記住我
ORIGINAL ARTICLE
Year : 2021 | Volume
: 46
| Issue : 4 | Page : 234-242
Significance of leukocyte β-glucocerebrosidase and plasma chitotriosidase in patients with β-thalassemia major
Fadime E Dursun
Department of Hematology, Prof. Dr Süleyman Yalçn City Hospital, Istanbul, Turkey
Correspondence Address:
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejh.ejh_47_21
Objective β-thalassemia major (β-TM) is an autosomal-recessive condition with various clinical presentations, including anemia, splenomegaly, and skeletal and cardiac involvement. The aim of this study was to analyze leukocyte β-glucocerebrosidase (β-GBA) levels and plasma chitotriosidase (PCT) activity in patients with β-TM and to determine the significance of these two enzymes in this disease.
Patients and methods This study included 40 patients, 18–55 years of age, who were under follow-up for β-TM in our clinic. Physical examination, ECG, echocardiography, laboratory findings, and the results of imaging tests obtained during routine control visits were recorded. Leukocyte β-GBA and PCT activity levels were analyzed in the blood using fluorometric methods.
Results The average age of the 40 patients, which included 24 (60%) women and 16 (40%) men, was 28.5±7.8 years. Leukocyte β-GBA levels were below 2.5 nmol/mg/h in 15 patients, and PCT activity was above 200 μmol/l/h in 10 patients. A positive correlation was detected for leukocyte β-GBA enzyme levels with cardiac T2* (P=0.024); however, a negative correlation was detected with intraventricular septum thickness (P=0.029) and left heart posterior wall thickness (P=0.030).
Conclusion Lower leukocyte β-GBA levels and higher PCT activity may be present in patients with β-TM. There may be an increase in cardiac iron load, intraventricular septum thickness, and left ventricle posterior wall thickness, especially in patients with lower leukocyte β-GBA levels and higher PCT activity. Therefore, leukocyte β-GBA levels and PCT activity may be associated with cardiac complications in patients with β-TM.
Keywords: cardiac T2FNx01, chitotriosidase, β-glucocerebrosidase, β-thalassemia major
β-thalassemia major (β-TM) and Gaucher disease (GD) are two autosomal-recessive conditions with similar clinical presentations, including anemia, splenomegaly, and skeletal involvement [1],[2],[3],[4]. The co-occurrence of these two diseases may cause diagnostic difficulties and may only be discovered if it is considered as a possibility. There are a few case reports on the concomitant appearance of GD and β-TM in the literature [5],[6]. In GD, β-glucocerebrosidase (β-GBA) levels are below 1 nmol/mg/h and plasma chitotriosidase (PCT) levels are above 200 μmol/l/h [7],[8]. Therefore, leukocyte β-GBA levels and PCT activity may be effective differential tests in patients with β-TM.
β-glucosidases (systematic name β-D-glucoside glucohydrolase) are enzymes that hydrolyze the β-glucoside bonds in oligosaccharides or other glucose compounds. Acid-β-glucosidase, or glucosylceramidase, cleaves glycosylceramide into glucose and ceramide [7]. Deficiency of these vital enzymes causes an accumulation of undigested substrates, glucocerebroside, and glucosylsphingosine in the monocyte–macrophage system and leading to various clinical presentations [8],[9].
Chitotriosidase (EC 3.2.1.14) is a functional chitinase that breaks down chitin, which is an important component of fungi (including various fungal pathogens) and various other pathogens [10],[11]. Chitotriosidase is synthesized by specifically activated macrophages and neutrophil precursors and is encoded by a gene on chromosome 1q31; it is regulated via an as yet unknown molecular mechanism [12],[13],[14],[15],[16].
The aim of this study was to review leukocyte β-GBA levels and PCT activity in patients with β-TM to detect possible GD as well as the association of these enzymes with clinical findings, organ damage, and laboratory and radiological findings.
Patients and methodsStudy design
This prospective cohort study was carried out in the Hematology Department of Prof. Dr Suleyman Yalcin City Hospital and included 40 patients (24 women and 16 men) diagnosed with β-TM between the ages of 18 and 55 years. All patients typically received regular erythrocyte suspension transfusions every 3 weeks and continuous oral iron-chelation therapy. Patients with another hematological disease or chronic disease, who did not agree to participate in the study, did not come to control visits, and did not regularly receive chelation treatment were excluded.
MethodsSystemic examination findings, cardiologic examination findings, ECG, echocardiography (ECHO), complete blood counts, biochemical analyses, ferritin levels, vitamin levels, thyroid function test results, and imaging test results collected during routine control patient examinations were retrospectively obtained from patient files and the hospital automation system in the Thalassemia Center of Prof. Dr Süleyman Yalçın City Hospital. Heart (T2*) and liver (R2) MRI findings for iron load were also obtained from patient files and hospital automation systems. Cardiac iron load in the heart by MRI was considered normal if T2* was more than or equal to 20 msn and was considered increased if T2* was less than 20 msn. Hepatic iron load by MRI was considered normal if R2 was less than or equal to 7 mg/g and was considered increased if R2 was more than 7 mg/g.
Detection of PTC by the fluorometric method
For plasma chitotriosidase (PCT) levels, 10 ml of blood was collected into EDTA tubes before blood transfusion. The blood samples were rapidly transferred to the laboratory. PCT activity was measured using a fluorometric method as described previously [10],[12],[13]. In brief, 5 ml of plasma in an EDTA tube was incubated with 100 ml of 4-methylumbelliferyl chitotrioside (Sigma Chemical Co, St Louis, Missouri, USA) at 37°C in citrate/phosphate buffer (0.1/0.2 M; pH 5.2) for 15 min. The reaction was stopped by adding 2 ml of glycine/NaOH buffer (0.3 M; pH 10.6). The fluorescence of 4-methylumbelliferon was measured at 445 nm, and the results are expressed in μmol/l/h.
Detection of leukocyte β-glucocerebrosidase by the fluorometric method
For leukocyte β-GBA levels, 10 ml of blood was collected into EDTA tubes before blood transfusion. The blood samples were rapidly transferred to the laboratory. Leukocyte β-GBA tests were performed using the fluorometric method [17],[18] at pH 5.5 with 4-methylumbelliferyl β-D-glucopyranoside (M3633; Sigma Chemical Co.) and sodium taurocholate (86339; Sigma Chemical Co) as the substrate solution. The substrate solution was incubated with a 3 mm DBS punch at 37°C for 17 h, and then the reaction was stopped. Fluorescence was recorded at excitation and emission wavelengths of 366 and 442 nm, respectively. A calibration curve was created using 4-methylumbelliferone (Sigma Chemical Co., 1381), and the results were evaluated. The results obtained are expressed in nmol/mg/h.
DNA was obtained from patients with leukocyte β-GBA levels below 2.5 nmol/mg/h to test for GBA gene mutations by next-generation sequencing. Selective specific PCR primers were designed to amplify regions of the β-GBA gene from the isolated DNA and to distinguish it from the pseudogene.
Ethical considerations
This study was approved by the local ethics committee of the Medeniyet University Faculty of Medicine (No.: 2020/0119). Written informed consent was obtained from each study participant after a data-oriented explanation was provided about the aims and scope of the study in accordance with the principles of the World Medical Association Declaration of Helsinki.
Statistical analysis
Statistical analyses were carried out using SPSS software (version 25.0; SPSS Inc., Chicago, Illinois, USA). Normally distributed continuous variables are presented as mean±SD (P>0.05, Kolmogorov–Smirnov or Shapiro–Wilk test; n<30). Non-normally distributed continuous variables are presented as the median (minimum–maximum). Intergroup comparisons were performed using Student’s t test or one-way analysis of variance for normally distributed data, and the Mann–Whitney U test or the Kruskal–Wallis test was used to examine non-normally distributed data. Categorical variables were compared between groups using the χ2 test or Fisher’s exact test. Correlations between variables were assessed using Spearman’s correlation coefficient. Statistical significance was set at P values less than 0.05.
ResultsForty patients [24 (60%) females and 16 (40%) males], with a mean age of 28.5±7.8 years (range, 18–55 years), were enrolled in the study. Laboratory findings, cardiac iron load, and hepatic iron load of the patients are presented in [Table 1]. Leukocyte β-GBA and PCT levels were evaluated and compared with normal reference values. The mean leukocyte β-GBA level was 3.3±1.7 nmol/mg/h [3.2 (0.6–7.6 nmol/mg/h)], and the mean PCT activity was 135.4±169.6 μmol/l/h [135.6 (0.1–744.4 μmol/l/h)]. Evaluation of other laboratory results revealed that hemoglobin and 25-OH-vitamin D3 levels were lower than normal, whereas ferritin, lactate dehydrogenase, and total bilirubin levels were higher.
Table 1 Laboratory findings and heart/liver iron overload values of the patientsThe patient distribution according to the results of pathological and nonpathological enzyme, ferritin, T2*, and R2 assessments is presented in [Table 2]. Leukocyte β-GBA levels were below 2.5 nmol/mg/h in 15 (37.5%) patients, which was low. PCT activity was more than 200 μmol/l/h in 10 (25%) patients, which was above the normal reference value and was high. Six (15%) patients had both low leukocyte β-GBA levels and high PCT activity. Cardiac T2*, obtained by MRI of the heart, was more than 20 msn in 10 (25%) patients, and hepatic R2, obtained by MRI of the liver, was more than 7 mg/g in 11 (27.5%) patients. In addition, 10 of the 11 patients with hepatic R2 more than 7 mg/g also had cardiac T2* less than 20 msn. The leukocyte β-GBA levels in these 10 patients were less than 2.5 nmol/mg/h, and their PCT activity levels were more than 200 μmol/l/h. Furthermore, the plasma ferritin levels of these patients were more than 1000 ng/ml.
Table 2 Patient distribution according to pathological and nonpathological enzyme, ferritin, T2*, and R2 resultsWe also reviewed the ECG and ECHO findings of the patients in our study ([Table 3]). No abnormal findings or rhythm disturbances were found on the ECG of any patient. The heart rate, P, PR, QR, QT, and QTc durations of ECGs were found to be within normal limits. When the cardiac ECHO findings of the patients were examined, the ejection fraction (EF) was less than 55% in one patient; the EF results of the other patients were normal. The intraventricular septum thickness was more than 1.0 cm, which is thicker than normal, in two patients; it was normal in all other patients. The leukocyte β-GBA levels of these two patients with increased intraventricular septum thickness were below 2.5 nmol/mg/h, and their PCT activity levels were above 200 μmol/l/h. The left ventricular posterior wall thickness (LVPWT) was within normal limits in all patients. Similarly, the left ventricular diastolic diameter (LVDD) and left ventricular systolic diameter (LVSD) were within normal limits in all patients.
The results of the correlation analyses of leukocyte β-GBA, PCT, and ferritin levels as well as other laboratory findings are shown in [Table 4]. A significant positive correlation was found between leukocyte β-GBA enzyme and blood white blood cell (WBC) counts (r=0400, P=0.011), and a negative correlation was found between ferritin levels (r=−0.317, P=0.046). Furthermore, a positive correlation was detected between leukocyte β-GBA levels and cardiac T2* (r=0.362, P=0.024). Positive correlations were also found for PCT activity with ferritin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (r=0.547, P=0.001; r=0.360, P=0.023; r=0.430, P=0.006; and r=0.340, P=0.033, respectively). Blood ferritin levels were negatively correlated with leukocyte β-GBA levels (r=−0.317, P=0.046) and were positively correlated with PCT activity (r=0.547, P=0.0001). In addition, a positive correlation was detected between ferritin and hemoglobin levels (r=0.315, P=0.048), a negative correlation was detected between ferritin and platelet levels (r=−0.400, P=0.011), and positive correlations were detected with AST (r=0.436, P=0.005) and ALT (r=0.360, P=0.023). Finally, there was a positive correlation between ferritin levels and hepatic R2 (r=0.314, P=0.049).
Table 4 Relations of PTC and leukocyte β-glucocerebrosidase with other laboratory findingsThe correlation of leukocyte β-GBA and PCT levels with cardiac ECG and ECHO findings was examined ([Table 5]). No correlation was found between leukocyte β-GBA levels and heart rate, P, PR, QRS, QT, and QTc (P=NS). However, statistically significant negative correlations were detected for leukocyte β-GBA levels with IVST and LVPWT (r=−0.35, P=0.029, and r=−0.35, P=0.030, respectively). However, no correlation was detected for leukocyte β-GBA enzyme levels with EF, LVDD, and LVSD (P=NS). In addition, no associations were found for PCT activity with EF, IVST, LVPWT, LVDD, or LVSD (P=NS). Positive correlations were detected for blood ferritin levels with IVST and LVPWT (r=0.325, P=0.049, and r=0.327, P=0.044, respectively). There was no association of ferritin with EF, IVS, PWT, LVDD, and LVSD (P=NS).
Table 5 Relations of PTC, leukocyte β-glucocerebrosidase, and ferritin with ECG and echocardiography findingsGBA gene sequencing was performed on the 15 patients with low leukocyte β-GBA levels (<2.5 nmol/mg/h), and no gene mutation was detected in any of the patients.
DiscussionWe analyzed leukocyte β-GBA levels and PCT levels in patients with β-TM. Leukocyte β-GBA levels were below the reference value (<2.5 nmol/mg/h) in 15 patients, and PCT levels were above the normal value (>200 μmol/l/h) in 10 patients. We detected lower concomitant leukocyte β-GBA levels and higher PCT levels in six patients. Cardiac T2* levels were below 20 msn in 10 of the 15 patients with low leukocyte β-GBA levels. These 10 patients also had higher PCT levels and ferritin levels more than 1000 ng/ml. Another important result was that blood ferritin levels were negatively correlated with leukocyte β-GBA and positively correlated with PCT activity.
Since the samples used for the tests were taken before blood transfusion, the hemoglobin levels were lower, as expected, in some of our patients. Similarly, the ferritin levels of the patients were higher than normal, except in two patients, despite regular chelation therapy. The higher than normal total bilirubin levels in our patient cohort were also attributed to repeated blood transfusions and hemolysis. In addition, 25-OH-vitamin D3 and vitamin B12 levels were also below normal limits in some patients due to insufficient nutrition and impaired nutrient absorption ([Table 1]). β-TM and GD present similar symptoms and laboratory features. Therefore, physicians should be careful in their differential diagnosis and be aware of the possibility of this rare association for suitable investigations, timely diagnosis, and prompt and proper treatment. Could we have missed a diagnosis of GD in some patients followed up due to a diagnosis of β-TM? Fatigue, nutritional problems, weakness, pale or yellow skin, irritability, slow growth, a swollen abdomen, and enlarged facial bones are usually observed in β-TM [19]. Since these symptoms can also be present in patients with GD, without proper testing, its co-occurrence with β-TM may be missed.
The patients’ cardiac function was evaluated using ECG and ECHO. None of the patients presented with an arrhythmia on ECG. The EF was less than 55% in one patient, and the IVST was thicker than normal in two patients. The LVPWT was within normal limits in all patients. Similarly, LVDD and LVSD were within normal limits in all patients ([Table 3]). These findings were attributed to the fact that our patients attended their control visits and regularly received iron-chelation therapy. Ferritin levels were above 5000 ng/ml in five patients. One of these five patients had an EF less than 55%. The cardiac T2* of this patient was below 20 msn. The lower leukocyte β-GBA levels and higher PCT activities and blood ferritin levels in our patients with cardiac T2* less than 20 ms are important results. β-TM is characterized by increased erythroferrone production by erythroid precursors, abnormal iron metabolism, and increased iron absorption through decreased hepatic hepcidin production [20],[21]. Patients with transfusion-independent thalassemia may develop iron overload, even without transfusion, through increased dietary iron absorption; however, they always have faster iron loading. Accumulation of iron in the liver, heart, and endocrine organs is the most important cause of morbidity in β-TM. Heart disease, which is the leading cause of death from iron overload, induces left ventricular dysfunction, heart failure, and arrhythmias [22]. Hepatic fibrosis, cirrhosis, and hepatocellular carcinoma due to cumulative iron exposure usually do not occur until advanced age [23],[24]. Therefore, monitoring and management of iron overload, particularly cardiac and hepatic iron loads, are an important aspect of thalassemia treatment [25],[26],[27],[28],[29],[30].
We aimed to answer the question, ‘What kind of association may exist between leukocyte β-GBA levels and other laboratory findings in patients with β-TM?’ For this purpose, we performed correlation tests of leukocyte β-GBA levels with other laboratory findings ([Table 4]). A positive correlation was found between leukocyte β-GBA levels and WBC counts. No study in the literature has investigated leukocyte β-GBA enzyme activity levels in patients with β-TM. Therefore, examining the association between leukocyte β-GBA levels and other parameters in our patients is important. Its correlation with the WBC count was an expected finding, as the level of β-GBA is measured in WBCs. Another important result is the negative correlation between leukocyte β-GBA and ferritin levels. Based on this result, blood ferritin levels are higher in patients with lower leukocyte β-GBA levels, and vice versa. These results suggest that lower leukocyte β-GBA levels and higher ferritin levels may be effective for predicting the poor prognosis.
Interestingly, leukocyte β-GBA levels were positively associated with cardiac T2* in our patients ([Table 4]). Patients with higher leukocyte β-GBA levels are more likely to have a cardiac T2* more than or equal to 20 ms, and this was the expected finding. However, the decrease in leukocyte β-GBA levels is probably associated with a cardiac T2* value more than or equal to 20 ms, indicating an increased cardiac iron load. Our results suggest that leukocyte β-GBA levels may be useful as a therapeutic target for reducing cardiac iron burden in patients with transfusion-dependent thalassemia and/or a prognostic marker to predict the clinical course.
A positive correlation was found for PCT activity with AST, ALT, gamma-glutamyl transferase, and ferritin levels ([Table 4]). Based on this result, higher PCT enzyme activity may facilitate hepatic iron accumulation, like ferritin. Some studies have reviewed PCT activity in β-TM [31],[32]. One of these studies showed that PCT activity was positively correlated with hepatic iron load [31]. In another study, PCT activity was found to be higher in adult patients with β-TM; similar to our study, PCT activity in these patients was found to be associated with blood ferritin levels [32].
The correlations of leukocyte β-GBA and PCT activity with cardiac ECG and ECHO findings were also examined ([Table 5]). Statistically significant negative correlations were found for the leukocyte β-GBA enzyme with IVST and LVPWT, which suggests an association between leukocyte β-GBA enzyme levels and cardiomyopathy. The positive correlations of ferritin levels with IVST and LVPWT suggest that ferritin levels are directly associated with cardiac iron accumulation. Based on the results of our study, leukocyte β-GBA had the opposite effect on ferritin. Accordingly, leukocyte β-GBA had a protective effect on the hearts of our patients with β-TM, whereas ferritin appeared to contribute to the development of cardiomyopathy. Cardiac complications are an important cause of mortality and morbidity in patients with β-TM. The most important complications observed in β-TM are dilated cardiomyopathy and arrhythmias [22]. Iron accumulation can cause cardiac dysfunction, which manifests as ventricular contractility, increased cardiac output, and enlargement of the ventricles due to chronic hemolytic anemia. Myocardial iron accumulation prevents left ventricular restriction and causes pulmonary hypertension [33],[34].
A GBA gene mutation analysis was carried out for the differential diagnosis of GD in the 15 patients with leukocyte β-GBA levels less than 2.5 nmol/mg/h, and no gene mutation was found in any of the patients. We could not explain the reason for the lower leukocyte β-GBA and higher PCT activities in these patients. Environmental factors or genetic factors other than GBA mutations may have led to these observed phenotypes. We believe that this issue will be clarified in future studies with a larger series.
This study has some limitations. First, we could not create a control group because of financial constraints. However, we used the normal reference values for leukocyte β-GBA and PCT levels as an alternative. The second limitation is the limited number of patients.
ConclusionsLeukocyte β-GBA and PCT activities may be, respectively, lower and higher than normal in patients with β-TM. However, we found that ferritin was related to the development of complications in these patients and was negatively correlated with leukocyte β-GBA levels. An important result of our study was that leukocyte β-GBA is more sensitive than ferritin as an indicator of cardiac iron accumulation. However, ferritin may be a more sensitive indicator of hepatic iron accumulation. We believe that these results will be supported by larger series and multicenter studies.
AcknowledgementsThe authors thank Ms Cagla Sarıturk for carrying out the statistical analyses. The authors also thank Sanofi-Genzyme Company for providing support for the analyses of leukocyte β-GBA levels, PCT activity, and GBA mutations.
Financial support and sponsorship
This work was supported by the Sanofi-Genzyme Company, Turkey.Conflicts of interest
There are no conflicts of interest.
References
留言 (0)