two determinants resulted in β-TI in five Italian subjects, whilein two other cases the phenotype remained that of β-TM. Kimura et al .(19) reported two members in a family who had the same α- and β-globingenotypes but presented different hematological manifestations.

In this study, we found two members of a family (Figure 2) with acombination of one triplicated α-globin locus and heterozygous for a β-thalallele but their clinical and hematological picture were not different fromthose of the other plain β-thal carriers in the same family. The lower Hblevel measured in case II-2 could be simply due to the female gender orgenetic factors. However, the interaction of the triplicated α-globin locusand heterozygous β-thal allele may play a role.

Conversely, our other case with co-inheritance of two additional α-globingenes and a β-globin gene mutation resulted in a mild β-TI condition (Hb

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The Frequency of α Triplications in Iran 329

9.8 g/dL, RBC 5.12 × 1012/L, MCV 59.2 fL, MCH 19.1 pg, Hb A2 5.8%and Hb F 4%). This is in agreement with Giordano et al . (14) who recentlyreported no significant effect to the phenotype of the β-thal carrier unlesstwo or more extra α genes are present (23). Indeed, in a number of studies(5,12,24) the combination of an homozygous α triplication (six genes intotal) with heterozygous β-thal alleles resulted in moderate-to-severe β-TIwith occasional blood transfusion requirements. For example, Fallah et al .(12) reported a child with an ααα/ααα genotype and heterozygous for theIVS-I-5 mutation, who had been transfused once for her anemia (12).

Of course, not all β mutations will behave in the same way, genotype/phenotype correlation, taking into consideration the high frequency of theαααanti 3.7 triplication, is essential in Iran, where the high consanguinity ratemakes it likely that homozygous individuals will be frequently found for bothβ and α defects. In conclusion, accurate determination of the frequency ofα-globin gene number and genotype/phenotype correlation is essential ingenetic counseling.

ACKNOWLEDGMENTS

The authors would like to thank the unknown reviewer for revising themanuscript. The authors also thank the individuals participated in this study.

Declaration of Interest: The authors report no conflicts of interest. The authorsalone are responsible for the content and writing of this article.

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