Impact of genotype on endocrinal complications of Children with Alpha-thalassemia in China

Impact of genotype on endocrinal complications of Children with Alpha-thalassemia in China www.nature.com/scientificreports OPEN Impact of genotype on endocrinal complications of Children with Alpha-thalassemia in China Received: 12 December 2016 1 2 3 1 1 Hong-Cheng Luo , Qi-Sheng Luo , Fu-Gao Huang , Chun-Fang Wang & Y e-Sheng Wei Accepted: 21 April 2017 Alpha-thalassemia occurs with high frenquency in China. Four common α-globin gene deletion Published: xx xx xxxx mutations (–SEA, -α3.7, and -α4.2, Haemoglobin Constant Spring (CS) mutation) were identified in Chinese patients. Individuals with alpha-thalassemia syndrome are more often of children. However report on endocrinal complications in children with alpha thalassemia in China are still absent. The present study aimed to investigate the impact of genotype on endocrinal complications in Chinese children. Association analysis between genotype and endocrinal compliaction development was conducted on 200 patients with 200 healthy controls. Hypogonadism was found to be the most prominent endocrinal complications (84.0%) leading to the growth retardation, hypogonadism, diabetes mellitus, hypothyroidism and hypoparathyroidism whose incidence were significantly higher CS in pateints. (α α/–SEA) was the main genotype of Alpha thalassemia identified in the patients (37.5%), and patients with the (-α4.2/–SEA) genotype had a higher prevalence of hypogonadism, diabetes mellitus and hypoparathyroidism (P = 0.001, P = 0.001, P < 0.001, respectively). Alpha-thalassemia (α-thalassemia) is caused by deletions or point mutations of the alpha-globin gene due to the complexity and diversity of genetic defects. The severity of the clinical phenotype of α -thal is diverse. Patients with severe α-thalassemia require frequent red blood cell transfusion for survival. As a result, many complications will occur in patients on regular blood transfusion with iron chelating therapy. Complications of α-thalassemia mainly result from chronic hemolysis and tissue hypoxia, causing iron overload and multiple organ dysfunction . A-thalassemia is a serious health problem worldwide, especially in Mediterranean areas, Southeast Asia and 2–4 Southern China . Guangxi Province is located in the southwest of China where the incidence of thalassemia is 24.51% . However, in the past decades, data on diagnose and treatment of α-thalassemia or related complications in children are still absent. In this study typical physical exam findings growth retardation, hypogonadism, thal - 6–9 assemic bone deformities, diabetes mellitus were included to identify the association between four genotype CS (SEA, -α3.7/–SEA, -α4.2/–SEA, α α/–SEA) and endocrine complications in children with α-thalassemia. Materials and Methods General. Two hundred Children (126 males and 74 females) with mean age of 9.64 ± 1.15 years (range, 3–12 years). Who were registered in The Affiliated Hospital of Youjiang Medical College for Nationalities from the period January 2010 to June 2016 were included in this research. α- thalassemia children were characterized with CS one of the genetype of SEA, -α3.7/–SEA, -α4.2/–SEA or α α/–SEA. Which was identified by the DNA sequenc- ing technique. The basic clinical information collected included Average Hematological Parameters of diagnosis, gender, age, age of start transfusion, age of start chelation, frequency of transfusion and related compliance. The study was approved by the Ethical committee of Youjiang Medical College for Nationalities and written informed consent was obtained from the subjects. The study was in compliance with the Helsinki declaration. We selected 200 cases of the same age group as a control group for research (113 males and 87 females, 9.35 ± 1.56 years, range, 3–12 years). The criteria for the control group are as follows: All individuals have a normal level of mean corpuscular volume (MCV) > 82.6 < 99.1fl, and mean corpuscular hemoglobin (MCH) > 26.9 < 33.3. A normal level of HbA (between 85% and 97.5%) and HbA2 (between 2.5% and 3.5%), the Department of Clinical Laboratory, the Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China. Department of neurosurgery, the Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China. Department of Ultrasound, the Aliated ffi Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China. Correspondence and requests for materials should be addressed to Y.-S.W. (email: weizhuren999@163.com) Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 1 www.nature.com/scientificreports/ Serum ferritin Genetype n RBC (*10 /L) HGB (g/L) MCV (fl) MCH (pg) MCHC (g/L) (ng/ml) SEA 65 5.21 ± 1.08 85.69 ± 23.44 62.43 ± 7.31 19.11 ± 2.29 305.11 ± 14.70 356.17 ± 25.76 3.7 -α /–SEA 37 5.09 ± 0.82 84.43 ± 15.63 56.28 ± 6.56 16.70 ± 1.67 299.61 ± 21.01 976.58 ± 79.11 4.2 -α /–SEA 23 5.32 ± 0.85 80.88 ± 14.67 52.49 ± 2.44 16.74 ± 2.90 294.50 ± 15.02 997.37 ± 78.69 CS a,b α α/–SEA 75 4.11 ± 0.96 79.17 ± 18.89 68.63 ± 8.38 18.64 ± 2.03 270.00 ± 25.01 1023.69 ± 81.55 Reference 3.50~5.50 110.00~160.00 80.00~100.00 27.00~34.00 320.00~360.00 15.00~250.00 Table 1. Clinical Average Hematological Parameters of the Study Population (x ± s). RBC, red blood cell; HGB, haemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; SEA: the Southeast Asian deletion; -α3.7, rightward deletion; -α4.2, leftward deletion; CS, Hb Constant Spring. a:compared with -α4.2/–SEA, P < 0.001; b:compared with -α3.7/– SEA, P < 0.001. Note: RBC, HGB, MCV, MCH and MCHC were the average hematological parameters level before the first blood transfusion. Normal levels serum ferritin, no one suffering from hemolytic anemia and malnutrition anemia. No cardiovascu- lar and blood infectious d disease. Its family without hypertension, diabetes. All the control group also were diag- nosed by the DNA sequencing technique, no one suffers from six common α - thalassemia (–SEA, -α3.7, -α4.2, CS WS QS α α, α α, α α) and seventeen β- thalassemia (17 M/N, CD41-42M/N, -28M/N, -29M/N, 31 M/N, -32M/N, 43 M/N, 654 M/N, -30M IVS-I-1M, IVS-I-5M 14–15 M, 27/28 M, 71–72 M/N, ΒeM/N, CAMP, IntM), which the common type of thalassemia in chinese people. Physical examination including. Red blood cells (RBC), Hemoglobin (HGBg/l), Mean corpuscular volume (MCV/fl), Mean hemoglobin content (MCH/pg), Mean hemoglobin concentration (MCHC), basal growth hor - mone, estradiol (in females) and testosterone (in males), thyroid-stimulating hormone (TSH), FT3, FT4, serum calcium concentration, serum phosphate and parathyroid hormone. Alpha globin mutations were analyzed using gap-PCR and reverse-hybridization assay according to the manufacturer. Classification of patients according to genotype. Children were divided into four groups according to their genotype based on the α-globin gene production. Group1–4: (SEA) deletions, (−3.7 kb merge SEA) deletions, (−4.2 kb merge SEA) deletions and (CS mutations merge SEA) deletions. Definitions. Short stature was defined as patient height > 2 standard deviation below the mean for age, gender and ethnicity . Short stature was evaluated by Children’s Health Rehabilitation Center (Affiliated Hospital of Youjiang Medical College for Nationalities, Guangxi, China). Hypogonadism was defined as low testosterone (in males or oestradiol (in females) level or subjects who had received testosterone or oestradiol therapy. Patients were diagnosed with diabetes mellitus based on WHO criteria or history of insulin therapy or oral antidiabetic therapy according to American Diabetes Association, World Health Organization Criteria and National Diabetes Health Group 1979. Hypothyroidism was defined according to TSH/FT3, FT4 or based on the history of treatment with levothy- roxine for previously diagnosed hypothyroidism. Hypoparathyroidism was defined as low serum calcium and low serum parathyroid hormone concentration, with increased serum phosphate. A hemoglobin level of less than 90 (g/L) was the standard for initiating transfusion in children with severe thalassemia. Infection, growth retardation, diabetes mellitus, hypogonadism, hypothyroidism, hypoparathy- roidism or other complications in thalassemia children, were the indications for transfusion at a relatively high level of haemoglobin. Statistical analysis. SPSS13.0 (SPSS, Inc., IL, USA) was used to conduct statistical analysis. χ test or Fisher’s exact test was used for comparation between different groups. Measurement data were represented as mean ± standard deviation (x ± s), and categorical data were represented as χ . P < 0.05 and P < 0.001 were considered to indicate statistically significant differences. Results Patient characteristics. All the patients were recruited from Ali ffi ated Hospital of Youjiang Medical College for Nationalities, Guangxi, China. The patients (126 males and 74 females) had a mean (SD) age of (9.64 ± 1.15) years. Hypogonadism was the most prominent endocrinal complications in patients (84.0%), followed by growth retardation (68.5%) and hypoparathyroidism (14.5%). A total 70.5% of patients start to use chelation in 3 years old. There was no significant difference in RBC, MCV, MCH and MCHC among the four groups ( P > 0.05). Clinical Average Hematological Parameters were summarized in (Table 1). Genetype of Thalassemia and endocrinal complications. Two major genetype identie fi d in the Alpha CS thalassemia patients were α α/–SEA (37.5%, 38.1% of males and 36.5% of females) and SEA (32.5%), followed CS by -α3.7/–SEA (18.5%) and -α4.2/–SEA (11.5%). A total of 94.1% of patients with the α α/–SEA genotype started earlier transfusion (≤3 year), 77.3% of patients received frequent transfusion (every 4–5 weeks) and CS 68.0% started earlier iron chelators (>3 years). In addition, patients with the α α/–SEA genotype had a higher Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 2 www.nature.com/scientificreports/ 3.7 4.2 CS SEA -α /–SEA -α /–SEA α α/–SEA Characteristics Patients (n = 65) (n = 37) (n = 23) (n = 75) P-value Gender   Male 126 31.7% 19.0% 11.1% 38.1% 0.98   Female 74 33.8% 17.6% 12.2% 36.5% Age of start transfusion (years)   ≤3 117 22.2% 13.7% 10.3% 53.8% <0.001   >3 83 47.0% 25.3% 13.3% 14.5% Frequency of transfusion (weeks)   Every 2–3 98 45.9% 19.4% 17.3% 17.3% <0.001   Every 4–5 102 19.6% 17.6% 5.9% 56.9% Age of start chelation (years)   ≤6 59 27.1% 15.3% 16.9% 40.7% 0.31   >6 141 34.8% 19.9% 9.2% 36.2% Growth retardation   Negative 63 50.8% 25.4% 14.3% 9.5% <0.001   Positive 137 24.1% 15.3% 10.2% 50.4% Hypogonadism   Negative 32 62.5% 15.6% 0.0% 1.9% 0.001   Positive 168 26.8% 19.0% 13.7% 40.5% Diabetes mellitus   Negative 186 33.9% 18.3% 9.1% 38.7% 0.001   Positive 14 14.3% 21.4% 42.9% 21.4% Hypothyroidism   Negative 174 36.8% 14.4% 8.6% 40.2% <0.001   Positive 26 3.8% 46.2% 30.8% 19.2% Hypoparathyroidism   Negative 171 38.0% 16.4% 9.4% 36.3% <0.001   Positive 29 0.0% 31.0% 24.1% 44.8% Table 2. Association between patient genotype and endocrinal complications. prevalence of growth retardation (92%), and patients with the -α4.2/–SEA genotype had a higher prevalence of hypogonadism and diabetes mellitus (100% and 73.9%, respectively) (Table 2). Growth retardation in patients. Growth retardation was identified in 75.2% of patients (≥ 6 years old) and 61.1% of patients (<6 years old), and no significant difference was identified between males and females. A total of 40.9% of patients with growth retardation started earlier blood transfusion (≤3 year), 69.3% received frequent transfusion (every 4–5 weeks), 89.8% started iron chelation (>3 years) and 17.5% were poor compliant (Table 3). Hypogonadism in patients. Hypogonadism was identified in 83.8% of patients (≥ 6 years old) and 84.2% of patients (<6 years old), and there was significant difference between males and females (P < 0.001). A total of 67.3% of patients with hypogonadism started earlier transfusion (≤3 years), 51.8% of them received frequent transfusion (every 2–3 weeks). 76.2% of patients with hypogonadism started iron chelation (>3 years) and 12.5% had a poor compliance (Table 4). Diabetes mellitus in patients. Diabetes mellitus was identified in 14 patients and 71.4% of them were ≥6 years old with no significant difference identified between males and females. 92.9% of them received fre- quent transfusion (every 2–3 weeks), and 85.7% of patients with hypogonadism started iron chelation (>3 years) (Table 5). Hypothyroidism in patients. 26 patients (15 males and 11 males) were diagnosed with hypothyroidism and no significant difference was identified between males and females. All of these patients started earlier trans- fusion (≤3 years). Most of the patients (88.5%) were more than 6 years older and 96.2% had a poor compliant (Table 6). Hypoparathyroidism in patients. Hypoparathyroidism was identified in 29 patients and 82.8% of them were ≥6 years old, no significant difference was observed between males and females. All of these patients started earlier transfusion (≤3 years) and most of them had a poor compliant (Table 7). Endocrine complication between case group and control group. er Th e was no significant difference in the incidence of endocrine complication between male and female in case group and control group, alpha thalassemia patients are significantly more likely to have growth retardation, hypogonadism, diabetes mellitus, Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 3 www.nature.com/scientificreports/ Growth retardation (n = 200) Negative Positive Characteristics Patients, n (n = 63) (n = 137) P-value Gender   Male 126 31.0% 69.0% 0.83   Female 74 32.4% 67.6% Age (years)   ≥6 105 24.8% 75.2% 0.03   <6 95 38.9% 61.1% Frequency of transfusion (weeks)   Every 2–3 98 57.1% 42.9% <0.001   Every 4–5 102 6.9% 93.1% Age of start transfusion (years)   ≤3 117 52.1% 47.9% <0.001   >3 83 2.4% 97.6% Age of start chelation (years)   ≤6 59 76.3% 23.7% <0.001   >6 141 12.8% 87.2% Compliance, %   <60 43 44.2% 55.8% 0.04   ≥60 157 28.0% 72.0% Table 3. Association between growth retardation and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. Hypogonadism (n = 200) Negative Positive Characteristics Patients, n (n = 32) (n = 168) P-value Gender   Male 126 23.0% 77.0% <0.001   Female 74 4.1% 95.9% Age (years)   ≥6 105 16.2% 83.8% 0.94   <6 95 15.8% 84.2% Frequency of transfusion (weeks)   Every 2–3 98 11.2% 88.8% 0.07   Every 4–5 102 20.6% 79.4% Age of start transfusion (years)   ≤3 117 3.4% 96.6% <0.001   >3 83 33.7% 66.3% Age of start chelation (years)   ≤6 59 32.2% 67.8% <0.001   >6 141 9.8% 90.8% Compliance, %   <60 43 20.9% 79.1% 0.32   ≥60 157 14.6% 85.4% Table 4. Association between hypogonadism and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. hypothy- roidism and hypoparathyroidism compared with controls (P < 0.001) (Table 8). The HGB level lower in patients (81.17 ± 15.23 g/L, range, 13~95 g/L) than control subjects (126.21 ± 17.65 g/L, range, 55~167 g/L). We also identified a significant difference between RBC and MCV indices in case group and control group (P < 0.001). Discussion Thalassemia is a well-known inherited hematologic disorder caused by reduced or absence of globin produc- tion . In China, this disease is prevalent in areas near the southern bank of the Yangtze River, such as Guangdong, 12–14 Guangxi, Fujian and Yunnan Provinces . Endocrine dysfunction is a frequent complication in thalassemic Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 4 www.nature.com/scientificreports/ Diabetes mellitus (n = 200) Negative Positive P- Characteristics Patients, n (n = 186) (n = 14) value Gender   Male 126 95.2% 4.8% 0.11   Female 74 89.2% 10.8% Age (years)   ≥6 105 90.5% 9.5% 1.14   <6 95 95.8% 4.2% Frequency of transfusion (weeks)   Every 2–3 98 99.0% 1.0% 0.001   Every 4–5 102 87.3% 12.7% Age of start transfusion (years)   ≤3 117 95.7% 4.3% 0.07   >3 83 89.2% 10.8% Age of start chelation (years)   ≤6 59 96.6% 3.4% 0.32   >6 141 91.5% 8.5% Compliance, %   <60 43 88.4% 11.6 0.32   ≥60 157 94.3% 5.7% Table 5. Association between diabetes mellitus and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. Hypothyroidism (n = 200) Negative Positive Characteristics Patients (n = 174) (n = 26) P-value Gender   Male 126 88.1% 11.9% 0.70   Female 74 85.1% 14.9% Age (years)   ≥6 105 78.1% 21.9% <0.001   <6 95 96.8% 3.2% Frequency of transfusion (weeks)   Every 2–3 98 79.6% 20.4% 0.004   Every 4–5 102 94.1% 5.9% Age of start transfusion (years)   ≤3 117 77.8% 22.2% <0.001   >3 83 100.0% 0.0% Age of start chelation (years)   ≤6 59 67.8% 32.2% <0.001   >6 141 95.0% 5.0% Compliance, %   <60 43 41.9% 58.1% <0.001   ≥60 157 99.4% 0.6% Table 6. Association between hypothyroidism and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. patients who are on regular blood transfusions. Iron overload has been considered to be the major cause of endo- crine abnormalities of α-thalassemia . Growth retardation, hypogonadism, diabetes mellitus and hypoparath- yrodism represent the most common endocrinopathies in thalassemic patients . In this study, we evaluates the impact of genotype on endocrinal complications of Children with Alpha- thalassemia in China and demonstrates that hypogonadism is the most frequent endocrine complication in α-thalassemia (84.0%), followed by growth retardation (68.5%) and hypoparathyroidism (14.5%). CS Our survey showed that the MCV levels in group (α α/–SEA) were higher than those in group (-α3.7/– SEA) and group (-α4.2/–SEA)(P < 0.001, P < 0.001, respectively), there were no significant differences in RBC, Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 5 www.nature.com/scientificreports/ Hypoparathyroidism (n = 200) Negative Positive Characteristics Patients (n = 171) (n = 29) P-value Gender   Male 126 87.3 12.7 0.35   Female 74 82.4 17.6 Age (years)   ≥6 105 77.1 22.9 <0.001   <6 95 94.7 5.3 Frequency of transfusion (weeks)   Every 2–3 98 80.6 19.4 0.05   Every 4–5 102 90.2 9.8 Age of start transfusion (years)   ≤3 117 75.2 24.8 <0.001   >3 83 100.0 0.0 Age of start chelation (years)   ≤6 59 69.5 30.5 <0.001   >6 141 92.2 7.8 Compliance, %   <60 43 39.5 60.5 <0.001   ≥60 157 98.1 1.9 Table 7. Association between hypoparathyroidism and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. Alpha thalassemia Contral group Characteristics (n = 200) (n = 200) P-value Gender   Male 126 (63.0%) 113 (56.5%) 0.22   Female 74 (37.0%) 87 (43.5%) Growth retardation   Negative 137 (68.5%) 195 (97.5%) <0.001   Positive 63 (31.5%) 5 (2.5%) Hypogonadism   Negative 168 (84.0%) 197 (98.5%) <0.001   Positive 132 (16.0%) 3 (1.5%) Diabetes mellitus   Negative 186 (93.0%) 198 (99.0%) 0.005   Positive 14 (7.0%) 2 (1.0%) Hypothyroidism   Negative 174 (87.0%) 193 (96.5%) 0.001   Positive 26 (13.0%) 7 (3.5%) Hypoparathyroidism   Negative 171 (85.5%) 199 (99.5%) <0.001   Positive 29 (14.5%) 1 (0.5%) Table 8. Comparison of endocrine complications in patients with alpha thalassemia and control group. HGB, MCH and MCHC levels among the four groups (P > 0.05), similar to the previous study by Zhu et al. . CS Compared with the other three groups (α α/–SEA, -α3.7/–SEA, -α4.2/–SEA), the group SEA had a significant lower serum ferritin levels (P < 0.001, respectively), this may be due to patients with SEA genetype generally do not receive blood transfusion therapy frequently unless combined with iron deficiency anemia, vitaminD deficiency, infection caused by long-term malnutrition anemia. In consistent with report by Zhou Y. U. et al . CS no significant difference was observed among the three group (α α/–SEA, -α3.7/–SEA, -α4.2/–SEA) in Serum ferritin levels (P > 0.05, respectively). CS In the present study, we found that the patients with the genetype of (α α/–SEA) had significant higher 18–20 prevalence of growth retardation, hypogonadism (P < 0.001, P = 0.001, respectively). Just like previous report hypogonadism was identified as the most common endocrine complication in the patients (84.0%). Gender, age Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 6 www.nature.com/scientificreports/ of start transfusion or start Chelation had a significant impact on hypogonadism development. However a lower 21, 22 prevalence of hypogonadism was found in some study , which were mainly attributed to difference in the economic status of patients, Physicians’ strategies to optimize chelation therapy, promoting compliance, educat- 23–26 ing patients and different ethnic . The patients with the genetype of (-α 4.2/–SEA) had a significantly higher prevalence of diabetes mellitus (P = 0.001). And there was no significant differences in the incidence of genotypes between males and females (P = 0.98). Compared to the present study 68.5% of patients identified with growth retardation, Hattab, F. N. et al . found a higher prevalence of growth retardation (75.9%). This may be attributed to the difference in economy, most of the patients come in the latter study from poor families, received poor health care treatment, which resulted in multiple infections, thereby aggravating growth retardation or other potential endocrine complications develop- ment in Alpha- thalassemia during childhood. Futher more, the discrepancy of clinical manifestations may be 28–30 impacted by genetic and environmental factors . er Th e was significant association between growth retardation and older year (≥6 years), earlier age of start transfusion, chelation, frequency of blood transfusion or poor com- pliance (P = 0.03, P < 0.001, P < 0.001, P < 0.001, P = 0.04, respectively). But there was no significant association between growth retardation and gender (P = 0.83). In the present study, 7.0% of patients were diagnosed with diabetes mellitu, similar to 8.0% in report by Ong, C. K. et al. . Several previous study have report a lower prevalence of diabetes mellitu, which ranged from 2.5% 32–34 35–37 to 4.9% , while Other had report a higer prevalence of diabetes mellitu, reaching 13% to 17.0% . These discrepancies can be attributed to differences in the age of patients and severity of Hepatitis C virus infection, 38, 39 transfusion rates and chelation therapies, male sex, liver iron concentration . There was significant association between diabetes mellitu and frequency of blood transfusion (P = 0.001), but there was no signic fi ant association between diabetes mellitu and gender, age, age of start transfusion, chelation, frequency blood transfusion or com- pliance (P = 0.11, P = 1.14, P = 0.07, P = 0.32, P = 0.32, respectively). Hypothyroidism was identified in 26 patients (13.0%), which was similar to the result reported by Eshragi, P . et al . . 41–43 While, other studies reported a lower prevalence of hypothyroidism, which ranged from 1.0% to 10.0% . The results of different studies vary widely, these discrepancies can be attributed to differences in genotype of thalas- semia, the age of patients or treatment protocols. Hypogonadism (84.0%), growth retardation (68.5%) and hypoparathyroidism (14.5%) were the first and the most frequent endocrine complications diagnosed in our present study. Today, many patients can benefit from modern treatment, improve the quality of life of patients dut to adopting in early and regular chelation therapy. er Th efore, prevention of the endocrine complications may be influenced by the improvement of medical diagno- sis and treatment. Monitoring compliance is essential in such conditions. er Th e are a few limitations need to be mention here. Firstly, the sample size is small, and the age of these patients too early which may result in limited power. Secondly, the type of iron chelation used could not be figured out, rare genetype of α-thalassemia were not included in our study. Thirdly, none of the analyses take into account the age effect properly. e Th incomplete medical records could prevent us from identifying predictive complication. Further studies are needed on the complications of all α-thalassemic and older patients in the region. CS In conclusion, our present study show that α α/–SEA, SEA, -α3.7/–SEA, and -α4.2/–SEA are the main genetype identified in α-thalassemia children in Guangxi Province, and hypogonadism, growth retardation and hypoparathyroidism are the most common endocrine complications in children with α-thalassemia. References 1. De Sanctis, V., Eleftheriou, A. & Malaventura, C. Prevalence of Endocrine Complications and Short Stature in Patients with a Th lassaemia Major: A Multicenter Study by the Thalassaemia International Federation (TIF). Pediatr Endocrinol Rev. 2(Suppl 2), 249–255 (2004). 2. Weatherall, D. J. Thalassemia as a Global Health Problem: Recent Progress Toward its Control in the Developing Countries. 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Biomed Rep 4, 728–736, doi:10.3892/br.2016.646 (2016). Author Contributions H.C.L. designed and wrote the manuscript. Q.S.L., F.H.H. and C.F.W. collected clinical data. Y.S.W. directed the writing of manuscript. All authors have reviewed and approved the final version of this manuscript. Additional Information Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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Impact of genotype on endocrinal complications of Children with Alpha-thalassemia in China

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www.nature.com/scientificreports OPEN Impact of genotype on endocrinal complications of Children with Alpha-thalassemia in China Received: 12 December 2016 1 2 3 1 1 Hong-Cheng Luo , Qi-Sheng Luo , Fu-Gao Huang , Chun-Fang Wang & Y e-Sheng Wei Accepted: 21 April 2017 Alpha-thalassemia occurs with high frenquency in China. Four common α-globin gene deletion Published: xx xx xxxx mutations (–SEA, -α3.7, and -α4.2, Haemoglobin Constant Spring (CS) mutation) were identified in Chinese patients. Individuals with alpha-thalassemia syndrome are more often of children. However report on endocrinal complications in children with alpha thalassemia in China are still absent. The present study aimed to investigate the impact of genotype on endocrinal complications in Chinese children. Association analysis between genotype and endocrinal compliaction development was conducted on 200 patients with 200 healthy controls. Hypogonadism was found to be the most prominent endocrinal complications (84.0%) leading to the growth retardation, hypogonadism, diabetes mellitus, hypothyroidism and hypoparathyroidism whose incidence were significantly higher CS in pateints. (α α/–SEA) was the main genotype of Alpha thalassemia identified in the patients (37.5%), and patients with the (-α4.2/–SEA) genotype had a higher prevalence of hypogonadism, diabetes mellitus and hypoparathyroidism (P = 0.001, P = 0.001, P < 0.001, respectively). Alpha-thalassemia (α-thalassemia) is caused by deletions or point mutations of the alpha-globin gene due to the complexity and diversity of genetic defects. The severity of the clinical phenotype of α -thal is diverse. Patients with severe α-thalassemia require frequent red blood cell transfusion for survival. As a result, many complications will occur in patients on regular blood transfusion with iron chelating therapy. Complications of α-thalassemia mainly result from chronic hemolysis and tissue hypoxia, causing iron overload and multiple organ dysfunction . A-thalassemia is a serious health problem worldwide, especially in Mediterranean areas, Southeast Asia and 2–4 Southern China . Guangxi Province is located in the southwest of China where the incidence of thalassemia is 24.51% . However, in the past decades, data on diagnose and treatment of α-thalassemia or related complications in children are still absent. In this study typical physical exam findings growth retardation, hypogonadism, thal - 6–9 assemic bone deformities, diabetes mellitus were included to identify the association between four genotype CS (SEA, -α3.7/–SEA, -α4.2/–SEA, α α/–SEA) and endocrine complications in children with α-thalassemia. Materials and Methods General. Two hundred Children (126 males and 74 females) with mean age of 9.64 ± 1.15 years (range, 3–12 years). Who were registered in The Affiliated Hospital of Youjiang Medical College for Nationalities from the period January 2010 to June 2016 were included in this research. α- thalassemia children were characterized with CS one of the genetype of SEA, -α3.7/–SEA, -α4.2/–SEA or α α/–SEA. Which was identified by the DNA sequenc- ing technique. The basic clinical information collected included Average Hematological Parameters of diagnosis, gender, age, age of start transfusion, age of start chelation, frequency of transfusion and related compliance. The study was approved by the Ethical committee of Youjiang Medical College for Nationalities and written informed consent was obtained from the subjects. The study was in compliance with the Helsinki declaration. We selected 200 cases of the same age group as a control group for research (113 males and 87 females, 9.35 ± 1.56 years, range, 3–12 years). The criteria for the control group are as follows: All individuals have a normal level of mean corpuscular volume (MCV) > 82.6 < 99.1fl, and mean corpuscular hemoglobin (MCH) > 26.9 < 33.3. A normal level of HbA (between 85% and 97.5%) and HbA2 (between 2.5% and 3.5%), the Department of Clinical Laboratory, the Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China. Department of neurosurgery, the Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China. Department of Ultrasound, the Aliated ffi Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China. Correspondence and requests for materials should be addressed to Y.-S.W. (email: weizhuren999@163.com) Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 1 www.nature.com/scientificreports/ Serum ferritin Genetype n RBC (*10 /L) HGB (g/L) MCV (fl) MCH (pg) MCHC (g/L) (ng/ml) SEA 65 5.21 ± 1.08 85.69 ± 23.44 62.43 ± 7.31 19.11 ± 2.29 305.11 ± 14.70 356.17 ± 25.76 3.7 -α /–SEA 37 5.09 ± 0.82 84.43 ± 15.63 56.28 ± 6.56 16.70 ± 1.67 299.61 ± 21.01 976.58 ± 79.11 4.2 -α /–SEA 23 5.32 ± 0.85 80.88 ± 14.67 52.49 ± 2.44 16.74 ± 2.90 294.50 ± 15.02 997.37 ± 78.69 CS a,b α α/–SEA 75 4.11 ± 0.96 79.17 ± 18.89 68.63 ± 8.38 18.64 ± 2.03 270.00 ± 25.01 1023.69 ± 81.55 Reference 3.50~5.50 110.00~160.00 80.00~100.00 27.00~34.00 320.00~360.00 15.00~250.00 Table 1. Clinical Average Hematological Parameters of the Study Population (x ± s). RBC, red blood cell; HGB, haemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; SEA: the Southeast Asian deletion; -α3.7, rightward deletion; -α4.2, leftward deletion; CS, Hb Constant Spring. a:compared with -α4.2/–SEA, P < 0.001; b:compared with -α3.7/– SEA, P < 0.001. Note: RBC, HGB, MCV, MCH and MCHC were the average hematological parameters level before the first blood transfusion. Normal levels serum ferritin, no one suffering from hemolytic anemia and malnutrition anemia. No cardiovascu- lar and blood infectious d disease. Its family without hypertension, diabetes. All the control group also were diag- nosed by the DNA sequencing technique, no one suffers from six common α - thalassemia (–SEA, -α3.7, -α4.2, CS WS QS α α, α α, α α) and seventeen β- thalassemia (17 M/N, CD41-42M/N, -28M/N, -29M/N, 31 M/N, -32M/N, 43 M/N, 654 M/N, -30M IVS-I-1M, IVS-I-5M 14–15 M, 27/28 M, 71–72 M/N, ΒeM/N, CAMP, IntM), which the common type of thalassemia in chinese people. Physical examination including. Red blood cells (RBC), Hemoglobin (HGBg/l), Mean corpuscular volume (MCV/fl), Mean hemoglobin content (MCH/pg), Mean hemoglobin concentration (MCHC), basal growth hor - mone, estradiol (in females) and testosterone (in males), thyroid-stimulating hormone (TSH), FT3, FT4, serum calcium concentration, serum phosphate and parathyroid hormone. Alpha globin mutations were analyzed using gap-PCR and reverse-hybridization assay according to the manufacturer. Classification of patients according to genotype. Children were divided into four groups according to their genotype based on the α-globin gene production. Group1–4: (SEA) deletions, (−3.7 kb merge SEA) deletions, (−4.2 kb merge SEA) deletions and (CS mutations merge SEA) deletions. Definitions. Short stature was defined as patient height > 2 standard deviation below the mean for age, gender and ethnicity . Short stature was evaluated by Children’s Health Rehabilitation Center (Affiliated Hospital of Youjiang Medical College for Nationalities, Guangxi, China). Hypogonadism was defined as low testosterone (in males or oestradiol (in females) level or subjects who had received testosterone or oestradiol therapy. Patients were diagnosed with diabetes mellitus based on WHO criteria or history of insulin therapy or oral antidiabetic therapy according to American Diabetes Association, World Health Organization Criteria and National Diabetes Health Group 1979. Hypothyroidism was defined according to TSH/FT3, FT4 or based on the history of treatment with levothy- roxine for previously diagnosed hypothyroidism. Hypoparathyroidism was defined as low serum calcium and low serum parathyroid hormone concentration, with increased serum phosphate. A hemoglobin level of less than 90 (g/L) was the standard for initiating transfusion in children with severe thalassemia. Infection, growth retardation, diabetes mellitus, hypogonadism, hypothyroidism, hypoparathy- roidism or other complications in thalassemia children, were the indications for transfusion at a relatively high level of haemoglobin. Statistical analysis. SPSS13.0 (SPSS, Inc., IL, USA) was used to conduct statistical analysis. χ test or Fisher’s exact test was used for comparation between different groups. Measurement data were represented as mean ± standard deviation (x ± s), and categorical data were represented as χ . P < 0.05 and P < 0.001 were considered to indicate statistically significant differences. Results Patient characteristics. All the patients were recruited from Ali ffi ated Hospital of Youjiang Medical College for Nationalities, Guangxi, China. The patients (126 males and 74 females) had a mean (SD) age of (9.64 ± 1.15) years. Hypogonadism was the most prominent endocrinal complications in patients (84.0%), followed by growth retardation (68.5%) and hypoparathyroidism (14.5%). A total 70.5% of patients start to use chelation in 3 years old. There was no significant difference in RBC, MCV, MCH and MCHC among the four groups ( P > 0.05). Clinical Average Hematological Parameters were summarized in (Table 1). Genetype of Thalassemia and endocrinal complications. Two major genetype identie fi d in the Alpha CS thalassemia patients were α α/–SEA (37.5%, 38.1% of males and 36.5% of females) and SEA (32.5%), followed CS by -α3.7/–SEA (18.5%) and -α4.2/–SEA (11.5%). A total of 94.1% of patients with the α α/–SEA genotype started earlier transfusion (≤3 year), 77.3% of patients received frequent transfusion (every 4–5 weeks) and CS 68.0% started earlier iron chelators (>3 years). In addition, patients with the α α/–SEA genotype had a higher Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 2 www.nature.com/scientificreports/ 3.7 4.2 CS SEA -α /–SEA -α /–SEA α α/–SEA Characteristics Patients (n = 65) (n = 37) (n = 23) (n = 75) P-value Gender   Male 126 31.7% 19.0% 11.1% 38.1% 0.98   Female 74 33.8% 17.6% 12.2% 36.5% Age of start transfusion (years)   ≤3 117 22.2% 13.7% 10.3% 53.8% <0.001   >3 83 47.0% 25.3% 13.3% 14.5% Frequency of transfusion (weeks)   Every 2–3 98 45.9% 19.4% 17.3% 17.3% <0.001   Every 4–5 102 19.6% 17.6% 5.9% 56.9% Age of start chelation (years)   ≤6 59 27.1% 15.3% 16.9% 40.7% 0.31   >6 141 34.8% 19.9% 9.2% 36.2% Growth retardation   Negative 63 50.8% 25.4% 14.3% 9.5% <0.001   Positive 137 24.1% 15.3% 10.2% 50.4% Hypogonadism   Negative 32 62.5% 15.6% 0.0% 1.9% 0.001   Positive 168 26.8% 19.0% 13.7% 40.5% Diabetes mellitus   Negative 186 33.9% 18.3% 9.1% 38.7% 0.001   Positive 14 14.3% 21.4% 42.9% 21.4% Hypothyroidism   Negative 174 36.8% 14.4% 8.6% 40.2% <0.001   Positive 26 3.8% 46.2% 30.8% 19.2% Hypoparathyroidism   Negative 171 38.0% 16.4% 9.4% 36.3% <0.001   Positive 29 0.0% 31.0% 24.1% 44.8% Table 2. Association between patient genotype and endocrinal complications. prevalence of growth retardation (92%), and patients with the -α4.2/–SEA genotype had a higher prevalence of hypogonadism and diabetes mellitus (100% and 73.9%, respectively) (Table 2). Growth retardation in patients. Growth retardation was identified in 75.2% of patients (≥ 6 years old) and 61.1% of patients (<6 years old), and no significant difference was identified between males and females. A total of 40.9% of patients with growth retardation started earlier blood transfusion (≤3 year), 69.3% received frequent transfusion (every 4–5 weeks), 89.8% started iron chelation (>3 years) and 17.5% were poor compliant (Table 3). Hypogonadism in patients. Hypogonadism was identified in 83.8% of patients (≥ 6 years old) and 84.2% of patients (<6 years old), and there was significant difference between males and females (P < 0.001). A total of 67.3% of patients with hypogonadism started earlier transfusion (≤3 years), 51.8% of them received frequent transfusion (every 2–3 weeks). 76.2% of patients with hypogonadism started iron chelation (>3 years) and 12.5% had a poor compliance (Table 4). Diabetes mellitus in patients. Diabetes mellitus was identified in 14 patients and 71.4% of them were ≥6 years old with no significant difference identified between males and females. 92.9% of them received fre- quent transfusion (every 2–3 weeks), and 85.7% of patients with hypogonadism started iron chelation (>3 years) (Table 5). Hypothyroidism in patients. 26 patients (15 males and 11 males) were diagnosed with hypothyroidism and no significant difference was identified between males and females. All of these patients started earlier trans- fusion (≤3 years). Most of the patients (88.5%) were more than 6 years older and 96.2% had a poor compliant (Table 6). Hypoparathyroidism in patients. Hypoparathyroidism was identified in 29 patients and 82.8% of them were ≥6 years old, no significant difference was observed between males and females. All of these patients started earlier transfusion (≤3 years) and most of them had a poor compliant (Table 7). Endocrine complication between case group and control group. er Th e was no significant difference in the incidence of endocrine complication between male and female in case group and control group, alpha thalassemia patients are significantly more likely to have growth retardation, hypogonadism, diabetes mellitus, Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 3 www.nature.com/scientificreports/ Growth retardation (n = 200) Negative Positive Characteristics Patients, n (n = 63) (n = 137) P-value Gender   Male 126 31.0% 69.0% 0.83   Female 74 32.4% 67.6% Age (years)   ≥6 105 24.8% 75.2% 0.03   <6 95 38.9% 61.1% Frequency of transfusion (weeks)   Every 2–3 98 57.1% 42.9% <0.001   Every 4–5 102 6.9% 93.1% Age of start transfusion (years)   ≤3 117 52.1% 47.9% <0.001   >3 83 2.4% 97.6% Age of start chelation (years)   ≤6 59 76.3% 23.7% <0.001   >6 141 12.8% 87.2% Compliance, %   <60 43 44.2% 55.8% 0.04   ≥60 157 28.0% 72.0% Table 3. Association between growth retardation and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. Hypogonadism (n = 200) Negative Positive Characteristics Patients, n (n = 32) (n = 168) P-value Gender   Male 126 23.0% 77.0% <0.001   Female 74 4.1% 95.9% Age (years)   ≥6 105 16.2% 83.8% 0.94   <6 95 15.8% 84.2% Frequency of transfusion (weeks)   Every 2–3 98 11.2% 88.8% 0.07   Every 4–5 102 20.6% 79.4% Age of start transfusion (years)   ≤3 117 3.4% 96.6% <0.001   >3 83 33.7% 66.3% Age of start chelation (years)   ≤6 59 32.2% 67.8% <0.001   >6 141 9.8% 90.8% Compliance, %   <60 43 20.9% 79.1% 0.32   ≥60 157 14.6% 85.4% Table 4. Association between hypogonadism and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. hypothy- roidism and hypoparathyroidism compared with controls (P < 0.001) (Table 8). The HGB level lower in patients (81.17 ± 15.23 g/L, range, 13~95 g/L) than control subjects (126.21 ± 17.65 g/L, range, 55~167 g/L). We also identified a significant difference between RBC and MCV indices in case group and control group (P < 0.001). Discussion Thalassemia is a well-known inherited hematologic disorder caused by reduced or absence of globin produc- tion . In China, this disease is prevalent in areas near the southern bank of the Yangtze River, such as Guangdong, 12–14 Guangxi, Fujian and Yunnan Provinces . Endocrine dysfunction is a frequent complication in thalassemic Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 4 www.nature.com/scientificreports/ Diabetes mellitus (n = 200) Negative Positive P- Characteristics Patients, n (n = 186) (n = 14) value Gender   Male 126 95.2% 4.8% 0.11   Female 74 89.2% 10.8% Age (years)   ≥6 105 90.5% 9.5% 1.14   <6 95 95.8% 4.2% Frequency of transfusion (weeks)   Every 2–3 98 99.0% 1.0% 0.001   Every 4–5 102 87.3% 12.7% Age of start transfusion (years)   ≤3 117 95.7% 4.3% 0.07   >3 83 89.2% 10.8% Age of start chelation (years)   ≤6 59 96.6% 3.4% 0.32   >6 141 91.5% 8.5% Compliance, %   <60 43 88.4% 11.6 0.32   ≥60 157 94.3% 5.7% Table 5. Association between diabetes mellitus and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. Hypothyroidism (n = 200) Negative Positive Characteristics Patients (n = 174) (n = 26) P-value Gender   Male 126 88.1% 11.9% 0.70   Female 74 85.1% 14.9% Age (years)   ≥6 105 78.1% 21.9% <0.001   <6 95 96.8% 3.2% Frequency of transfusion (weeks)   Every 2–3 98 79.6% 20.4% 0.004   Every 4–5 102 94.1% 5.9% Age of start transfusion (years)   ≤3 117 77.8% 22.2% <0.001   >3 83 100.0% 0.0% Age of start chelation (years)   ≤6 59 67.8% 32.2% <0.001   >6 141 95.0% 5.0% Compliance, %   <60 43 41.9% 58.1% <0.001   ≥60 157 99.4% 0.6% Table 6. Association between hypothyroidism and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. patients who are on regular blood transfusions. Iron overload has been considered to be the major cause of endo- crine abnormalities of α-thalassemia . Growth retardation, hypogonadism, diabetes mellitus and hypoparath- yrodism represent the most common endocrinopathies in thalassemic patients . In this study, we evaluates the impact of genotype on endocrinal complications of Children with Alpha- thalassemia in China and demonstrates that hypogonadism is the most frequent endocrine complication in α-thalassemia (84.0%), followed by growth retardation (68.5%) and hypoparathyroidism (14.5%). CS Our survey showed that the MCV levels in group (α α/–SEA) were higher than those in group (-α3.7/– SEA) and group (-α4.2/–SEA)(P < 0.001, P < 0.001, respectively), there were no significant differences in RBC, Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 5 www.nature.com/scientificreports/ Hypoparathyroidism (n = 200) Negative Positive Characteristics Patients (n = 171) (n = 29) P-value Gender   Male 126 87.3 12.7 0.35   Female 74 82.4 17.6 Age (years)   ≥6 105 77.1 22.9 <0.001   <6 95 94.7 5.3 Frequency of transfusion (weeks)   Every 2–3 98 80.6 19.4 0.05   Every 4–5 102 90.2 9.8 Age of start transfusion (years)   ≤3 117 75.2 24.8 <0.001   >3 83 100.0 0.0 Age of start chelation (years)   ≤6 59 69.5 30.5 <0.001   >6 141 92.2 7.8 Compliance, %   <60 43 39.5 60.5 <0.001   ≥60 157 98.1 1.9 Table 7. Association between hypoparathyroidism and each of the demographic, frequency of transfusion, age of start transfusion, Age of start chelation, compliance. Alpha thalassemia Contral group Characteristics (n = 200) (n = 200) P-value Gender   Male 126 (63.0%) 113 (56.5%) 0.22   Female 74 (37.0%) 87 (43.5%) Growth retardation   Negative 137 (68.5%) 195 (97.5%) <0.001   Positive 63 (31.5%) 5 (2.5%) Hypogonadism   Negative 168 (84.0%) 197 (98.5%) <0.001   Positive 132 (16.0%) 3 (1.5%) Diabetes mellitus   Negative 186 (93.0%) 198 (99.0%) 0.005   Positive 14 (7.0%) 2 (1.0%) Hypothyroidism   Negative 174 (87.0%) 193 (96.5%) 0.001   Positive 26 (13.0%) 7 (3.5%) Hypoparathyroidism   Negative 171 (85.5%) 199 (99.5%) <0.001   Positive 29 (14.5%) 1 (0.5%) Table 8. Comparison of endocrine complications in patients with alpha thalassemia and control group. HGB, MCH and MCHC levels among the four groups (P > 0.05), similar to the previous study by Zhu et al. . CS Compared with the other three groups (α α/–SEA, -α3.7/–SEA, -α4.2/–SEA), the group SEA had a significant lower serum ferritin levels (P < 0.001, respectively), this may be due to patients with SEA genetype generally do not receive blood transfusion therapy frequently unless combined with iron deficiency anemia, vitaminD deficiency, infection caused by long-term malnutrition anemia. In consistent with report by Zhou Y. U. et al . CS no significant difference was observed among the three group (α α/–SEA, -α3.7/–SEA, -α4.2/–SEA) in Serum ferritin levels (P > 0.05, respectively). CS In the present study, we found that the patients with the genetype of (α α/–SEA) had significant higher 18–20 prevalence of growth retardation, hypogonadism (P < 0.001, P = 0.001, respectively). Just like previous report hypogonadism was identified as the most common endocrine complication in the patients (84.0%). Gender, age Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 6 www.nature.com/scientificreports/ of start transfusion or start Chelation had a significant impact on hypogonadism development. However a lower 21, 22 prevalence of hypogonadism was found in some study , which were mainly attributed to difference in the economic status of patients, Physicians’ strategies to optimize chelation therapy, promoting compliance, educat- 23–26 ing patients and different ethnic . The patients with the genetype of (-α 4.2/–SEA) had a significantly higher prevalence of diabetes mellitus (P = 0.001). And there was no significant differences in the incidence of genotypes between males and females (P = 0.98). Compared to the present study 68.5% of patients identified with growth retardation, Hattab, F. N. et al . found a higher prevalence of growth retardation (75.9%). This may be attributed to the difference in economy, most of the patients come in the latter study from poor families, received poor health care treatment, which resulted in multiple infections, thereby aggravating growth retardation or other potential endocrine complications develop- ment in Alpha- thalassemia during childhood. Futher more, the discrepancy of clinical manifestations may be 28–30 impacted by genetic and environmental factors . er Th e was significant association between growth retardation and older year (≥6 years), earlier age of start transfusion, chelation, frequency of blood transfusion or poor com- pliance (P = 0.03, P < 0.001, P < 0.001, P < 0.001, P = 0.04, respectively). But there was no significant association between growth retardation and gender (P = 0.83). In the present study, 7.0% of patients were diagnosed with diabetes mellitu, similar to 8.0% in report by Ong, C. K. et al. . Several previous study have report a lower prevalence of diabetes mellitu, which ranged from 2.5% 32–34 35–37 to 4.9% , while Other had report a higer prevalence of diabetes mellitu, reaching 13% to 17.0% . These discrepancies can be attributed to differences in the age of patients and severity of Hepatitis C virus infection, 38, 39 transfusion rates and chelation therapies, male sex, liver iron concentration . There was significant association between diabetes mellitu and frequency of blood transfusion (P = 0.001), but there was no signic fi ant association between diabetes mellitu and gender, age, age of start transfusion, chelation, frequency blood transfusion or com- pliance (P = 0.11, P = 1.14, P = 0.07, P = 0.32, P = 0.32, respectively). Hypothyroidism was identified in 26 patients (13.0%), which was similar to the result reported by Eshragi, P . et al . . 41–43 While, other studies reported a lower prevalence of hypothyroidism, which ranged from 1.0% to 10.0% . The results of different studies vary widely, these discrepancies can be attributed to differences in genotype of thalas- semia, the age of patients or treatment protocols. Hypogonadism (84.0%), growth retardation (68.5%) and hypoparathyroidism (14.5%) were the first and the most frequent endocrine complications diagnosed in our present study. Today, many patients can benefit from modern treatment, improve the quality of life of patients dut to adopting in early and regular chelation therapy. er Th efore, prevention of the endocrine complications may be influenced by the improvement of medical diagno- sis and treatment. Monitoring compliance is essential in such conditions. er Th e are a few limitations need to be mention here. Firstly, the sample size is small, and the age of these patients too early which may result in limited power. Secondly, the type of iron chelation used could not be figured out, rare genetype of α-thalassemia were not included in our study. Thirdly, none of the analyses take into account the age effect properly. e Th incomplete medical records could prevent us from identifying predictive complication. Further studies are needed on the complications of all α-thalassemic and older patients in the region. CS In conclusion, our present study show that α α/–SEA, SEA, -α3.7/–SEA, and -α4.2/–SEA are the main genetype identified in α-thalassemia children in Guangxi Province, and hypogonadism, growth retardation and hypoparathyroidism are the most common endocrine complications in children with α-thalassemia. References 1. De Sanctis, V., Eleftheriou, A. & Malaventura, C. Prevalence of Endocrine Complications and Short Stature in Patients with a Th lassaemia Major: A Multicenter Study by the Thalassaemia International Federation (TIF). Pediatr Endocrinol Rev. 2(Suppl 2), 249–255 (2004). 2. Weatherall, D. J. Thalassemia as a Global Health Problem: Recent Progress Toward its Control in the Developing Countries. 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Biomed Rep 4, 728–736, doi:10.3892/br.2016.646 (2016). Author Contributions H.C.L. designed and wrote the manuscript. Q.S.L., F.H.H. and C.F.W. collected clinical data. Y.S.W. directed the writing of manuscript. All authors have reviewed and approved the final version of this manuscript. Additional Information Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Repo R ts | 7: 2948 | DOI:10.1038/s41598-017-03029-9 8

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