CASE SERIES
Hippokratia 2016, 20(2):153-159
Vargiami E, Dimitriadou M, Economou M, Christoforidis A, Zafeiriou DI
1st Department of Pediatrics, Hippokratio General Hospital, Aristotle University, Thessaloniki, Greece
Abstract
Background: Gaucher disease (GD) is a lysosomal storage disorder characterized by severe skeletal complications. Bone complications are an important cause of morbidity of GD and are thought to result from imbalance in bone remodeling.The objective of this case series was to analyze the long-term effect of enzyme replacement therapy on chemokines MIP-1a and MIP-1b, cytokines IL-3, IL-6, IL-10, and IL-12, osteoprotegerin (OPG) and osteocalcin (BGP), chitotriosidase, quantitative ultrasound sonography (QUS), bone magnetic resonance imaging (MRI) and dual-energy X-ray absorptiometry (DXA) in patients with GD in Northern Greece. In addition, the study aimed in investigating possible relationship between the above mentioned parameters.
Patients and Methods: Seven patients with GD type I (three males and four females) were included in the study. Mean age was 26.29 ± 15.34 years (range 7-47 years). Six patients were receiving enzyme replacement therapy (ERT), with 40-60 IU/kg of imiglucerase weekly, for a mean period of 36 months prior to study initiation. One patient started ERT after his inclusion in the study. The levels of MIP-1a, MIP-1b, IL-3, IL-6, IL-10, IL-12, OPG, BGP, chitotriosidase, bone imaging parameters assessed with two different techniques (QUS and DXA) and MRI data were estimated at baseline (T0) and after two years on ERT.
Results: Chitotriosidase, MIP-1a, and IL-6 levels decreased in all patients after two years of ERT (p =0.05). In contrast, OPG and BGP levels increased (p =0.04 and p =0.02, respectively). Bone mineral density (BMD) demonstrated a progressive improvement with regards to the Z-score in all patients (p =0.05). The decrease in the plasma levels of MIP-1a strongly correlated with a decrease in the plasma levels of chitotriosidase. Additionally, decreased plasma levels of IL-6 were correlated with increased Z-score both at baseline (T0) as well as two years later, in all patients. There was no correlation between MRI findings and any inflammatory biomarker.
Conclusions: Measurement of serum markers in patients with GD under ERT could be used as an auxiliary tool in the monitoring of bone involvement, in combination with MRI imaging and BMD. However, larger studies involving higher numbers of GD patients are needed to confirm these conclusions. Hippokratia 2016, 20(2): 153-159
Key words: Gaucher disease type I, biomarkers, serum cytokines, skeletal complications, enzyme replacement therapy
Corresponding author: Professor Dimitrios I Zafeiriou, MD, PhD, Professor in Child Neurology and Developmental Pediatrics, 1st Department of Pediatrics, Aristotle University of Thessaloniki, 106 Egnatia str., 54622 Thessaloniki, Greece, tel/fax: +302310241845, e-mail: jeff@med.auth.gr
Introduction
Gaucher disease (GD) is the most common lysosomal storage disorder, known to be inherited in the autosomal recessive way1,2.It is caused by the deficiency of glucocerebrosidase leading to accumulation of glucosylceramide3-5.
Common clinical symptoms and manifestations include hepatomegaly, splenomegaly, hematological and various skeletal complications, whereas in the neuronopathic forms neurological complications of different severity and rate are also present. Almost all GD patients develop skeletal complications, consisting mainly of bone remodeling failure, osteopenia, osteoporosis, osteolytic lesions of various size, Ehrlenmeyer’s flask deformity, marrow infiltration and avascular necrosis. Substantial progress has been performed the last decade on the pathogenesis of bone changes in GD, which is currently attributed to multiple factors. Such factors include vascular compression due to increased intraosseous pressure, metabolic disturbance with increased bone resorption, cytokine and chemokine enhancement of osteoclast function, macrophage-derived factors’ effects, as well as interactions between Gaucher cells, osteoclasts, osteoblasts and mesenchymal cells6-9. Imaging methodologies for the evaluation of skeletal involvement are currently employed and provide accurate evaluation and staging of bone lesions in GD10. Practical limitations and increased costs of these imaging procedures have initiated the investigation of the use of biochemical markers for evaluation of osseous involvement11.
Enzyme replacement therapy (ERT) has changed the natural history of GD12.However, while some GD manifestations (anemia, thrombocytopenia, hepatosplenomegaly) rapidly improve with ERT, the skeletal tissue shows a slower response12-14.
Because Gaucher cells release proinflammatory cytokines and tartrate-resistant acid phosphatase, it has been hypothesized that complications result from a high turnover rate combined with high macrophage-mediated osteoclastic bone resorption8. However, reports studying biochemical markers of bone turnover have given variable results so far.
We performed a prospective study in a case-series of GD patients from Northern Greece under long-term ERT to investigate treatment effects on bone mineral density (BMD), biochemical marker levels and magnetic resonance imaging (MRI) findings, as well as possible relationships between them.
Patients and Methods
Seven GD type I patients (three males and four females) on ERT were enrolled in this prospective study. The diagnosis of GD was confirmed by demonstration of deficient glucocerebrosidase activity and genotyping. Mean age was 26.29 ± 15.34 years (range 7–47 years). Six patients were receiving ERT with 40-60 IU/kg of imiglucerase weekly for a mean period of 36 months prior to their enrollment in the study, while one patient started ERT after his inclusion.
Serum markers, macrophage inflammatory protein (MIP)-1a, MIP-1b, Interleukin (IL)-3, IL-6, IL-10, IL-12, osteoprotegerin (OPG), osteocalcin (BGP), as well as chitotriosidase, a marker of total disease burden, were investigated in this group of patients, at baseline (T0) and two years later. MIP-1a and MIP-1b levels in ethylenediaminetetraacetic acid (EDTA) plasma samples were measured by enzyme-linked immunosorbent assay (ELISA) (for MIP-1a, MIP-1b: R&D Systems, Inc., Minneapolis, US, for Osteoprotegerin: sRANKL BioVendor GmbH, Heidelberg, Germany, and for IL-6, IL-10, IL-3, IL-13: Osteocalcin Invitrogen, Life Technologies Corporation, Camarillo, CA). Chitotriosidase in plasma samples was measured by a standard enzyme activity assay. Serum OPG and BGP levels were assessed using commercial kits, based on ELISA methods, according to the instructions of the manufacturer.
BMD was measured using a dual-energy X-ray absorptiometry (DXA), technique using Cronos® bone densitometer (DMS, France) at two sites: the lumbar spine vertebrae (L2–L4) and the left proximal femur. For adult patients (older than 20 years) Z-score values were directly acquired from the scanner output, while for those aged 7 to 20 years the parameter was calculated normalizing crude BMD using the standard population proposed by Kelly et al15.According to the guidelines of the International Society of Clinical Densitometry (I.S.C.D.) a Z-score ≤2.0 was considered abnormal16,17. At the lumbar site, BMD was measured at the second, third and fourth lumbar vertebrae and the mean value expressed as grams per square centimeter (BMDs, g/cm2). At the proximal femur, BMD was measured at the femoral neck and the total hip. Results were also expressed as grams per square centimeter square (g/cm2). Z-scores were calculated based on BMD measurements of a normal age- and sex-matched Caucasian population18.
Quantitative ultrasound (QUS) is a bone health assessment technique, grown popular in recent years since its introduction in 1984. Compared to DXA, QUS is more widely accessible because of a portable device, low cost, and absence of ionizing radiation19. This technology has been used to determine the bone health status of women, men, children and, in certain cases, infants8. The ultrasound is a type of sound wave with a frequency exceeding the normal auditory range of humans (>20 kHz), while the frequency used in QUS usually lies between 200 kHz and 1.5 MHz19,20.
QUS measurements were performed using Omnisense® 7000 P (Sunlight Medical Ltd, Tel Aviv, Israel), equipped with a handheld probe, specifically designed to assess the axial speed of sound (SOS) along the surface of peripheral bones. Two sites on the non-dominant side were assessed: the distal third of the radius (SOSR) and the midshaft of the tibia (SOST). Calibration of the device was carried out on a daily basis using the manufacturer’s verification phantom, whereas repeatable positioning of the probe was achieved by the use of simple measuring gauges as instructed by the manufacturer of the device. Z-scores for SOS values were calculated according to normative data derived from a normal sex- and age-matched Greek population20.
Measures of QUS, as well as MRI of bones -representing bone marrow infiltration-, were investigated in this group of patients at baseline (T0) and subsequently two years later, and so was the relationship between these parameters.
The authors obtained ethical approval from hospital’s Bioethics Committee (protocol number 91, 1/6/2010). In addition, consent was obtained from all study subjects or their parents/legal representatives before study entry.
Statistical analysis
For statistical analysis and graphical demonstration, Excel® for Mac 2011 version 14.0.0 (Microsoft, Redmond, CA, USA) and the Statistical Package for Social Sciences (SPSS®) version 20 (SPSS IBM, Chicago, IL, USA) were employed. Results are presented as means ± standard deviation. The Shapiro-Wilk test was used to identify parameters with skewed distribution. In parameters with normal distribution, mean values were compared using paired Student t-test, whereas linear correlations were calculated with the Pearson’s correlation coefficient. In parameters with a skewed distribution, significance was assessed with Mann-Whitney test and Spearman’s correlation coefficient, respectively.
Results
All seven patients in this case series presented with a GD1 phenotype and were free of neurological signs. However, only five of them carried the common “neuroprotective” N370S mutation in homozygosity. One patient carried two heterozygous disease-causing mutations (N409S/L483P), and the seventh patient carried the L444P mutation in homozygosity – presenting, however, with a GD1 neurological-free phenotype. All patients had developed skeletal complications, consisting mainly of osteopenia and marrow infiltration. These changes predominantly affected long bones and the vertebrae. Failing of remodeling of the distal femora producing the characteristic “Erlenmeyer flask” appearance, a common skeletal abnormality in patients with GD, was already present in four patients of the cohort before initiation of ERT, while none of them developed additional skeletal complications while on ERT.
Two years after baseline, plasma chitotriosidase activity, MIP-1a and IL-6 levels were decreased in all patients (p =0.05 and p =0.03, respectively) (Table 1) (Figure 1, Figure 2). In contrast, other biochemical markers, such as OPG and BGP, were increased in all patients after two years of ERT therapy (p =0.04 and p =0.02, respectively) (Table 1) (Figure 3, Figure 4). Hip BMD demonstrated a progressive improvement in all patients after two years of treatment, according to the Z-score (p =0.05) (Table 1) (Figure 5).
Figure 1: Plasma levels of macrophage inflammatory protein (MIP)-1a were decreased in all patients after two years of enzyme replacement therapy.
Figure 2: Plasma levels of Interleukin-6 were decreased in all patients after two years of enzyme replacement therapy.
Figure 3: Plasma levels of Osteoprotegerin (OPG) were increased in all patients after two years of enzyme replacement therapy.
Figure 4: Plasma levels of Osteocalcin (BGP) were increased in all patients after two years of enzyme replacement therapy.
Figure 5: Bone mineral density (based on Z-score) showed a progressive improvement in all patients after two years of enzyme replacement therapy.
Regarding plasma levels of chemokines and chitotriosidase activity studied, a statistically significant correlation between the decrease in MIP-1a plasma levels and chitotriosidase could be demonstrated (Table 2) (Figure 6). Other markers (MIP-b, BGP, OPG) were weakly correlated with plasma chitotriosidase activity (Table 2). Additionally, decreased plasma levels of IL-6 strongly correlated with increased Z-score in DXA in all patients (Table 2) (Figure 7). The correlation of all serum markers with bone MRI findings was poor.
Figure 6: Decreased plasma levels of macrophage inflammatory protein (MIP)-1a and chitotriosidase were strongly correlated after two years of enzyme replacement therapy.
Figure 7: Decreased plasma levels of Interleukin-6 were strongly correlated with increased Z-score, in all patients, after two years of enzyme replacement therapy.
Discussion
GD is a lysosomal storage disorder affecting multiple organ systems1,2. Bone involvement in GD is known to be frequent. According to existing literature, it occurs in approximately 75 % of GD type 1 patients21,22.Pathophysiologically, bone marrow is infiltrated by lipid-laden macrophages, called Gaucher cells21,22.The skeletal manifestations of GD include a variety of bone pathologies due to the progressive glucocerebroside storage, changes of vascularity, and impaired bone remodeling. Bone manifestations in GD include bone infarcts, avascular bone necrosis, cortical thinning, lytic bone lesions, osteosclerosis, fractures due to osteopenia or osteoporosis22. Under ERT these pathologies are, at least, partly reversible22-24.Evaluation of response to enzyme replacement requires accurate, rapid and non-invasive monitoring of disease activity11,22,25-26. Changes in osteoclast and osteoblast activity can be measured by markers of bone metabolism. Different markers of bone turnover have been evaluated in patients on ERT11,22,25-26. These changes seem to be reversible after initiation of ERT due to an increase in bone formation and a decrease of bone resorption26,27.
The extent of bone involvement in GD cannot be estimated only by clinical examination. Imaging of bone and bone marrow in GD aims to evaluate the skeletal complications of the disease and to follow up treatment effects on bone disease. MRI is the method of choice to evaluate the extent of bone disease prior to therapy and during follow-up in patients on ERT22,28.
With regards to the underlying pathology, GD is regarded as a chronic inflammatory disorder, with macrophages producing proinflammatory cytokines that could promote bone resorption. Several clinical manifestations, such as gammopathies, predisposition to infections, and lymphoid malignancies, suggest a more diverse dysregulation of the immune system, lying beyond the macrophage6,7,29-30.Previous studies have evaluated changes in several enzymatic biomarkers and newly-described chemokines6,7,29-30. These changes may explain the chronic inflammatory reaction and the increased incidence of lymphoid malignancies that have been repeatedly reported among GD patients, and seem to be reversible after initiation of ERT6,7,29-30.In particular, variable elevations of proinflammatory cytokines IL-6, IL-8, IL-1, osteoclast-activating cytokines such as macrophage inflammatory protein-1, and anti-inflammatory cytokines and factors such as IL-10 have been reported. Various investigations revealed that plasma levels of the chemokines MIP-1a and MIP-1b are markedly increased in GD patients. Particularly plasma MIP-1b levels tend to be higher in untreated patients with skeletal disease compared to untreated patients without skeletal disease8,31.In patients under ERT therapy, the plasma levels of MIP-1b have been reported to be lower after some years of therapy31. The most consistent finding is a difference in osteocalcin levels between patients who experienced bone complications and those who did not8.
In the present study, serum chitotriosidase activity, MIP-a and IL-6 levels were decreased in all GD patients. Other biochemical markers such as OPG and BGP levels were increased in all patients after two years of ERT therapy. Decreased plasma levels of both MIP-1a and chitotriosidase were strongly correlated. Additionally, decreased plasma levels of IL-6 were strongly correlated with increased Z-score, in all GD patients. Bone mineral density showed a progressive improvement in all patients after two years of ERT based on the Z-score. No correlation was found between QUS measurements and studied markers in GD patients under ERT.
The results of the study suggest that biochemical markers can illustrate various metabolic changes occurring in the osseous tissue in GD patients. Therefore, biochemical markers could be used in the evaluation of skeletal involvement in type 1 GD patients. In particular, osteocalcin and MIP-a are useful tools, when combined with imaging methodologies, in the evaluation of skeletal involvement in type 1 GD during ERT – and their monitoring can provide insights into the evolution of bone lesions.
Limitations of the current study are the small number of patients, as well as the fact that six out of the seven patients were already on ERT at baseline – probably not allowing the observation of most prominent alterations of the biomarkers studied. Future research should be addressed at larger GD cohorts and focus on the applications and usefulness of biomarkers in the evaluation of osseous tissue response to specific treatments in patients with GD.
Conflict of interest
The present study was funded by Genzyme. In addition, EV, ME and DIZ have received travel and research grants from Genzyme.
Acknowledgement
We would like to thank Eleni Michelakaki, Evangelia Dimitriou and Marina Moraitou, Department of Enzymology, Institute of Child Health, Athens, Greece, as well as Anne Katrine Giese, MD, and Arndt Rolfs, MD, Albert-Kossel-Institute for Neurodegeneration, Rostock, Germany, for their help in performing molecular studies in the study patients. Anna Taparkou, Biologist, 1st Department of Pediatrics AUTH, we thank for her help in performing biochemical measurements. Athanassia Anastasiou, Radiologist, Hippokratio General Hospital of Thessaloniki, we thank for the interpretation of the MRI findings. Helen Michelakakis, Department of Enzymology, Institute of Child Health, Athens, we would like to thank for chitotriosidase measurements. Sofia Vakalopoulou and Eudoxia Mandala, Hematologists, Hippokratio General Hospital of Thessaloniki, we thank for providing clinical data of their patients. Nikolaos Gombakis, Pediatrician-Hematologist, 1st Department of Pediatrics AUTH, we would like to thank for his help in the initial designing of the study. Finally, we would like to thank Genzyme for funding the study.
References
1. Brady RO, Kanfer JN, Bradley RM, Shapiro D. Demonstration of a deficiency of glucocerebroside-cleaving enzyme in Gaucher’s disease. J Clin Invest. 1966; 45: 1112-1115.
2. Beutler E, Grabowski GA. Gaucher disease. Scriver C, Beaudet AL, Sly WS, Valle D (eds). The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York, 2001, 3635-3668.
3. Meikle PJ, Hopwood JJ, Clague AE, Carey WF. Prevalence of lysosomal storage disorders. JAMA. 1999; 281: 249-254.
4. Poorthuis BJ, Wevers RA, Kleijer WJ, Groener JE, de Jong JG, van Weely S, et al. The frequency of lysosomal storage diseases in The Netherlands. Hum Genet. 1999; 105: 151-156.
5. Pinto R, Caseiro C, Lemos M, Lopes L, Fontes A, Ribeiro H, et al. Prevalence of lysosomal storage diseases in Portugal. Eur J Hum Genet. 2004; 12: 87-92.
6. Liu J, Halene S, Yang M, Iqbal J, Yang R, Mehal WZ, Chuang WL, et al. Gaucher disease gene GBA functions in immune regulation. Proc Natl Acad Sci U S A. 2012; 109: 10018-10023.
7. Sotiropoulos C, Theodorou G, Repa C, Marinakis T, Verigou E, Solomou E, et al. Severe impairment of regulatory T-cells and Th1-lymphocyte polarization in patients with Gaucher disease. JIMD Rep. 2015; 18: 107-115.
8. van Dussen L, Lips P, Everts VE, Bravenboer N, Jansen ID, Groener JE, et al. Markers of bone turnover in Gaucher disease: modeling the evolution of bone disease. J Clin Endocrinol Metab. 2011; 96: 2194-2205.
9. Campeau PM, Rafei M, Boivin MN, Sun Y, Grabowski GA, Galipeau J. Characterization of Gaucher disease bone marrow mesenchymal stromal cells reveals an altered inflammatory secretome. Blood. 2009; 114: 3181-3190.
10. Komninaka V, Kolomodi D, Christoulas D, Marinakis T, Papatheodorou A, Repa K, et al. Evaluation of bone involvement in patients with Gaucher disease: a semi-quantitative magnetic resonance imaging method (using ROI estimation of bone lesion) as an alternative method to semi-quantitative methods used so far. Eur J Haematol. 2015; 95: 342-351.
11. Drugan C, Jebeleanu G, Grigorescu-Sido P, Caillaud C, Craciun AM. Biochemical markers of bone turnover as tools in the evaluation of skeletal involvement in patients with type 1 Gaucher disease. Blood Cells Mol Dis. 2002; 28: 13-20.
12. Weinreb NJ, Charrow J, Andersson HC, Kaplan P, Kolodny EH, Mistry P, et al. Effectiveness of enzyme replacement therapy in 1028 patients with type 1 Gaucher disease after 2 to 5years of treatment: a report from the Gaucher Registry. Am J Med. 2002; 113: 112-119.
13. Poll LW, Koch JA, Willers R, Aerts H, Scherer A, Häussinger D, et al. Correlation of bone marrow response with hematological, biochemical, and visceral responses to enzyme replacement therapy of nonneuronopathic (type 1) Gaucher disease in 30 adult patients. Blood Cells Mol Dis. 2002; 28: 209-220.
14. Ciana G, Addobbati R, Tamaro G, Leopaldi A, Nevyjel M, Ronfani L, et al. Gaucher disease and bone: laboratory and skeletal mineral density variations during a long period of enzyme replacement therapy. J Inherit Metab Dis. 2005; 28: 723-732.
15. Kelly TL, Specker BL, Binkely T, Zemel BS, Leonard MB, Kalkwarf HJ, et al. Pediatric BMD reference database for US. white children. Bone. 2005; 36: S30.
16. Kalkwarf HJ, Zemel BS, Gilsanz V, Lappe JM, Horlick M, Oberfield S, et al. The bone mineral density in childhood study: bone mineral content and density according to age, sex, and race. J Clin Endocrinol Metab. 2007; 92: 2087-2099.
17. Lewiecki EM, Gordon CM, Baim S, Binkley N, Bilezikian JP, Kendler DL, et al. Special report on the 2007 adult and pediatric Position Development Conferences of the International Society for Clinical Densitometry. Osteoporos Int. 2008; 19: 1369-1378.
18. Fiore CE, Barone R, Pennisi P, Pavone V, Riccobene S. Bone ultrasonometry, bone density, and bone turnover markers in type 1 Gaucher disease. J Bone Miner Metab. 2002; 20: 24-38.
19. Chin KY, Ima-Nirwana S. Calcaneal quantitative ultrasound as a determinant of bone health stutus: what properties of bone does it reflect? Int J Med Sci. 2013; 10: 1778-1783.
20. Christoforidis A, Papadopoulou E, Dimitriadou M, Stilpnopoulou D, Gkogka C, Katzos G, et al. Reference values for quantitative ultrasonography (QUS) of radius and tibia in healthy greek pediatric population: clinical correlations. J Clin Densitom. 2009; 12: 360-368.
21. Germain DP. Gaucher’s disease: a paradigm for interventional genetics. Clin Genet. 2004; 65: 77-86.
22. Mikosch P, Hughes D. An overview on bone manifestations in Gaucher disease. Wien Med Wochenschr. 2010; 160: 609-624.
23. Yoshino M, Watanabe Y, Tokunaga Y, Harada E, Fujii C, Numata S, et al. Roles of specific cytokines in bone remodelling and hematopoiesis in Gaucher disease. Pediatr Int. 2007; 49: 959-965.
24. Sipos W, Pietschmann P, Rauner M, Kerschan-Schindl K, Patsch J. Pathophysiology of osteoporosis. Wien Med Wochenschr. 2009; 159: 230-234.
25. Parisi MS, Mastaglia SR, Bagur A, Goldstein G, Zeni SN, Oliveri B. Body composition and bone metabolism in young Gaucher disease type I patients treated with imiglucerase. Eur J Med Res. 2008; 13: 31-38.
26. Deegan PB, Moran MT, McFarlane I, Schofield JP, Boot RG, Aerts JM, et al. Clinical evaluation of chemokine and enzymatic biomarkers of Gaucher disease. Blood Cells Mol Dis. 2005; 35: 259-267.
27. Sims KB, Pastores GM, Weinreb NJ, Barranger J, Rosenbloom BE, Packman S. Improvement of bone disease by imiglucerase (Cerecyme) therapy in patients with skeletal manifestations of type 1 Gaucher disease: results of a 48-month longitudinal cohort study. Clin Genet. 2008; 73: 430-440.
28. Vom Dahl S, Poll L, Di Rocco M, Ciana G, Denes C, Mariani G, et al. Evidence-based recommendations for monitoring bone disease and the response to enzyme replacement therapy in Gaucher patients. Curr Med Res Opin. 2006; 22: 1045-1064.
29. Micheva I, Marinakis T, Repa C, Kouraklis-Symeonidis A, Vlacha V, Anagnostopoulos N, et al. Dendritic cells in patients with type I Gaucher disease are decreased in number but functionally normal. Blood Cells Mol Dis. 2006; 36: 298-307.
30. Rogowski O, Shapira I, Zimran A, Zeltser D, Elstein D, Attias D, et al. Automated system to detect low-grade underlying inflammatory profile: Gaucher disease as a model. Blood Cells Mol Dis. 2005; 34: 26-29.
31. van Breemen MJ, de Fost M, Voerman JS, Laman JD, Boot RG, Maas M, et al. Increased plasma macrophage inflammatory protein (MIP)-1alpha and MIP-1beta levels in type 1 Gaucher disease. Biochim Biophys Acta. 2007; 1772: 788-796.