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HBB genotype in TDT in Cyprus
regular transfusion in these cases because of concerns about the consequences of long-term transfusion, notably transfu- sion iron overload. The result of not correcting the tha- lassemia phenotype in earlier life includes a variety of chronic complications which can become apparent later. These include liver disease, pulmonary hypertension, endocrine dysfunction, bone disease and splenomegaly leading to splenectomy. Quality of life is significantly impaired and life expectancy may be reduced.12-15 In the middle of the phenotypic spectrum are TDT patients who are not absolutely transfusion-dependent early in their life, and regular transfusion may be delayed in the age range 2– 10 years,16,17 during which interval they may be at risk of developing complications of untreated thalassemia.
Over 400 HBB variants causing β-thalassemia have been reported to date18 and are generally classified as β0, in which no functional β-globin chains can be produced; β+, in which β-globin chain production is severely reduced; and β++, in which β-globin chain production is mildly reduced.19 A pre- vious study evaluated the influence of HBB pathogenic vari- ants and other genetic modifiers on time to initiate regular transfusion, and developed a predictive model derived from a composite score. HBB genotype was found to be the most powerful predictor of severity.16,17 In routine practice, genet- ic diagnosis is often not available to the clinician and is not currently used for clinical decision-making. Further evi- dence of different outcomes associated with different geno- types is needed to recommend routine determination of genotype at birth and to make use of genotypic information in clinical decision-making about the initiation of transfu- sion and changing other aspects of standard practice.
Cyprus is a Mediterranean island with a relatively stable indigenous population at high risk of thalassemia. Following introduction of a national carrier screening pro- gram in 1974, all new diagnoses of infants on the govern- ment-controlled southern part of the island have been doc- umented and standard care is given in four treatment cen- ters,20 based on shared protocols for transfusion and chela- tion developed over the past 30 years. The decision on the start of transfusion has been based purely on clinical obser- vation according to best standard practice. The patients have remained stable within each clinic with very little immigration or emigration. The Cyprus thalassemia cohort consists of TDT patients born between 1960 and 2000, and their follow-up to 2004 was published previously.1 Herein, we present updated information with longer follow-up from this cohort, in which we generate a more robust esti- mate of survival of thalassemia with standard treatment, we confirm changing causes of mortality, and we explore the association of genotype with long-term outcomes and survival.
Methods
Patients and data preparation
This study is a follow-up of a previously described cohort of patients.1 Briefly, we included all patients who were transfusion- dependent at the time of the first study, who were born in Cyprus between 1960 and 2000, and who were treated using standard care in the four thalassemia centers. Data were collected and vali- dated for each treatment center up to September 30, 2018. Causes of death were classified as cardiac, liver (including hepatocellular carcinoma), malignancy, infection and other, using standard clini- cal criteria. Considering the introduction of the Cyprus tha-
lassemia screening and prevention program,20 which resulted in identification soon after birth of all affected children from 1974 onwards, year of birth was categorized as pre-1974 or 1974 onwards. Based on commonly agreed categories of severity of pathogenic variants,21 HBB genotype was classified as severe, moderate and mild, as shown in Table 1, assuming equally spaced severity across categories.
Chelation treatment was categorized based on the predominant chelator used during follow-up from year 2000 onwards: (i) defer- oxamine only, (ii) deferiprone alone or in combination with defer- oxamine, and (iii) deferasirox. Deferoxamine was given by subcu- taneous or intravenous infusion, whereas deferiprone and deferasirox were administered orally. For splenectomy, the effect on survival was assumed to be dependent on whether and when it had been performed, and was categorized as (i) before 16 years of age, and (ii) after 16 years of age or not done.
To evaluate genotype associations with the transfusion regi- mens, we used data previously collected for 336 patients from the cohort, as part of the THALAMOSS project.22 These data com- prised the age of the first regular transfusion, and annual blood usage and pre-transfusion hemoglobin levels, between 2014 and 2016.
Statistical analysis
A univariate analysis was performed to estimate the overall sur- vival and the association of different risk factors with mortality using Kaplan-Meier analysis. The log-rank test was used to evalu- ate the statistical significance of the effect of the risk factors on survival. For the multivariate analysis, we used Cox proportional- hazards regression with a backwards approach in which all poten- tial factors that were significant in the univariate analysis were included in the initial regression model and then sequentially removed to obtain the best-fit model. All models were tested for whether they met the proportional hazards assumption for a Cox proportional-hazards model.
Proportions were compared in contingency tables using the Pearson χ2 test. Mortality rates and confidence intervals were cal- culated overall and for the periods 1980-89, 1990-99, 2000-09, and 2010-18. The Kruskal-Wallis test and the Wilcoxon rank-sum test for post-hoc analyses were utilized to study the association between HBB genotype and transfusion data. All statistical analy- ses were performed using R (version 3.6.1) and R packages sur- vival and survminer.
Ethical considerations
The study was approved by the Cyprus National Bioethics Committee and was initiated by PT as part of a project commis- sioned by the Cyprus Ministry of Health. Subsequently, informed consent was obtained for clinical follow-up of the cohort as part of the THALAMOSS project (EU FP7 grant agreement 306201), which included collection of transfusion data.
Results
A summary of the clinical characteristics of the 537 cohort patients are documented in Table 2. There were two fewer patients than in the original study. Both of these patients were found to have been ineligible, one because of birth prior to 1960, and the other with a diagnosis of Hb H disease. Eight patients had been treated with sibling allo- geneic stem cell transplant, at a mean age of 12.7 years. Follow-up on these eight patients was censored at the date of transplantation.
The total number of patient-years of follow up was
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