Abstract
We describe 27 cases of mild-to-severe α-thalassemia (α-thal) syndrome caused by interaction of Hb Adana [α59(E8)Gly→Asp, GGC>GAC (α2)] with deletional and nondeletional α+-thal mutations in Indonesian patients. Hematological profiles and clinical manifestations of all patients were assessed by routine procedures. The genotypes were generated by a multiplex-polymerase chain reaction (m-PCR), PCR-RFLP (restriction fragment length polymorphism)-based method, and DNA sequencing. The α-thal patients who had Hb Adana in combination with the 3.7 kb deletion mostly have mild-to-moderate anemia. In contrast, patients who were compound heterozygotes for Hb Adana and nondeletional mutations, generally showed a more severe anemia and it mostly presented in childhood. Thus, accurate diagnosis of α-thal disorders is not only important for future management of these patients but also for providing proper genetic counseling to the family.
Hb Adana is a highly unstable variant hemoglobin (Hb) resulting from a mutation at codon 59 on the α1- or α2-globin gene (HBA1: c.179G>A or HBA2: c.179G>A) (Citation1,2). In Indonesia, we found Hb Adana on the α2-globin gene (Citation3,4). The most severe phenotype due to homozygous Hb Adana, manifesting as hydrops fetalis, has been previously reported (Citation3). Study of the frequency of Hb Adana trait in the Indonesian population has not yet been finished; however, our preliminary population study showed that this Hb variant was detected at a relatively low frequency (0.4%) in the Javanese population, although it is quite common (16.0%) in Indonesian patients (Citation4). It contributes to many kinds of α-thalassemia (α-thal) syndromes, especially in compound heterozygosity with the 3.7 kb deletion, which is the most common α+-thal deletion in Indonesia (Citation4). Interestingly, we also found the combination of Hb Adana with other nondeletional mutations such as Hb Constant Spring (Hb CS, HBA2: c.427T>C (Citation5), codon 22 (α2, HBA2: c.69C>T) (Citation6), a regulatory single nucleotide polymorphism (rSNP) 149709T<C (GenBank DQ431198) in a non genic region between the ζ- and α-globin genes (Citation7) that creates a new promoter causing α-thal [Online Mendelian Inheritance in Man (OMIM) 141800.0218] and IVS-II-142 (α2, HBA2: c.440+2G>A) (Citation8). In this study, we report the hematological and clinical features of patients having Hb Adana in combination with either deletional or nondeletional α+-thal mutations.
Selection of Patients and Features
Data used in this study was retrospectively evaluated including patients who were referred to the GenNeka Clinic (Yayasan GenNeka, Eijkman Institute for Molecular Biology, Jakarta, Indonesia) and were diagnosed with α-thal based on hematological parameters and molecular diagnosis. Clinical features including age of first presentation, clinical course and physical examination were recorded.
Hematology Analysis and Molecular Studies
Hematological analysis consisted of complete blood counts (CBC), blood smear and Hb analysis. The CBCs were measured using Cell Dyne 1700 (Abbot Diagnostics, Abbott Park, IL, USA), erythrocyte morphology was assesed by blood smear examination under light microscope and Hb analysis was performed using high performance liquid chromatography (HPLC) on a VARIANT™ Hemoglobin Testing System (β-Thalassemia Short Program; Bio-Rad Laboratories, Hercules, CA, USA). Genomic DNA was isolated from leukocytes using modified Gentra Puregene Blood Kit (Qiagen Inc., Valencia, CA, USA) and used as a template for the polymerase chain reaction (PCR)-based mutation detection.
Single α Gene Deletions [–α3.7 (rightward) and –α4.2 (leftward) types]
We performed multiplex-PCR (m-PCR) using primers as previously described (Citation4). Each 25 μL reaction mixture contained 5× KAPA2G GC Buffer (with MgCl2), 200 μM dNTPs (Invitrogen, Life Technologies Corp., Carlsbad, CA, USA), 0.2 μM of primers SA3P, XYHA and XY1, 0.1 μM of primers 3R1 and 2/3P (), 4.0% DMSO, 0.5 units of KAPA2G Robust HotStart DNA polymerase (5 U/μL) (KAPA Biosystems Inc., Woburn, MA, USA), and 100 ng of genomic DNA as template. The PCR was carried out on a Veriti™ 96-well thermal cycler (Applied Biosystems, Foster City, CA, USA) using the following conditions: initial denaturation at 95°C for 3 min.; 35 cycles at 95°C for 30 seconds, 62°C for 45 seconds, 72°C for 2 min.; then final extension at 72°C for 5 min. Product sizes of PCR were analyzed by electrophoresis on 1.0% agarose gel (LE Agarose, Roche Applied Science, Mannheim, Germany). The gel was photographed using Molecular Imager Gel Doc™ XR System (Bio-Rad Laboratories). Product sizes of PCR were 1940 bp for the normal band, 2220 bp for the –3.7 kb mutant band, and 1673 bp for the –4.2 kb mutant band. For nondeletional mutation detection, we first performed DNA sequencing and then developed and applied direct mutation detection [PCR-RFLP (restriction fragment length polymorphism)].
TABLE 1 List of Primers Used to Detect Single α-Globin Gene Deletions (Ivy Ng, personal communication)
We carried out PCR-RFLP to detect the codon 59 mutation as previously described (Citation3). We developed a PCR-RFLP-based method to detect Hb CS, codon 22 and the IVS-II-142 mutations. For the Hb CS mutation detection, 25 μL reaction mixture contained 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8, 200 μM dNTPs, 0.4 μM of each primer (), 0.625 units of Taq DNA polymerase (New England BioLabs Inc, Ipswich, MA, USA) and 100 ng of genomic DNA. Polymerase chain reactions were carried out on a 9700 thermal cycler (Applied Biosystems) using the following conditions: initial denaturation at 95°C for 5 min.; 35 cycles at 95°C for 30 seconds, 68°C for 30 seconds, 72°C for 1 min.; then a final extension at 72°C for 5 min.
TABLE 2 List of Primers, Polymerase Chain Reaction Product Sizes, Restriction Enzymes and Restriction Fragment Length Polymorphism Product Sizes to Detect Nondeletional α-Thalassemia Mutations
For the detection of the codon 22 and IVS-II-142 mutations, each 25 μL reaction contained 5× Phusion GC Buffer containing 7.5 mM MgCl2 as 1× final concentration, 200 μM dNTPs, 0.4 μM of each primer (), 6.0% DMSO, 0.5 Units of Phusion DNA Polymerase (New England BioLabs Inc.) and 100 ng of genomic DNA. The PCR conditions were as follows: initial denaturation at 95°C for 5 min.; 35 cycles at 95°C for 1 min., 62°C for 30 seconds, 72°C for 1 min.; then a final extension at 72°C for 5 min.
For the detection of the rSNP 149709T<C mutation, each 25 μL reaction contained 1× PCR buffer (Qiagen), 1× Q solution (Qiagen), 200 μM dNTPs, 0.2 μM of each primer (), 1 unit of HotStar Taq Polymerase (Qiagen) and 100 ng of genomic DNA. The PCR conditions were as follows: initial denaturation at 95°C for 15 min.; 35 cycles at 95°C for 1 min., 54°C for 1 min., 72°C for 1 min.; then a final extension at 72°C for 5 min.
Polymerase chain reactions to detect the nondeletional mutations were carried out on a 9700 thermal cycler (Applied Biosystems). Digestion, using appropriate restriction enzymes, was according to the manufacturer’s instructions. All digested products were electrophoresed in 2.0% agarose. The gels were photograped using Gel Doc™ XR (Bio-Rad Laboratories). The PCR product sizes, restriction enzymes and RFLP product sizes are given in .
Twenty-seven cases were studied, 25 were of Indonesian ethnic background and two were Chinese. Eighteen cases (67.0%) were compound heterozygotes for Hb Adana and either the –α3.7 (17 cases) or –α4.2 (one case) mutations (), and nine cases (33.0%) were compound heterozygotes for Hb Adana and other nondeletional mutations ().
TABLE 3 Hematological and Clinical Data of Hb Adana Patients in Combination With One Gene Deletion at Diagnosis
TABLE 4 Hematological, Molecular and Clinical Data of Patients Carrying Hb Adana in Combination With Nondeletional α+-Thalassemia at Diagnosis
Although the clinical manifestations in patients with Hb Adana in combination with single gene deletions were mostly mild, the onset varied widely from infant to late adult (1 month to 49 years old) with the steady state Hb levels ranging from 6.1 to 11.9 g/dL (9.4 ± 1.2) (). Only two patients required regular blood transfusions, one of whom started at 3 months of age and one at 3 years old. Two patients first presented at quite a late age (42 and 49 years). Several patients had a slight increase in Hb F levels (patients 3, 4, 6 and 7 in ) as previously described (Citation1,9). This phenomenon might be related with the increase of γ-globin gene expression, which should be further elucidated.
The nondeletional mutations in trans to Hb Adana were Hb CS (four cases), codon 22 (three cases), rSNP 149709T>C mutation (one case) and IVS-II-142 (one case) (). The first presentation varies from 7 days to 28 years old, with the steady state Hb level ranging from 4.9 to 11.1 g/dL. Most patients (five out of nine) required regular blood transfusions, two patients required irregular blood transfusions, and only one patient did not require any blood transfusions. One patient, a compound heterozygote for Hb Adana and rSNP 149709T>C required regular blood transfusions since 2 months of age, but did not require them after a splenectomy at age 9 years. Two related patients (case 6 and 7) with the same genotype (Hb Adana and codon 22) showed very different clinical severity as thalassemia major and mild thalassemia intermedia. This situation was also found in patients with a compound heterozygosity for Hb Adana and Hb CS ().
Although Indonesian α-thal patients who had Hb Adana in combination with the 3.7 kb deletion generally have mild-to-moderate anemia, a few of them could manifest as severe anemia. This phenotype variation has been reported previously (Citation2,9). Patients who had Hb Adana in combination with nondeletional mutations mostly showed more severe symptoms than those who had it with single gene deletion as we can see from their blood transfusion status, and who presented at an earlier age. The clinical severities varied widely between patients, even for those with the same genotype. The diversity of the hematology profiles and clinical manifestations might be due to genetic or non genetic factors, further research is required to explain this phenotype variation. Moreover, traits of the Hb Adana (Citation3) and the other nondeletional α-thal mutations in this study exhibit a normal range of Hb level with mild reduction of red cell indices, such as codon 22 (MCV >70.0 fL, MCH >25.0 pg), rSNP 149709T>C (MCV >70.0 fL, MCH >22.0 pg) and IVS-II-142 (MCV >79.0 fL, MCH >26.0 pg). Therefore, routine DNA analysis, regardless of the hematology feature, is strongly suggested for diagnosis of α-thal in Indonesia, especially during antenatal screening.
ACKNOWLEDGMENTS
The authors thank Professor Suthat Fucharoen, (Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Thailand) for valuable comments, suggestions and proofreading the manuscript; our thanks also to Dr. Trevor Jones for proofreading the manuscript. The authors also thank Dr. Pustika A Wahidiyat and Teny T Sari from the Department of Child Health, Cipto Mangunkusumo Hospital, Jakarta; Dr. Dasril Daud from the Department of Child Health, Medical Faculty, Hasanuddin University, Makassar, Intan Russianna from Gatot Subroto Hospital, Jakarta; Udjiani E. Pawitro from Tangerang Regional General Hospital, Tangerang, Banten, for referring the patients.
Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
REFERENCES
- Çürük MA, Dimovski AJ, Baysal E, . Hb Adana or α259(E8)Gly→Aspβ2, a severely unstable α1-globin variant, observed in combination with the –(α)20.5 kb α-thal-1 deletion in two Turkish patients. Am J Hematol. 1993;44(4):270–275.
- Traeger-Synodinos J, Metaxotou-Mavrommati A, Karagiorga M, . Interaction of an α+-thalassemia deletion with either a highly unstable α-globin variant (α2, codon 59, GGC→GAC) or a nondeletional α-thalassemia mutation (AATAAA→AATAAG): comparison of phenotypes illustrating “dominant” α-thalassemia. Hemoglobin. 1999;23(4):325–337.
- Nainggolan IM, Harahap A, Setianingsih I. Hydrops fetalis associated with homozygosity for Hb Adana [α59(E8)Gly→Asp (α2)]. Hemoglobin. 2010;34(4):394–401.
- Setianingsih I, Harahap A, Nainggolan IM. α Thalassaemia in Indonesia: phenotypes and molecular defects. Adv Exp Med Biol. 2003;531:47–56.
- Eng LI, Baer A, Lewis AN, Welch QB. Hemoglobin Constant Spring (slow-moving hemoglobin X components) and Hemoglobin E in Malayan aborigines. Am J Hum Genet. 1973;25(4):382–387.
- Harteveld CL, Wijermans PW, van Delft P, Rasp E, Haak HL, Giordano PC. An α-thalassemia phenotype in a Dutch Hindustani, caused by a new point mutation that creates an alternative splice donor site in the first exon of the α2-globin gene. Hemoglobin. 2004;28(3):255–259.
- De Gobbi M, Viprakasit V, Hughes JR, . A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter. Science. 2006;312(5777):1215–1217.
- Noguera NI, Gonzalez FA, Davoli RA, Milani AC, Villegas A. A novel splice acceptor site mutation of the α2-globin gene causing α-thalassemia. Hemoglobin. 2001;25(3):311–315.
- Douna V, Papassotiriou I, Garoufi A, . A rare thalassemic syndrome caused by interaction of Hb Adana [α59(E8)Gly→Asp] with an α+-thalassemia deletion: clinical aspects in two cases. Hemoglobin. 2008;32(4):361–369.