Introduction

Beta Thalassemia major is a group of hereditary blood disorders caused by defective b-globin synthesis leading to an imbalance between a-and b-globin chains [1]. This imbalance causes ineffective red blood cell production, hemolysis and increased red blood cell turnover in the bone marrow. The manifestations of this ineffective erythropoiesis will vary, depending upon the severity of an array of genotypic variations, and ranges from clinically asymptomatic to a severely anemic phenotype who are transfusion dependent [2].

The management of this anemia necessitates regular blood transfusions and chelation to offset the inevitable iron overload, which is also potentiated by an increased intestinal iron absorption [3]. Further, although regular blood transfusions and iron chelation have greatly increased the life expectancy of transfusiondependent beta thalassemia major (TDTM) patients, osteoporosis and osteopenia remain a serious complications, even in optimally transfused and chelated patients [4]. Chronic endocrinopathies and metabolic bone disease are thus the major health issues in these groups of patients [5]. Multiple endocrine dysfunctions including delayed puberty with growth failure, hypogonadism, diabetes, hypothyroidism, hypoparathyroidism and, less frequently, adrenal insufficiency [6]. Numerous factors contribute to the genesis of bone disease in TDTM including bone marrow expansion [7], hypogonadism [8], defective growth hormone-insulin-like growth factor-1 (GH-IGF-1) axis [9], altered pattern of cytokines [10], and iron deposition in bone [6,11].

According to the World Health Organization (WHO), diagnosis of osteoporosis is based on the T- or Z score for bone mineral density (BMD), assessed at the lumbar spine or the femoral neck. Osteoporosis is defined by a BMD that is 2.5 standard deviation (SD) below the mean value for a young adult female (T-score less than or equal to -2.5 SD) [12]. While the T score compares an individual’s BMD to that of a healthy young adult of the same gender, the Z score compares the individual’s BMD to that of other people of the same age, gender, and body size. Similarly, osteopenia is described and Z-score 1.5 SD below the mean value.

In this study, endocrine dysfunction namely delayed puberty with hypogonadism, impaired glucose tolerance with diabetes mellitus, hypothyroid, and hypo-parathyroid along with low BMDs in adults with TDTM are reported. Changes in BMDs due to administration of calcium/vitamin D along with bisphosphonate therapy during follow-up are also reported. Lastly, improvements in iron overload over the study period in spite of several endocrinopathies is also reported.

Materials & Methods

This study was conducted in the Department of Hematology, Sultan Qaboos University Hospital, Oman, by enrolling the current cohort of adult patients with TDTM. Ninety-one patients, who are alive today from amongst the total number of TDTM patients (n=145), aged more than thirteen years, and who were on regular follow ups with blood transfusions and chelation, were included in the study. During their day care visits for blood transfusion, these patients underwent evaluation for underlying endocrine and metabolic bone disease in addition to the routine full blood counts, serum ferritin levels (SF) and blood chemistry studies. The height, weight, and body mass index (BMI) of all participants were measured by standard techniques and were interpreted against WHO standards [13]. All patients underwent screening for underlying delayed puberty with hypogonadism, altered glucose metabolism and diabetes mellitus, hypothyroidism, hypoparathyroidism and adrenal insufficiency on a six-monthly basis. Apart from hormonal evaluations, they also underwent screening for metabolic bone disease with serum 25(OH) vitamin D and dual energy X-ray absorptiometry (DEXA) scan of lumbar spine on a yearly basis.

Additionally, serial serum calcium, alkaline phosphatase and liver transaminases as well as renal function studies were performed every three months while serum ferritin levels were done every month. Further, iron overload was also assessed by in house 1.5 and 3 Tesla MRI machine (Siemens LTD, MAGNETOM Aera and MAGNETOM Vida respectively) and the cardiac and liver T2* values were calculated using the licensed CMR tools software [14].

Females with breast stage 1 after 13 years of age, or males with testicular volume <4 mL after 14 years of age were diagnosed to have delayed puberty, whereas, short stature was defined as height less than 2 standard deviations [5]. Failure to progress to the next Tanner stage in 6 months was labelled as arrested puberty. Patients without any sign of puberty beyond 16 years were diagnosed to have hypogonadism and evaluated with specific hormonal studies [5]. Diagnoses of impaired glucose tolerance, impaired fasting glucose with elevated fructosamine test for glycated albumin was used to diagnose diabetes mellitus, while the diagnosis of hypothyroidism, hypoparathyroidism, and vitamin D deficiency were made as per standard guidelines using the serum hormonal levels and other diagnostic tests [15-17]. Hormonal assays were performed on Cobas E 601 fully automated analyzer using electrochemiluminescence (ECL) technology for immunoassay analysis including Cortisol, Estradiol, FSH, LH, PTH, hCG, Prolactin, Testosterone, T3, T4, & TSH levels. Liver function tests, Renal function test, Serum electrolytes, S. calcium, phosphorous, alkaline phosphatase were tested by spectrophotometric methods using the fully automated clinical chemistry analyzer (COBAS C 501 analyzer) and plasma glucose was measured by the hexokinase method. DEXA scan of the lumbar spine was done in the these patients, using the Hologic Horizon W machine, and BMD was interpreted as per International Society for Clinical Densitometry (ISCD 2019) criteria, after adjusting for height [18].

Statistical Analysis:

Clinical and laboratory parameters were compared and quantitative data were expressed as mean ± standard deviation and range. The students T test was used but the Wilcoxon–MannWhitney test was used when data was not normally distributed and expressed as median with interquartile range. Spearman’s rank of correlation coefficient (rS) was used to study the correlations of the various SF levels in different categories of endocrinopathies. A correlation was considered poor if rS was <0.4, moderate if rS was between 0.4-0.6, good or substantial if rS was between 0.6-0.8 and excellent if rS was > 0.8. Multiple means were compared using the ANOVA test and a p value <0.05 was considered as statistically significant. All data recording, statistical analysis and results extraction were achieved using program of Statistical Package for the Social Sciences (IBM SPSS, USA, version 23).

Results

The baseline demographic characteristics of the TDTM patients (n=91) are described in Table I. Although the median BMI was 23.8 kg/m², 3 (3.3%) and 8 (8.8%) patients were severely underweight and underweight respectively, whereas, 13 (14.2%) patients were obese. Data of metabolic bone disorder and endocrinopathies are shown in Table II and Table III respectively. Seventeen patients (18.7%) had at least one endocrinopathy, whereas 49 (53.8%) had two or multiple endocrine deficiencies, but twenty-five patients (27.5%) did not have any endocrinopathy.

Table 1: Demographic characteristics, in Adults with Transfusion-dependent Thalassaemia cohort (n=91).

Table 2: Metabolic Bone Disease in Adults with Transfusion-Dependent Thalassemia (n=91).

Table 3: Endocrinopathies in Adults with Transfusion-Dependent Thalassemia (n=91) Although bone mineral density was normal in the majority of patients (62.6%), osteopenia increased from 8 (23.5%) to 26 (76.5) patients, whereas osteoporosis reduced from 24 (70.5%) to 10 (29.5%) patients over the study period on bisphosphonate and regular iron chelation. This difference was statistically significant (Wilcoxon-Mann-Whitney test, p<0.001). Six patients (17.6%) had documented non pathological fractures. Moreover, S. Alkaline Phosphatase and S. Calcium levels also showed a statistically significant improvement in terms of the initial versus the last results in this subset with a p value of <0.001 and p=0.001(paired student’s t test) respectively. However, both these parameters showed a negative correlation with Z-scores from the DEXA scans (Figure 1a & 1b) indicating that treatment with oral calcium and 25OH Vitamin D was showing the desired improvement in the BMD parameters in terms of improvement in the Z-scores. Further, z-scores were also negatively correlated with SF levels (Figure 1c).

Figure 1a: Correlation between S. Calcium and Z-score for Lumbar L1-L4 BMD. Figure1b: Correlation between S. Alkaline Phosphatase and Z-Score for Lumbar L1-L4 BMD. Figure 1c: Correlation between S. Ferritin and Z-score for Lumber L1-L4 BMD

Similarly, 25OH Vitamin D levels was deficient, insufficient and sufficient, respectively in 24 (70.5%), 8 (23.5%) and 2 (6%) initially, which improved to 1 (3%), 8 (23.5%) and 23 (73.5%) respectively on regular 25OH vitamin D supplementation. Further, this change with improved 25OH Vitamin D levels over the study period was also statistically significant (paired students T test, p<0.001).

Hypogonadism was the commonest endocrinopathy in 47 (51.6%), followed by impaired glucose metabolism in 43 (47.3%), with IDDM in 10 (10.9%) and NIDDM in 7 (7.7%), delayed puberty in 31 (34.1%), impaired thyroid function in 17 (18.7%), and hypoparathyroidism in 9 (9.9%) patient. We did not find any case of overt adrenal insufficiency, although one patient showed subnormal cortisol levels, but the results of the ACTH stimulation test (Synacthen) were normal with no necessity of hormone replacement therapy. Although multiple endocrinopathies were seen in more than half of these patients, respectively, two, three, four, five and six endocrinopathies were seen in 16(17.6%), 11(12.1%), 14(15.4%), 7(5.5%), and 1(1.1%) patients with Table III giving the details of the associated endocrinopathies within the intra-individual categories of endocrinopathies.

Discussion

Major findings of our study was the significant prevalence of low BMD, with the early onset and high prevalence of multiple endocrine deficiencies. All this occurred in spite of regular blood transfusions along with iron chelation, including the state of art monitoring with MRI techniques, along with regular endocrine function evaluation by hormonal studies as well as BMD screening with DEXA scans.

In an overburdened system like our country, meeting the ideal transfusion and serum ferritin targets may be difficult, thus anemia and poor compliance to chelation, with resulting morbidity, including low BMD and several/multiple endocrinopathies in our patient cohort is likely to be common, especially considering the life-long treatment in absence to a recourse like bone marrow transplantation in these unfortunate patients. The median pretransfusion hemoglobin in our cohort was 9.5 with an IQR between 9.2 to 9.8g/dL and is well within the recommended range by the Thalassaemia international federation guidelines with the median transfusion index of 201 mL/kg/year [19]. The role of splenectomy is also important in the management of TDTM patients and 31(34%) patients underwent the procedure in this cohort and this was helpful in maintaining a narrow range of transfusion Index between the IQR range of 169 to 226 mL/kg/year.