{'fraction_non_alphanumeric': 0.0466624284806103, 'fraction_numerical': 0.03254926891290528, 'mean_word_length': 4.75, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 9, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Interaction of Hb Adana (HBA2: c.179G>A) with Deletional and Nondeletional α +-Thalassemia Mutations: Diverse Hematological and Clinical Features

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 α1or α2-globin gene (HBA1: c.179G>A or HBA2: c.179G>A) (1,2). In Indonesia, we found Hb Adana on the α2-globin gene (3,4). The most severe phenotype due to homozygous Hb Adana, manifesting as hydrops fetalis, has been previously reported (3). 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 (4). It contributes to many kinds of αthalassemia (α-thal) syndromes, especially in compound heterozygosity with the 3.7 kb deletion, which is the most common α þ -thal deletion in Indonesia (4). Interestingly, we also found the combination of Hb Adana with other nondeletional mutations such as Hb Constant Spring (Hb CS, HBA2: c.427T>C (5), codon 22 (α2, HBA2: c.69C>T) (6), a regulatory single nucleotide polymorphism (rSNP) 149709T<C (GenBank DQ431198) in a non genic region between the ζand α-globin genes (7) that creates a new promoter causing α-thal [Online Mendelian Inheritance in Man (OMIM) 141800.0218] and IVS-II-142 (α2, HBA2: c.440þ2G>A) (8). In this study, we report the hematological and clinical features of patients having Hb Adana in combination with either deletional or nondeletional α þ -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.
For the detection of the codon 22 and IVS-II-142 mutations, each 25 μL reaction contained 5Â Phusion GC Buffer containing 7.5 mM MgCl 2 as 1Â final concentration, 200 μM dNTPs, 0.4 μM of each primer (Table 2), 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 1Â PCR buffer (Qiagen), 1Â Q solution (Qiagen), 200 μM dNTPs, 0.2 μM of each primer (Table 2), 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 Table 2.
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 -α 3.7 (17 cases) or -α 4.2 (one case) mutations (Table 3), and nine cases (33.0%) were compound heterozygotes for Hb Adana and other nondeletional mutations (Table 4).
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 AE 1.2) ( Table 3). 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 Table 3) as previously described (1,9). 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) ( Table 4). 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 (Table 4).
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 (2,9). 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 (3)