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1 ISSN: (print), X (electronic) Hemoglobin, 2014; 38(4): ! 2014 Informa Healthcare USA, Inc. DOI: / ORIGINAL ARTICLE A Case Series of a-thalassemia Intermedia Due to Compound Heterozygosity for Hb Adana [HBA2: c179g4a (or HBA1); p.gly60asp] With Other a-thalassemias in Malay Families Hafiza Alauddin 1, Noor-Adilah Jaapar 1, Raja Z. Azma 1, Azlin Ithnin 1, Noor-Farisah A. Razak 2, C-Khai Loh 3, Hamidah Alias 3, Zarina Abdul-Latiff 3, and Ainoon Othman 4 1 Haematology Unit, Department of Pathology, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia, 2 Diagnostic Medical Laboratory Department, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia, 3 Department of Paediatrics, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia, 4 Department of Medical Science II, Faculty of Medicine and Health Science, Universiti Sains Islam, Nilai, Malaysia Abstract Hb Adana [HBA2: c179g4a (or HBA1); p.gly60asp] is a rare hemoglobin (Hb) variant due to a mutation at codon 59 of the a2- or a1-globin gene resulting in a glycine to aspartic acid substitution. Two siblings with a unique coinheritance of Hb Adana and Hb Constant Spring (Hb CS, a142, Term!Gln, TAA4CAA; HBA2: c.427 T4C) (a codon 59 a/a CS a), were compared phenotypically with another two siblings carrying the Hb Adana mutation and a 3.7 kb deletion (a codon 59 a/ a 3.7 ). Although they all had a-thalassemia intermedia (a-ti), the former were clinically more severe than the latter. The first pair of siblings presented at a much younger age than the second pair and showed lower Hb levels and significant extramedullay hemopoiesis. Another case of a hydropic fetus as a result of Hb H/Hb Adana is also described. Their clinical phenotypes and hematological parameters are all presented for comparison. Introduction Nondeletional a-thalassemias can result from mutations of the a2 gene (a T a) or the a1 gene(aa T ). Nondeletional a-thalassemias are more commonly caused by mutations of the a2 gene rather than the a1 gene (1), while mutations affecting the former are believed to result in a more severe anemia because it produces 2 3 times more a-globin mrna than that of the a1 gene (2). In recent years, more nondeletional a-thalassemias have been recognized (3). Patients with nondeletional a-thalassemia (a-thal) are more likely to have splenomegaly and require blood transfusions (4,5). Hb Adana [HBA2: c179g4a (or HBA1); p.gly60asp] is a rare hemoglobin (Hb) variant that results from a mutation involving codon 59 of either the a1 or a2 gene and producing a highly unstable Hb molecule. The a-globin chain variant with a Gly!Asp substitution at codon 59, results in an introduction of a larger and charged aspartic acid molecule that compromises the stability of the protein (6). This Hb variant was first described in two related patients in Adana, Turkey. These patients inherited the mutation on the a1 gene in trans to an a 0 mutation [ (a) 20.5 ] that resulted in a a-thal Address correspondence to Dr. Hafiza Alauddin, Haematology Unit, Department of Pathology, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latiff, Cheras, Kuala Lumpur 56000, Malaysia. Tel: Fax: drhafiza@ppukm.ukm.edu.my Keywords a 3.7, SEA, Codon 59 mutation, Hb Adana, Hb Constant Spring (Hb CS) History Received 1 May 2013 Revised 8 December 2013 Accepted 11 December 2013 Published online 14 May 2014 intermedia (a-ti) phenotype (6). Since then a few other cases have been reported elsewhere involving patients of various ethnic groups (7 9). In contrast to the unstable b-globin variants, the highly unstable a-globin variants are usually identified in compound heterozygotes with other a-thal mutations leading to a-ti phenotypes (10). We report a series of cases involving interactions of Hb Adana (a codon 59 a) with other a-thalassemias encountered at our center with the emphasis on the interaction of Hb Adana with Hb Constant Spring (Hb CS, a142, Term!Gln, TAA4CAA; HBA2: c.427 T4C; p.x143gln) (a codon 59 a/a CS a) in a Malay family, which, to the best of our knowledge, has never been reported to date. Materials and methods Clinical, hematological and hemoglobin studies This study involved four Malay patients from two unrelated families and an aborted fetus. Blood samples were collected in EDTA-containing vacutainers. Red blood cell (RBC) indices were measured using an automated hematology analyzer, LH750 (Beckman Coulter, Miami, FL, USA). The peripheral blood films were automatically prepared and stained with LH slide maker and stainer (Beckman Coulter) and were reported by experienced hematologists. Hemolysates of the samples were analyzed with high performance liquid chromatography (HPLC) using a

2 278 H. Alauddin et al. Hemoglobin, 2014; 38(4): VARIANT TM Hemoglobin Testing system with the b-thalassemia Short Program (Bio-Rad Laboratories, Hercules, CA, USA) to determine the Hb subtypes described in the instruction manual. The H-inclusion test was carried out using standard methods and was evaluated by an experienced laboratory technician. Genomic DNA was extracted from white blood cells (WBC) of the EDTA-anticoagulated peripheral blood samples using the Blood Mini DNA Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer s guidelines. The DNA concentration and purity was determined spectrophotometrically at 260 and 280 nm and DNA samples were stored at 30 C before use. We optimized a gap-polymerase chain reaction (gap-pcr) assay capable of detecting any combination of the six common a-globin gene deletions utilizing the primer sets published by Chong et al. (11) that was able to detect single a gene deletions [ a 3.7 (rightward), a 4.2 (leftward)] and two a gene deletions ( SEA, FIL, MED, THAI and (a) 20.5 ]. The samples were then subjected to a second phase single tube multiplex amplification refractory mutation system-pcr (ARMS-PCR) assay to screen for nondeletional a-globin gene mutations that used the method published by Eng et al. (12). This method allowed detection of the nondeletional a-globin mutations on the a2 gene. The mutations screened included: initiation codon (ATG4A G), codon 30 (DGAG), codon 35 (TCC4CCC), codon 59 (GGC4GAC), codon 125 (CTG4CCG) or Hb Quong Sze [Hb QS, HBA2: c.377 T4C (or HBA1)] and termination codon TAA4CAA or Hb CS. The standard reaction mix contained 1 mg DNA, 10 pmol of each primer and 1.5 U of Taq DNA polymerase (Qiagen) and was I-1 I-2 II-1 II-2 Alleles α 59 α α CS α Figure 1. An interaction between Hb Adana and Hb CS in Family A. αα subjected to PCR amplification at 94 C for 12 min. for initial denaturation, followed by 32 cycles of denaturation at 94 C for 40 seconds, annealing at 62 C for 20 seconds and extension at 72 C for 3 min. Amplicons were size fractionated with the standard agarose gel electrophoresis. Results The probands were siblings (II-1 and II-2) from Family A and another set of siblings (II-1 and II-5) from Family B. The stillborn fetus (III-2) was the offspring of proband II-1 from Family B. Family A The probands II-1 and II-2 from Family A were characterized with Hb CS (a CS a) in trans to a mutation at codon 59 (a codon 59 a) on the a2-globin gene predicted to lead to the hyperunstable Hb variant combination. Their mother was heterozygous for Hb CS (a CS a/aa), while their asymptomatic father inherited a mutation at codon 59 (a codon 59 a/aa) (Figure 1, Table 1). Both siblings presented at the age of 3 years old with fever, anemia and hepatosplenomegaly. Initial investigations concluded that they were both heterozygotes for Hb CS. However, this did not explain the severity of their disease. The elder sister developed cholelithiasis at the age of 8 years old and had undergone cholecystectomy. Unfortunately, both siblings were not on any regular follow-up and finally presented to our center with maxillary hyperplasia and massive hepatosplenomegaly (spleen of 10 cm and liver of 3 cm, respectively). Their growth was not affected. Their Hb levels ranged from 6.5 to 7.5 g/dl without the need for blood transfusions. Their peripheral blood films showed hypochromic microcytic red cells with marked anisopoikilocytosis, polychromasia, basophilic stippling, target cells and occasional nucleated red blood cells. High performance liquid chromatography revealed a peak at the Hb CS retention time and no Hb H (b4) inclusion bodies were detected (Table 1). Family B The probands II-1 and II-5 inherited the 3.7 kb a + -thal gene deletion in trans to the codon 59 mutation (a codon 59 a/ a 3.7 ). The stillborn hydropic fetus (III-2) was characterized with a two a gene deletion and the codon 59 mutation Table 1. The hematology parameters of the probands and carriers with Hb Adana. Cases Family A Family B Parameters I-1 II-1 II-2 II-1 II-5 III-2 Sex-age M-37 F-11 M-9 M-31 M-17 stillborn a Hb (g/dl) NA RBC (10 12 /L) MCV (fl) MCH (pg) Hb A 2 (%) ND Hb F (%) ND Hb X (%) ND 2.3 (Hb CS) 2.0 (Hb CS) H-inclusion ND negative negative negative negative a Genotype a codon 59 a/aa a codon 59 a/a CS a a codon 59 a/a CS a a codon 59 a/ a 3.7 a codon 59 a/ a 3.7 a codon 59 a/ SEA Hb: hemoglobin; NA: not available; RBC: red blood cell; MCV: mean corpuscular volume; MCH: mean corpuscular Hb; ND: not done; codon 59: Hb Adana; Hb CS: Hb Constant Spring; a 3.7 : one a gene deletion; SEA :twoa gene deletion. a Gender unknown.

3 DOI: / Hb Adana Coinherited With Other -Thalassemias 279 Figure 2. Interactions between Hb Adana and a single a gene deletion ( a 3.7 ) in II-1 and II-5 as well as Hb Adana and a two a gene deletion ( SEA ) in III-2 of Family B. I-1 I-2 Alleles α 59 α II-1 II-2 II-3 II-4 II-5 -α SEA αα III-1: unknown status III-2 III-3: HbH disease (a codon 59 a/ SEA ). The wife of II-1 (II-2) carried two a gene deletions (aa/ SEA ), while the mother (I-2) of the probands was found to carry the 3.7 kb a + -thal gene deletion (aa/ a 3.7 ). Unfortunately, their father (I-1) had passed away earlier at the age of 81 years due to an unknown cause and was unable to be characterized, but he was presumed to carry the codon 59 mutation (Figure 2). They were both young male siblings, aged 31 and 17, who had a history of anemia and intermittent jaundice during acute febrile episodes but had only rarely required blood transfusions. These two brothers were only brought to medical attention when the wife of II-1 (II-2), aborted two hydropic fetuses (III-1 and III-2). Clinically, both brothers appeared pale and had splenomegaly. The fetus (III-2) was delivered stillborn at 28 weeks gestation and was confirmed to be hydropic by antenatal ultrasound and clinical postmortem. Investigations had ruled out infective or blood grouping incompatibility between the mother and the fetus. Their hematology parameters and DNA analysis results are also shown in Table 1. Discussion The mutation at codon 59 results in the formation of a hyperunstable molecule and has no product to be visualized by routine hematology studies (8). Therefore, the true frequency of this molecular anomaly is unknown as the defect can only be ascertained by DNA analysis. Moreover, Hb Adana carriers are asymptomatic or exhibit only mild anemia, with red cell indices quite similar to those with a + -thal trait due to one a-globin gene deletion (9), as can be seen in I-1 of Family A, who was clinically asymptomatic (Table 1). The siblings from family A provided a very rare finding of how the interaction between two different types of nondeletional a2 gene mutations resulted in an a-ti phenotype although the two a1 genes are still functional. Both presented in early childhood with symptomatic anemia and jaundice that was aggravated by fever. Pyrexia has been shown to enhance intra-erythrocytic inclusion body formation that might lead to acute hemolytic crisis associated with infections (5,13). Hemolysis is thought to play a more dominant role in causing anemia than ineffective erythropoiesis in patients with Hb H disease (14). In terms of laboratory findings, the brother (II-2) of Family A, showed a low mean corpuscular volume (MCV) and mean corpuscular Hb (MCH), while in his sister (II-1), the MCH was normal and MCV was near normal (25.6 pg). In a review article by Chui et al. (4), the MCV in nondeletional a-thal was higher than in deletional a-thal. However, anemia was more pronounced in nondeletional a-thal and so was reticulocytosis. These findings might have accounted for the higher MCV values, especially in the sister who had a near normal MCV level. a-thalassemic erythrocytes are hyperhydrated, resulting in the increased red cell volume relative to the Hb content. This is due to the insertion of oxidized a CS -globin chain into the red cell membrane and its cytoskeleton, similar to the effect caused by oxidized b-globin chain in Hb H disease (15). It is postulated, although unproven, that the early closing of the K-Cl cotransporter in a-thalassemic erythrocytes, in contrast to b-thalassemic erythrocytes, prevents loss of K-Cl and water and leads to erythrocyte hyperhydration and higher MCV (16). The peripheral blood films of siblings from Family A revealed significant basophilic stippling that was not seen in the films of siblings from Family B. In a study that looked at homozygous Hb CS cases, significant basophilic stippling in the peripheral blood film was also reported. The authors postulated that there was a possibility that the mrnas of the unstable a CS precipitate and aggregate resulting in the basophilic stippling seen under light microscopy (17). The siblings from Family B showed an interaction of the codon 59 mutation with the more common single a gene deletion ( a 3.7 ) that resulted in a milder clinical phenotype with their Hb levels ranging from g/dl. They presented with a history of symptomatic anemia and jaundice during acute infection episodes that seldom necessitated blood transfusion. Inheritance of a two a gene deletion usually results in a trait clinical phenotype but when combined with the codon 59 mutation, the clinical phenotype became worse, similar to the first case series published by Çürük et al. (6), although in their cases, the a1 gene was affected. Douna et al. (8) and George et al. (18) also reported cases with the codon 59 mutation and a 3.7 in trans, who also showed a-ti phenotypes. The severity of these cases as compared to cases with two gene deletions were postulated to

4 280 H. Alauddin et al. Hemoglobin, 2014; 38(4): be due to exarcerbation of the abnormal red cell physiology that already has excess b-globin chains with more pronounced and ineffective erythropoiesis (5). Similarly, Traeger- Synodinos et al. (19) also described an interaction of Hb Adana with a single a gene deletion presenting with a-ti in a patient at the age of 10 years old. Furthermore, the authors also demonstrated the occurrence of both ineffective erythropoiesis and peripheral hemolysis in the probands studied (19). Comparing the siblings from Family A to the siblings from Family B, it is noteworthy that the former were more severely affected than the latter. The involvement of two nondeletional a gene mutations has resulted in a much more severe phenotype than a combination of nondeletional and deletional mutations. This is expected because a nondeletional mutation is known to cause more severe effects on the red cells than the deletional ones. They tend to have a higher red cell volume and inappropriate to the degree of anemia because of the cellular changes (14,15). The aborted fetus (III-2), who was the baby of II-1, was severely hydropic and was miscarried at 28 weeks gestation. It was later confirmed that the fetus carried Hb H disease who had also inherited the codon 59 mutation in trans to the more common two a gene deletion ( SEA ). Similarly, in 2008, Henderson et al. (20) also reported two cases of hydrops fetalis associated with the codon 59 mutation and a two a gene deletion ( FIL ) in a Filipino couple. Fucharoen et al. (21) commented that the clinical course of nondeletional Hb H is highly variable, even involving the same genotypes, and it is difficult to predict the clinical course of the disease. In terms of management, repeated blood transfusions should be considered in selected patients (usually with the nondeletional forms) with severe anemia, massive erythroid expansion causing bony changes and extramedullary erythropoiesis as in the brother (II-2) in Family A. However, there are no clear guidelines for managing a-ti, especially with regards to the frequency of transfusions and management of complications (22,23). Having said that, even for thalassemia patients with common globin gene mutation interactions, the genotype-phenotype correlation are not always clear due to other modifying factors (18,24). Severe phenotypes associated with this mutation in a homozygous state is a very important issue in the prevention and management program of thalassemia. In our patient with two nondeletional a gene mutations, where we anticipate a more severe phenotype, close monitoring of clinical features and early transfusions should be encouraged to avoid the irreversible bony expansion and splenectomy. This is the first case report of a-ti resulting from compound heterozygosity for nondeletional a-thal (Hb CS and Hb Adana) detected in a Malay family. This case report highlights the need to consider a hyperunstable a Hb variant (Hb Adana) in patients with a clinical phenotype of a-ti in the absence of a strongly positive H-inclusion test and with normal Hb electrophoresis. We also describe the variable clinical spectrum (mild and severe) forms of Hb Adana. Routine laboratory screening programs, however, cannot easily facilitate their detection since definitive diagnosis is often dependent on DNA analysis. Declaration of interest The authors report no conflicts of interest. 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