European Journal of Cardiovascular Medicine

Thalassemia is a group of genetic disorders characterized by an imbalance in alpha and beta globin chain production, leading to hypochromic, microcytic red blood cells with a shorter half-life and resulting anemia. Beta thalassemia occurs due to decreased beta globin chain synthesis, causing an excess of alpha globin chains¹. First recognized in 1925 by Dr. Thomas Cooley as "Cooley’s anemia," it was later renamed "Thalassemia" by Wipple and Bradford due to its prevalence around the Mediterranean region. These hereditary anemias result from mutations that reduce or eliminate the production of either α or β globin chains, affecting hemoglobin synthesis².People of Mediterranean, Middle Eastern, African, and Southeast Asian descent are at higher risk of carrying thalassemia genes, with β-thalassemia being the most common single-gene disorder in India. India contributes 10% of the world's thalassemia births annually, with higher prevalence among Sindhis, Gujaratis, Punjabis, and Bengalis, ranging from 1% to 17%. Thalassemia is classified into α and β types, with β-thalassemia further divided into β⁰ (no β-globin synthesis) and β⁺ (partial β-globin synthesis)³. Clinically, β-thalassemia presents as minor (trait), intermedia, or major, with the minor form being asymptomatic and the major form causing severe transfusion-dependent anemia⁴. β-thalassemia intermedia is of moderate severity, usually not requiring regular transfusions. Affected infants appear normal at birth due to fetal hemoglobin (α₂γ₂), but anemia develops around six months when adult hemoglobin (α₂β₂) production fails⁵. To sustain normal growth and survival, regular blood transfusions become essential.More than 200 deletions or point mutations affecting α- or β-globin mRNA transcription, processing, or translation have been identified, with most being point mutations in critical regions of the β-globin gene. Genetic modifier genes influence disease severity by reducing globin chain imbalance, leading to milder forms of β-thalassemia⁶. Certain mutations, such as deletional and non-deletional hereditary persistence of fetal hemoglobin (HbF), increase γ-globin production, mitigating disease severity. Genome-wide association studies have identified polymorphisms in genes like *BCL11A* and *HBS1L-CMYB*, which elevate HbF levels and modify β⁰-thalassemia severity. Additionally, secondary genetic modifiers, including *TA* polymorphisms linked to Gilbert syndrome and apolipoprotein E ε4 allele, contribute to complications like cholelithiasis, heart failure, and iron overload in thalassemia patients.Thalassemia leads to imbalanced globin chain production, ineffective erythropoiesis, and increased oxidative stress, contributing to anemia, bone deformities, and organ complications. Iron overload in beta-thalassemia major alters arterial structures, increasing carotid intima-media thickness (CIMT), a marker for early atherosclerosis and cardiovascular disease⁶. Carotid Doppler imaging is a valuable tool for detecting subclinical carotid atherosclerosis, aiding in stroke and coronary artery disease risk assessment^7,8.

AIM

To evaluate lipid profile changes, carotid intimal medial thickness, age correlation, and duration from first blood transfusion with atherosclerotic changes in beta thalassemia major cases compared to controls.

This case-control study was conducted in the Department of Paediatrics, JLN Medical College, Ajmer, from February 2023 until the completion of the required sample size. The study population included beta thalassemia major patients attending the thalassemic clinic at JLN Hospital as cases, while controls were healthy patients of the same age and sex admitted with other diseases. The inclusion criteria consisted of beta thalassemia major patients aged 3–18 years attending the clinic. Exclusion criteria included patients with familial hypercholesterolemia (confirmed by history), cardiovascular symptoms indicating heart failure, chronic systemic illnesses, or thyroid disease associated with thalassemia.

TABLE 1 AGE DISTRIBUTION

The above table shows the age distribution of subject. Out of 150 subjects, majority of the cases  are with in the 11-15 years age group while the majority of control are within 6-10 years of age group. There is no significant age distribution difference between cases and control p<0.0935. 

TABLE 2: RESIDENTIAL STATUS

The majority of subjects in both group are from rural area, with control having slightly higher number (76%) of rural subjects compared to cases (70.67%). There is significant difference found regarding residential status distribution between case and control p<0.0001.

Graph: DURATION OF ILLNESS, TIMING OF FIRST BLOOD TRANSFUSION  And FREQUENCY OF BLOOD TRANSFUSION

Among the 75 cases studied, the majority (31 cases) had a duration of illness between 6–10 years, while only 7 cases had an illness duration of more than 15 years. Most cases (40) received their first blood transfusion within 3–12 months of age, whereas only 13 cases had their first transfusion after 24 months. In terms of transfusion frequency, 53.33% (40 cases) required blood transfusions every 15 days, while only 20% (15 cases) needed transfusions at intervals longer than 30 days.

TABLE 3 VLDL LEVEL

The above table shows the VLDL level of subjects. VLDL level of < 30mg/dl found in 48 (64%) cases as compare to 75 (100%) control. There is a highly significant difference found regarding VLDL level between case and control P < 0.0001.

TABLE 4 HDL LEVEL

The above table shows the HDL level of subjects. 47 cases (62.67%) have value between 20-30 mg/dL whereas 49 control (65.33%) are having 31-40 mg/dL HDL levels. Cases showed significant lower HDL levels as compared to control. There is a highly significant difference found regarding HDL level between case and control P<0.0001.

TABLE 5 LDL LEVEL

The above table shows the LDL level of subjects. 75 cases (100%) have value < 110 mg/dl whereas 74 control (98.67%) are having <110 mg/dL LDL levels. There is a significant difference found regarding LDL level between case and control P<0.0001.

TABLE 6 SERUM CHOLESTEROL LEVEL

The  above table  shows that out of 75 cases 53 (70.67%) have cholesterol level between 50- 100 mg/dl while 48 (64%) control have 101 - 150 mg/dl. There is a significant decrease in cholesterol level in case as compared to control.  There is a significant difference found regarding serum cholesterol between case and control p <0.0010.

Graph showing: TRIGLYCERIDE LEVEL , HEMOGLOBIN LEVEL , SERUM FERRITIN

Cases had higher triglyceride levels than controls, with 49.33% (37 cases) having levels between 101–150 mg/dl, while 77.33% (58 controls) had levels between 50–100 mg/dl, showing a significant difference (*P<0.0001*). Hemoglobin levels were lower in cases, with 64% (48 cases) having <10 gm/dl, whereas 74.67% (56 controls) had levels >11 gm/dl, also showing a significant difference (*P<0.0001*). Serum ferritin levels were markedly higher in cases, with 25.33% (19 cases) having levels between 2001–4000 g/l, while all controls (100%) had levels below 2000 g/l, indicating a significant difference (*P<0.0001*).

TABLE 7 SERUM IRON

Above table shows higher serum iron level  in cases as compared to control.  Out of 75 cases, 48 (64%) have serum iron level between 151-250 while 72 control (96%) have there serum iron level between 50 - 150. There is a significant difference found regarding serum iron between case and control P<0.0001.

TABLE 8 CIMT

The above table shows CIMT value of subjects , 68 (90%) cases have CIMT in the range of 0.41 - 0.60 while 75 (100%)control have CIMT in range of 0.20 -0.40. The CIMT in the cases is significantly higher as compared to control. There is a significant difference found regarding CIMT between case and control P <0.0001.

GRAPH: MEAN CIMT VALUES IN VARIOUS AGE GROUP and ATHEROGENIC INDEXES OF PLASMA LEVEL

The mean (SD) carotid intima-media thickness (CIMT) was significantly higher in cases across all age groups compared to controls, with values ranging from 0.44 (0.05) mm to 0.56 (0.08) mm in cases versus 0.32 (0.03) mm to 0.38 (0.03) mm in controls (*P<0.0001*). Similarly, the mean (SD) atherogenic index of plasma (AIP) was elevated in cases across all age groups, ranging from 0.60 (0.09) to 0.62 (0.03), compared to 0.29 (0.05) to 0.39 (0.04) in controls (*P<0.0001*). These findings indicate a significant difference in CIMT and AIP values between cases and controls, suggesting increased cardiovascular risk in beta thalassemia major patients

The mean age  of cases in this study is  8.99+4.12 years, among which 38.67 percent of children are in 11-15 years group vs mean age of controls 10.1+3.93 years in which 37.33 percent of children are in the 6-10 years of age group. The mean age in other studies were  -  Egypt (Tantawy et al⁹., 2009; Ismail and El-Sherif,⁵ 2010) 8.05±3.12  years, which was in agreement with our study.

In our study the subjects are recruited more from rural area in both case (70.67%) and control (76.00%) as similar to study done in Egypt (Laila M. Sherief,  Osama Dawood 2017)⁷ where  cases 40 (61.5%) vs control 27 (54%) are more from rural area.

In our present study it is found that  among 75 cases 40 (53.33%) cases diagnosed at the age of <12 months, 31 (41.33%) cases have duration of illness  6-10 years, 40 (53.33%) cases have there first blood transfusion in the 3-12 month of age and 43 (57.33%) cases require blood transfusion in every  15 days.  Similar study   done by  Egypt (Ismail and El-Sherif, 2010)⁵ the duration since first transfusion ranged from 1.5–13 years with a mean of 7.26±3.7, among them thirty two patients (51.6%) had frequent blood transfusion. 

In our study VLDL level of < 30mg/dl found in 48 (64%) cases as compare to 75 (100%) control.   HDL level of  47 cases (62.67%) have value between 20-30 mg/dL whereas 49 control (65.33%) are having 31-40 mg/dL levels.  LDL level of <110mg/dl found in  75 cases (100%) whereas 74 control (98.67%) are having <110 mg/dL LDL levels.  Out of 75 cases 53 (70.67%) have cholesterol levels between 50- 100 mg/dl while 48 (64%) control have 101 - 150 mg/dl .  Triglyceride levels of  37 (49.33%) cases have  between 101 -150 mg/dl whereas 58 (77.33 %) controls have  between 50 - 100 mg/dl level. 

In our study it is found out that cases have lower cholesterol, HDL, LDL level then control where as cases have higher triglyceride level and VLDL level then control. Our results are in agreement with the results obtained by Egypt (Hamdy, Lamea 2012),West Bengal (Angshuman Dey⁶ 2016, Tarun Kumar¹⁰ 2017).

In our study 48 cases (64%) have low haemoglobin  <10 gm/dl while 56 (74.67%) control shows a higher haemoglobin level in the range of >11 gm/dl. There is  significant difference found  between patients and controls regarding measurements of haemoglobin. This finding is in concordance with study done in Iraq (Ahmed Abdul-Mohsin Alshammary, Sabih Salih Al-Fetlawi 2022)⁸ who studied on 60 patients and got  haemoglobin 7.11±1.28 gm/dl,  showed a significant difference between cases and controls regarding measurements of haemoglobin.

In our study, 19 cases (25.33%)  having serum ferritin level between 2001– 4000 g/l  where as 75 control (100%) have below 2000 g/l. Significantly higher serum ferritin is found among patients as compared to control.  Our result was in agreement with  study done in Pakistan (Talha Riaz,  Muhammad Ubaid Khan 2011)¹¹ they studied on 79 cases with mean age  was 10.8±4.5 years  got  mean serum ferritin 4236.5 (±2378.3) ng/dl followed by 2490±1579 ng/dl which is done in Egypt (Laila M. Sherief, Osama Dawood 2017)⁷, followed by 890±194.3ng/dl (Ismail and El-Sherif, 2010)⁵.  

In our study out of  75 cases, 48 (64%) have serum iron level between 151250 g/dl while 72 control (96%) have there serum iron level between 50 – 150 g/dl.  There is  significantly higher  value in cases as compared to control with mean (233.15 + 38.92) which is found similar in study done in West Bengal (Angshuman Dey, Goutam Chakraborti 2016)⁶ who  study on 50 thalassemia cases  and 47 control, got serum iron level significantly higher in  cases with multiple blood transfusions than control groups. 

Our data show that 68 (90%) cases have CIMT in the range of 0.41 - 0.60 while 75 (100%) control have CIMT in range of 0.20 -0.40. CIMT of thalassemic patients is significantly increased compared to controls. This was in concordance with other studies that had been done previously by Cheung¹² et al., 2006 (0.45±0.04 mm vs 0.39±0.02mm), Hahalis¹³ et al., 2006 (0.51± 0.07mm vs 0.46± 0.07mm).  The increased CIMT value in older age group in thalassemia patient  in our study  due to iron excess which causes changes in the arterial anatomy and carotid artery thickness. Increased CAIMT have been described as a mirror of atherosclerotic burden, and a predictor of subsequent events including myocardial infarction and stroke. CIMT shows a strong relationship with features of iron overload and atherosclerotic changes⁷. 

 In our study it is found that mean atherogenic index of plasma (AIP) of the controls are 0.29 (0.05), 0.30 (0.04),  0.31(0.04)  and 0.39 (0.04) in 1 to 5 years, 6 to 10 years,  11 to 15 years and 16-<18 years of  age groups respectively, as against 0.60 (0.09), 0.60 (0.10), 0.61 (0.012), 0.62 (0.03) in cases in the similar age groups, which is quite high, as compared to the controls. These findings align with those of Sanghamitra Ray (2021) at Chacha Nehru Bal Chikitsalaya, Delhi, where transfusion-dependent thalassemia patients had a higher mean AIP (0.57±0.25) than healthy controls. The elevated AIP in cases is attributed to hypertriglyceridemia-induced hepatic lipase activity, leading to HDL-C degradation and increased coronary atherosclerosis risk.

From our study we concluded that there is significant corelation of serum ferritin, serum iron level, triglyceride level, cholesterol level, duration since first blood transfusion with increase CIMT level in thalassemia patient. CIMT shows a strong relationship with features of iron overload and atherosclerotic changes. Children with Transfusion Dependent Thalassemia are at increased risk for premature atherosclerosis as evidenced by high CIMT in all age groups. Prevention of the progression of atherosclerosis in early stages is important by decreasing body iron load in the thalassaemic patients. Measurement of CIMT in children with TDT is a convenient and non-invasive tool for estimating the risk of premature atherosclerosis, early atherosclerotic changes as well as in the follow-up to prevent progression of atherosclerosis.  

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