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Graphical abstract. Xmn1 polymorphism (rs7482144) is a genetic polymorphism characterized by substitution (C>T) at the -158 position of γ-globin gene leading to the following 3 genotypes: homozygous for major allele (CC), heterozygous (CT), and homozygous for minor allele (TT). This study cohort consisted of only CT and CC genotypes without any TT genotype. The figure shows line diagrams demonstrating comparison between CC and CT genotypes in terms of median annual transfusion requirement (ATR) and median height before and after hydroxyurea in patients of HbE-β non-transfusion-dependent thalassemia. A statistically significant decrease in ATR and increase in height was noted following hydroxyurea therapy in both groups. Still, change in CT (median ATR drop: 33%, increase in median height: 3.7%, pCT=0.001) was more marked compared to that in CC genotype (median ATR drop: 28%, increase in median height: 2.8%, pCC=0.003).
Introduction
Thalassemia syndromes are a group of genetic disorders characterized by quantitative hemoglobinopathy, eventually leading to chronic hemolytic anemia. β-thalassemia results from the decreased production of β-globin chains and is one of the most common single-gene disorders worldwide. This condition is inherited in an autosomal recessive manner, with an exceptionally high prevalence in Southeast Asian and Mediterranean populations. Compound heterozygosity of β-thalassemia with fetal hemoglobin (HbE) leads to HbE-β-thalassemia, which is quite common in Eastern India. HbE-β-thalassemia is, thus, one of the predominant forms of symptomatic thalassemia apart from β-thalassemia major and intermedia in this part of the country [1]. Clinically, thalassemia can be categorized as the more severe transfusion-dependent thalassemia (TDT) and the relatively less severe non–transfusion-dependent thalassemia (NTDT). HbE-β-thalassemia has diverse clinical presentations ranging from TDT to NTDT. The global burden of TDT is considerable, and NTDT constitutes only a small indistinct proportion of patients [2].
Blood transfusion, together with iron chelators, has long since been the mainstay of treatment for symptomatic thalassemia syndrome. Transfusion provides symptomatic relief and improves growth; however, patients often face an elevated risk of blood-borne infections and iron overload. Although the latter can be attenuated to some extent with iron chelators, the overall quality of life is affected. This issue has prompted a search for alternative treatment modalities, of which the HbF inducer has gained widespread attention. Hydroxyurea is the most commonly used and studied medicine in this group of drugs, especially in patients with NTDT, and its use has been recommended by the Thalassemia International Federation (TIF) [3]. However, it remains an uncharted territory as knowledge gaps exist regarding its mechanism of action, efficacy, response modifiers, and safety profile.
The efficacy of hydroxyurea is considered to be influenced by certain modifiers, one of which is a single nucleotide polymorphism (SNP) known as Xmn1 polymorphism (rs7482144). This polymorphism is characterized by substitution (C>T) at -158 position of globin gene 2 (HBG2) in the β-globin locus (HBB). This polymorphism is present in approximately 30% of the individuals in most populations [4]. The SNP database is a free public archive of a broad range of SNPs. A specific nonredundant number, known as the reference SNP (rs) number, is assigned to individual SNPs, which facilitates easy and uniform identification of a specific SNP from the database. Xmn1 polymorphism is designated by rs7482144, and it results in 3 genotypes: homozygous for major allele (CC), heterozygous (CT), and homozygous for minor allele (TT). The presence of the T allele (CT/TT) has been shown to facilitate an improved response to hydroxyurea treatment compared with the absence of this allele. This substitution results in the reduced binding of transcription factors that silence the γ-globin gene production [5], thus increasing the HbF percentage. Therefore, in patients with the T allele, hydroxyurea plays a synergistic role in HbF induction and enhances the treatment outcome. However, hydroxyurea response in β-thalassemia is multifactorial and includes genetic factors (certain γ-globin gene mutations: IVSI_1, CD30) as well as environmental factors such as the age of the first transfusion [6]. This interplay of multiple factors can explain the variations in the response to hydroxyurea therapy in patients with thalassemia.
Among all the HbF inducers, hydroxyurea is the most commonly used. It was approved by the U.S. Food & Drug Administration for use in children with sickle cell anemia in 20177) and also has been recommended in a selected group of patients with NTDT by the TIF [3]. However, considering its drug toxicity profile and benefit only in a subset of patients, targeted therapy is desirable. Hence, there is an unmet need to evaluate the efficacy of hydroxyurea and determine the modifiers of its response to reap the maximum benefit with minimum risk. Only a limited number of studies have been conducted in this regard globally, and such studies in Eastern India are few and far apart. Moreover, most available studies are focused on TDT, with minimal information on NTDT. This study was conducted to fill the gaps in the existing research. The objectives included: (1) estimating the prevalence of Xmn1 polymorphism in the study cohort; (2) assessing the efficacy of hydroxyurea therapy in patients with NTDT in Eastern India; and (3) determining the effect of Xmn1 polymorphism on the efficacy of hydroxyurea therapy in these patients. This study tested the hypothesis that the T allele of Xmn1 polymorphism had a favorable impact on the efficacy of hydroxyurea therapy in children with NTDT.
Methods
This observational analytical ambispective cohort study was conducted from March 2020 to July 2021 in the Department of Pediatrics, IPGME&R, a tertiary hospital in Kolkata, West Bengal, located in Eastern India, with approval from the Institutional Ethics Committee (IPGME &R/IEC/2020/014 dated January 28, 2020). Although the total study duration was 16 months, patients were enrolled only in the initial 10 months so that a minimum of 6-month follow-up could be ensured in all participants. All consecutive eligible children with NTDT who had been recently initiated on hydroxyurea therapy (<1 month ago) at the thalassemia control unit of the institution were included in this study after obtaining informed consent from guardians of all participants and informed assent from children aged 7–12 years.
1. Inclusion criteria
(1) Children with NTDT confirmed via hemoglobin high-performance liquid chromatography or molecular analysis; (2) Age group of 2–12 years; (3) Hydroxyurea therapy started within 1 month before inclusion in the study; (4) Adequate medical records available to support data collection.
NTDT was defined as thalassemia requiring infrequent transfusions, less than 8 per year. These patients usually present after 2 years of age and include β-thalassemia intermedia, mild, and moderate phenotypes of HbE-β-thalassemia and α-thalassemia intermedia (HbH disease) [3].
2. Exclusion criteria
(1) Patients who received blood transfusion within the last 4 weeks (to avoid compromising the molecular analysis); (2) Patients who were unwilling to undergo regular follow-up for 6 months after the therapy.
At the time of enrolment in the study, baseline data before commencing hydroxyurea therapy, including ATR (defined as the average number of transfusions over the year), height, and HbF levels, were obtained from their past medical records. The children were examined for any adverse clinical effects of the drug using laboratory tests such as complete blood count (CBC), liver function tests (LFTs), and renal function tests (RFTs). Furthermore, relevant molecular genetic analysis was performed free of charge at Genetics Service Unit, BRIC-National Institute of Biomedical Genomics, a collaborative genetic laboratory of the institute (methodological details have been described in the following subsection). The dose of hydroxyurea was adjusted to 10–15 mg/kg/day as a single dose. The children also received folic acid supplementation during the entire length of the study and were transfused when the hemoglobin level fell below the range of 5–6 g/dL. Children on hydroxyurea therapy were followed up prospectively in the outpatient department during the 6 months of treatment. During this time, patients were periodically evaluated for any adverse effects via monthly outpatient department visits and scheduled laboratory tests, such as CBC, LFTs, and RFTs, every 2 weeks for the first 3 months and then monthly for the remaining 3 months. This follow-up was supported by telephonic interviews when needed.
The drug was temporarily halted if any of the following effects were noted: (1) absolute neutrophil count <1,500/μL; (2) platelet count <1,00,000/μL; (3) more than a twofold increase in serum hepatic transaminases from the baseline; (4) >50% rise in serum creatinine from the baseline [8]. After the normalization of parameters, the drug was restarted and the patient was closely monitored. However, the drug was permanently discontinued if the adverse effects reappeared. At the end of 6 months of therapy, the ATR was calculated by extrapolating data from 6 months postonset of hydroxyurea and the height of the children was recorded using a stadiometer. HbF (%) was estimated using the cation exchange method with a Bio-Rad Variant II machine, which was the same as the one used for the estimation of pretreatment HbF values to facilitate comparison. HbF was estimated at any timepoint only in patients with a minimum gap of 2 months from the last transfusion. The investigator was blinded to the results of genetic polymorphism until the completion of final data collection, whereas the analyst was blinded to the study hypothesis. These measures were adopted to eliminate interviewer and analyst bias in this study.
3. Molecular methods
Polymerase chain reaction (PCR)-restriction fragment length polymorphism was performed to detect Xmn1 polymorphism (Fig. 1), and HbE mutation and β-thalassemia genotypes were detected using the amplification refractory mutation system PCR (ARMS-PCR). The common β-thalassemia genotypes were: IVS1-5 (G>C), Cd 8/9 (+G), Cd 41/42 (-CTTT), and 619-bp deletion (Fig. 2). Moreover, β-thalassemia mutations that could not be identified using ARMS-PCR were determined using Sanger sequencing. The primer sequences used for these tests have been provided in the supplementary material.
4. Statistical analysis
Data were entered in a Microsoft Excel spreadsheet. Categorical variables were expressed as the number and percentage of patients and compared across the groups using Pearson chi-square test for the independence of attributes or the Fisher exact test as appropriate. Quantitative variables were expressed as mean, median, and standard deviation and compared across the groups using the Mann-Whitney U test. Comparison over time was performed using the Wilcoxon signed-rank test. The statistical software IBM SPSS Statistics ver. 22.0 (IBM Co., Armonk, NY, USA) was used for analysis. An α-level of 5% was considered, i.e., if a P value was <0.05, it was considered statistically significant.
Results
1. Study flow and patient characteristics
A total of 50 patients with NTDT were evaluated for eligibility, of which 16 patients did not fulfill the inclusion and/or exclusion criteria and 6 refused to participate. The remaining 28 patients who were already receiving hydroxyurea therapy for <1 month were enrolled in the study; of these, 1 patient was lost to follow-up and the drug had to be discontinued in 2 patients because of adverse effects. The prevalence of Xmn1 polymorphism was estimated in all 28 patients, the adverse effect of hydroxyurea therapy was studied in 27 patients, and the efficacy of this therapy and its comparative analysis between the genotypes was examined in 25 patients. Although baseline HbF was estimated in all patients, comparative HbF study pre- and posttherapy could be performed in only 13 patients who had a gap of at least 2 months from the last transfusion at the time of HbF assay (Fig. 3). The baseline characteristics of the study population are summarized in Table 1.
2. Genotypic characteristics of the study population
All patients in the study population exhibited HbE-β-thalassemia, and the commonest genotype was IVS1-5 (G>C), which was seen in 21 patients (75%). Thus, majority of the population harbored IVS1-5 (G>C) and HbE mutation in the compound heterozygous state. The prevalence rates of CC and CT genotypes of Xmn1 polymorphism were 12 (43%) and 16 (57%), respectively, with none having the TT genotype.
3. Hydroxyurea exposure and adverse effects
Hydroxyurea was administered at a median dose of 10 +1.40 mg/kg/day (10–15 mg/kg/day) for a total duration of 6 months. Drug toxicity developed in 6 patients (22%), and permanent discontinuation was required only in 2 patients (7%). Myelosuppression was the most common adverse effect observed, followed by transaminitis and elevated creatinine levels (Fig. 4). A statistically significant association was not found between Xmn1 polymorphism and drug toxicity (P=0.357) in this study.
4. Overall efficacy of hydroxyurea therapy
In 25 patients who completed 6 months of hydroxyurea therapy, the median ATR was significantly lower (P<0.001) and the median height was significantly higher (P<0.001) at the end of the therapy. HbF was compared over time in 13 patients, and a statistically significant increase in its level was noted at the end of the treatment (P=0.023).
5. Efficacy of hydroxyurea therapy in relation to Xmn1 polymorphism
The response was analyzed in the 2 groups of patients with CC and CT genotypes at the end of the study period in terms of ATR, height, and HbF (%) and compared across time before and after the study. The results of the analysis are summarized in Table 2. A statistically significant decrease in transfusion requirement and increase in height were observed after hydroxyurea therapy in both groups. Nonetheless, the change in CT genotype (median ATR drop: 33%, increase in median height: 3.7%, pCT=0.001) carried more weightage than that in the CC genotype (median ATR drop: 28%, increase in median height: 2.8%, pCC=0.003) (ref: graphical abstract). In contrast, a statistically significant association was not found between Xmn1 polymorphism and the change in HbF level following hydroxyurea therapy in this study (pCT=0.139, pCC=0.068).
Discussion
Since the recognition of thalassemia as a distinct entity, blood transfusion has been the cornerstone of treatment.; however, the advent of HbF inducers has transformed the scenario. Hydroxyurea is the most used drug in this category [3]. Unfortunately, it is not free of adverse effects; hence, it is essential to strike a balance between the risks and benefits of using this medicine. Certain modifiers of hydroxyurea response have been studied in the past, but this study attempted to explore whether Xmn1 polymorphism plays a role in modifying the response to hydroxyurea, especially in the less-explored NTDT population. The ultimate objective was to guide the selection of candidates for hydroxyurea therapy.
In this study cohort, all patients were compound heterozygous for HbE-β-thalassemia, indicating that it might be the commonest form of NTDT in Eastern India. Another study previously performed at the same location, i.e., Kolkata, West Bengal, has reported similar findings [1]. In our study population, the commonest β-thalassemia genotype was IVS1-5 (G>C), which was encountered in 75% of the patients. This result has been corroborated by the findings of 2 other studies, which also reported IVS1-5 (G>C) to be the commonest HBB gene mutation, with prevalence rates of approximately 33% and 44% [9,10].
At the end of 6 months of hydroxyurea therapy, the median ATR was significantly lower (P<0.001) and median height (P<0.001) and median HbF (%) were significantly higher (P=0.023) in our study population. The positive response to hydroxyurea noted in this study agrees with the observations of Keikhaei et al. [11]. The study found a significant decrease in the transfusion rate (P=0.001) and a significant increase in HbF (%) (P=0.03) in patients with thalassemia intermedia treated using hydroxyurea. Approximately 22% of our patients developed drug toxicity, and permanent discontinuation was needed in 7% of the patients. A previous study from Rajasthan, India, also reported adverse effects in approximately 14% of the patients, with a clinically significant problem in only 4% [8]. The commonest adverse effect in our cohort was myelosuppression, which is indeed a potentially hazardous condition. However, all adverse effects were reversed after discontinuation of the therapy. Interestingly, adverse effects were noted at a lower dose (median dose: 10 mg/kg/day) in our study than at a higher dose (20 mg/kg/day) in the abovementioned study [8], which could be attributed to genetic polymorphisms in drug-metabolizing enzymes [12].
On analyzing the response to hydroxyurea therapy among different genotypes of Xmn1 polymorphism, a statistically significant decrease in median ATR and an increase in median height were observed following hydroxyurea therapy in both groups. Nonetheless, the change in CT (median ATR drop: 33%, increase in median height: 3.7%, pCT=0.001) was more critical than that in the CC (median ATR drop: 28%, increase in median height: 2.8%, pCC=0.003) genotype. Thus, patients with the T allele of Xmn1 polymorphism indeed had an edge over those without it. This observation confirmed the findings of some previous investigations in India and abroad [13-15]. Notably, a statistically significant association was not found between the Xmn1 genotypes and either HbF (%) increase or adverse effects in our cohort. This finding helps us to draw an inference that although Xmn1 polymorphism modifies the clinical response to hydroxyurea, it does not apply to its biochemical response or adverse effect profile.
Most similar studies conducted till date have focused on either on β-TDT [14,15] and β-NTDT [5,15] patients or on HbE-βTDT patients [13,16]. Only one study was conducted by Italia et al. [16] on 11 patients with HbE-β-TDT and 2 patients with HbE-β-NTDT in Mumbai, India. Thus, our study is the first of its kind in Eastern India to evaluate the impact of Xmn1 polymorphism on the efficacy of hydroxyurea in a relatively larger group of patients with non–transfusion-dependent HbE-β-thalassemia.
This study, like most others, has certain limitations, which could be summarized as follows: (1) Small sample size with a short follow-up period, which was inadequate to evaluate the long-term efficacy. (2) Inability to determine the effectiveness of higher permissible dosage of the drug. (3) The lack of the TT genotype of Xmn1 polymorphism in the study population. (4) The presence of a certain selection bias, for instance, the fact that only patients with moderate HbE-β-NTDT who required transfusions attended the hospital and were enrolled and not the milder ones as they often do not seek medical attention. (5) Other parameters likely to influence the response to hydroxyurea therapy in these patients were not determined. Nevertheless, considering the lower prevalence of NTDT compared with TDT, we believe that our study has added substantially to the prevailing knowledge on the rational use of hydroxyurea in patients with HbE-β-NTDT in Eastern India and has emphasized the possible role of Xmn1 polymorphism as a modifier of hydroxyurea response.
Hydroxyurea is an effective adjunctive treatment for patients with NTDT. However, due to its potential adverse effects, cautious and targeted use of this drug is warranted, for which Xmn1 polymorphism can function as a guide. As patients possessing the T allele of this polymorphism exhibited a slightly better response to hydroxyurea, the benefits of using this drug, especially in these patients, outweigh the risks. Hence, Xmn1 polymorphism analysis can guide us in carefully selecting patients with HbE-β-NTDT for hydroxyurea therapy. However, hydroxyurea therapy showed a beneficial response in both groups, with relatively better results in those with the CT genotype, and the absence of the T allele did not substantially aggravate the risk of drug toxicity. Therefore, in case of non-availability of data on Xmn1 polymorphism, all patients with HbE-β-NTDT, particularly those in Eastern India, deserve a trial of hydroxyurea while closely monitoring for possible adverse effects.
In conclusion, Xmn1 polymorphism study can aid in selecting patients with HbE-β-NTDT for hydroxyurea therapy by rationalizing the benefit–risk assessment. However, further investigations with a larger sample size and multicentric studies encompassing all other modifiers are required to substantiate the results of this study.
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