A Lysosomal storage disease mimicking common paediatric symptoms?

Transcription

1 A Lysosomal storage disease mimicking common paediatric symptoms? Dominique Roland 1, François Eyskens 2 1 Institut de Pathologie et de Génétique, Génétique Humaine, Centre Agréé des maladies héréditaires du métabolisme, 25, Avenue Georges Lemaître, 6041 Gosselies (Charleroi) 2 Department of Paediatrics, Centre for Inherited Metabolic diseases CEMA, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem dominique.roland@ipg.be Campaign Metabolic diseases Abstract Introduction: Recently the Belgian recommendations for diagnosis, treatment and monitoring of Gaucher disease (GD) that already dated from 2004, were revised. This expert opinion was inspired by a working group focusing on a profound revising of the former Guidelines according to the recent knowledge. Increasing awareness of this rare lysosomal storage disease among paediatrician is a need in order to anticipate early diagnosis in paediatric patients, to avoid a time consuming diagnostic odyssey and to prevent irreversible organ damages. Methodology: Based on a clinical history to illustrate the subject, we wanted to emphasise the clinical and biochemical diagnosis of this rare disease in paediatric population as it is widely recognised that an early treatment in lysosomal storage diseases is the clue to a better outcome. Enzyme replacement therapy is the treatment of choice in children under 18 years of age. In adults, new oral therapies are now available and could be implemented in children in the future. Discussion: This text based on Belgian expert opinion aims at paediatricians and treating physicians with respect to the necessary assessment(s) at diagnosis for GD, the treatment according to disease severity as well as the monitoring of the patient during the follow up. It reflects not only international recommendations and literature but also the knowledge and expertise of Belgian experts. Key words Gaucher Disease, glucocerebrosidase deficiency, lysosomal storage disease, hepatomegaly, enzyme replacement therapy. Introduction Gaucher disease (GD) is the most common lysosomal storage disease (LSD), a class of inborn errors of metabolism implicating the defect of one of 50 enzymes contained in the lysosome. Although GD is a rare disease, some characteristics could be misinterpreted during paediatric period with frequent symptoms as anaemia, fatigue and bone pain delaying a proper diagnosis and a correct management. In this paper, we would like to highlight the signs and symptoms in paediatric patients and present the necessary assessment(s) at diagnosis, the right time for treatment according disease severity as well as the recommended monitoring of patients during the follow up. These considerations reflect not only international recommendations and literature but also the knowledge and expertise of Belgian experts. GD is due to a decreased levels of activity of the lysosomal enzyme glucocerebrosidase or acid-ß glucosidase, a membrane-associated lysosomal enzyme. This deficiency results in a decreased breakdown of the glycosphingolipid glucocerebroside, which accumulates in the lysosomes of the monocytemacrophage system 1,2. This accumulation results in various symptoms such as anaemia, thrombocytopenia, splenomegaly, hepatomegaly and a complex bone disease which are the result of infiltration of spleen, liver and bone marrow by Gaucher cells. The incidence of GD varies between 1/ in Central Europe and 1/2.000 in some non- European countries such as Israel. The acute and chronic neuronopathic forms, also known as types 2 and 3 GD, are much rarer and account for 5-10% of all Gaucher patients in Central Europe compared with the more frequent non-neuronopathic form also known as GD type 1 1,3,4,5,6,7. Three clinical forms have been defined, based on absence (type 1) or presence (type 2 -acute, infantile-neonatal and type 3 subacute, chronic, childhood) of neurological signs 8. These include myoclonic epilepsy, oculomotor apraxia and progressive neurodegeneration 1,4,8. Nevertheless, as in most LSDs, the clinical picture is more a phenotypic continuum from a perinatal lethal neuronopathic form to an asymptomatic chronic, non neuronopathic form; however, this classification is useful when talking about management options and genetic counselling. Clinical variability of disease expression among siblings underscores our lack of understanding and could be explained by genetic modifiers, however this theory still has to be consolidated. To illustrate the complexity of diagnosis and management of a patient with GD, we report on the history of a young Turkish girl and then expose the Belgian recommendations for diagnosis and management of paediatric patients. The recent update was achieved by the collaboration of several Belgian experts. Material Case report A Turkish girl, first child of consanguineous parents, was referred to a Belgian hospital for a second opinion around the question of splenectomy at the age of 9 years. Besides an enormous enlarged spleen, the clinical examination showed an extreme growth retardation (length 10 cm below third centile for age); the clinical neurologic examination was normal. Laboratory results revealed a thrombocytopenia /mm3 accompanied by a slight anaemia and leukopenia. CT-scan of the abdomen showed a hepatosplenomegaly (liver 1134 cc; spleen 1218 cc). The diagnosis of GD was confirmed by a low enzymatic activity of ß-glucocerebrosidase in lymphocytes (1.9 µmol/g/h - Range Value: ) and an elevated plasma chitotriosidase activity nmol/ml/h. DNA-analysis demonstrated that the patient was homozygous for the L444P mutation and thus affected by the neuronopathic type 3 GD. A supranuclear ophtalmoplegia however became clear by neuro-ophtalmologic examination only one year after the initial diagnosis. She started up with enzyme replacement therapy (ERT), imiglucerase (60 U/kg body weight/2 weeks; this dose was adjusted towards 120 U/kg/2 weeks when the neurological signs became apparent). After 12 months of treatment by ERT, the thrombocytopenia stabilized at /mm 3, the liver volume normalized, the spleen volume reduced to 800 cc and the plasma chitotriosidase diminished to nmol/ml/h. The patient had a catch up growth, a normal puberty spurt and reached her target height at the age of 15 years. DXA scan of bone after puberty indicated an osteopenia at the lumbar vertebrae (Z-score -1.35). Despite ERT with very high dose, the neurological 16

2 state further deteriorated with development of hearing problems, vestibular dysfunction, neurologic regression (Full scale IQ 60, performance <verbal IQ) and epilepsy (generalized convulsions). Methods Recommendations have been recently discussed amongst Belgian experts in LSD and new experts opinion have been edited concerning clinical and biochemical diagnosis, management and therapy for Gaucher patients. The clinical diagnosis First symptoms in a child can be as subtle as chronic fatigue due to anaemia, bruising/bleeding or growth/puberty delay. There is a wide variability in the clinical presentation of type 1 GD, with occasional discordance between visceral and bone involvement. Early signs and symptoms that a primary care provider may observe also include abdominal pain. As the disease progresses, common features may include hepatosplenomegaly. Pronounced splenomegaly is nearly universal in symptomatic children while bone disease characterized by osteopenia, osteonecrosis, and bone pain is present in the advanced stage. Children are less likely to present with osteonecrosis or pathologic fracture than adults, but often have growth retardation and chronic bone pain related to growth. Bone X rays are useful in the initial assessment to identify bone deformities but are not sensitive to visualize changes in bone marrow which are better seen on bone MRI and QSCI (Quantitative chemical shift imaging), a quantitative MRI technique measuring fat content in the axial bone marrow. This last technic is now available in Belgium in a specialized radiologic centre. Serious pulmonary disease occurs mostly in children with a more severe course of GD. Recurrent pulmonary infections and progressive dyspnoea with an evolution to respiratory insufficiency are the hallmark of a severe disease. In these cases, infiltration of the alveolar spaces and/or the inter- and intralobular septa by Gaucher cells leads to air space and/or interstitial disease. Pulmonary hypertension and intrapulmonary shunts related to the hepatopulmonary syndrome have also been reported. Lung evaluation should include imaging studies to diagnose air space and/or interstitial disease (X rays and CT), and pulmonary function test (PFT). Finally, GD patients should be investigated through non-invasive tests such as echocardiography to evaluate pulmonary hypertension and intrapulmonary shunts 9,10. GD is a multisystemic disease. Every suspected or newly diagnosed paediatric patient should undergo an initial assessment which is given in the Table 1. Initial evaluation of the patient evaluates the impact of the lysosomal disease on the different organ systems involved (bone marrow, liver, spleen, lungs, central nervous system). In case of identification of neurologic symptoms (or a least one L444P mutation) during the evaluation, the child should undergo a neuroophthalmologic examination to exclude a supranuclear gaze palsy (SNGP) which is a characteristic sign of a neurologic form of GD type 2 or 3 (Table 2). Horizontal saccadic eye movement abnormalities are the hallmark of a severe systemic involvement in neurologic GD. Saccade initiation failure are observed in horizontal eye movements but also (and to a lesser extend) in vertical eye movements, as a result of cerebral impairment 2. Enzymatic activity, biomarkers and molecular diagnosis The diagnosis of Gaucher disease is classically confirmed by the measurement of the activity of the enzyme glucocerebrosidase in leukocytes or fibroblasts1. Dried blood spot (DBS) can be used as screening method. If DBS is negative but clinical symptoms is suggestive of GD, do retest enzyme activity on peripheral blood cells. On the other hand, false-positive rate is high for DBS. An increased plasma chitotriosidase activity, secreted by activated human macrophages is present in various lysosomal storage diseases. In GD, this protein is used as biomarker for the diagnosis as well as for therapeutic monitoring of non chitotriosidase deficient patients 11,12. But more than 40 % of Caucasian population display an heterozygous or homozygous common mutation in the chitotriosidase gene CHIT1 gene responsible for chitotriosidase deficiency in about 5-6% of individuals, including GD patients 12,13. In carriers for the common null mutation c.1049_1072 dup24 in CHIT1 gene, chitotriosidase activity falls to 50% of normal and is absent or very low in homozygous state. In case of low chitotriosidase, mutation search in CHIT1 is recommended and chitotriosidase activity cannot be used to monitor the patient. Other biomarkers than chitotriosidase can be used in children and adults (e.g. CCL18 /PARC). These biomarkers however are not specific for GD. Recently a Tabel 1: Evaluation for a suspected or newly diagnosed patient General Liver Bone Pulmonary Genetic counselling - Clinical symptoms : bleeding symptoms, hepatosplenomegaly evaluation and neurologic exam - Growth/puberty assessment - Laboratory findings including : blood count, coagulation tests, hepatic function, transaminases, renal function, serum iron, ferritin, TIBC, chitotriosidase, Vitamin D status - Ultrasound, non-invasive elastography (Fibroscan) : screening for advanced fibrosis or cirrhosis; esp. in splenectomised patients - Advanced liver fibrosis and cirrhosis : referral to a Hepatology clinic Work-up for portal hypertension and associated complications; Hepatocellular carcinoma (HCC) screening by highend-ultrasound, serum alpha fetoprotein and transient elastography (in adults) - X-rays of the femora, pelvis, full spine and any other symptomatic bone - Bone MRI of the lower extremities of lumbar spine, femur, tibia, and humerus (depending on symptoms) + BMB or Dusseldorf Gaucher score *. - QCSI (see text). - Dual-Energy X-ray Absorptiometry (DXA) to evaluate Bone Mineral Density (BMD) - Signs of Cor pulmonale : Doppler Echocardiography, lung X ray, high-resolution CT of the lungs, oxygen saturation, right-heart catheterization; esp. in splenectomised (female) patients. - In case of unproductive cough, dyspnoea, decreased exercise tolerance : Doppler echocardiography, lungs X ray, pulmonary function tests, chest high resolution CT - Family evaluation : both parents and especially all siblings should be tested for carrier or affected status when the mutations are identified in the index patient. * Semi-quantitative methods of bone marrow scoring (BMB or Dusseldorf Gaucher score) evaluate both on MRI, the lumbar spine and femoral for the first method and the extent of Gaucher bone marrow infiltration in the lower extremities for the second. A higher BMB score means more severe bone marrow involvement. Tabel 2: Acute and chronic neuronopathic forms. Additional considerations in diagnosis and monitoring Genetic testing Acute form, type 2 Chronic form, type 3 Follow up when treated What should be tested - Determination of genotype : some patients are at high risk of developing neuronopathic disease (e.g. homozygote for L444P or D409H mutations) - Clinical-neurological examination - Examination of eye movements to identify supranuclear gaze palsy (SNGP) - Electro-Encephalogram (EEG); if required Auditory Brainstem Evoked potentials (AEP) - No ERT/Supportive/palliative follow-up - Neurological examination - Examination of eye movements to identify SNGP - MRI, EEG, AEP, vestibular testing - Psychological examination including testing for : IQ, attention memory, apraxia - Thorax CT if abnormalities on X-ray - BMD (DXA) - Every 3 months: clinical and neurological examination, including eye movements - Every 3 months: haematology, coagulation test,.. and chitotriosidase - Every 6 months: thorax X-ray (CT thorax if abnormalities) - Every 12 months: BMD (DXA) - EEG if epileptic seizures occur, every 12 months, AEP and psychometry - Clinic neurology : development of myoclonic seizures is a bad prognostic factor 17

3 highly sensitive and specific biomarker for diagnosis and follow-up monitoring of GD patients has been found and described, namely glucosylsphingosine or lyso-gl1, in plasma of GD patients by LC-tandem mass spectrometry. This one is considered a key biomarker of GD 14,15. As GD is inherited as an autosomal recessive disorder, a molecular genetic testing on peripheral blood cells, fibroblasts or from mouth swabs can be used to confirm the diagnosis and further to confirm inheritance in the family. Recurrent mutations are identified through genetic analysis. N370S, L444P, 84GG, IVS2+1G>A pathogenic variant in GBA1 gene represent 90% and % of the mutant alleles in Ashkenazi Jewish individuals and non-jewish individuals with GD type 1 respectively. A negative screening for these common mutations, however, does not exclude GD. Sequencing the entire GBA1 gene is therefore strongly recommended when clinical suspicion is high. Over 300 pathogenic mutations are known to cause GD 4,5,6,7,8. In non-neurologic GD type 1, N370S mutation has been usually recognized with a milder phenotype or clinical course. Patients with at least one N370S allele do not develop neurologic symptoms. Asymptomatic patients have also been described with the N370S/N370S genotype. The frequency of the N370S allele is higher among the Jewish (carrier frequency 1/17), Portuguese and Spanish population 1,5. Homozygosity for L444P is the genotype most frequently associated with neuronopathic GD and presents itself as a severe pathology with the probability of neurological impairment as observed in the Turkish patient described in this article. L444P mutation is more frequent in the Swedish population. In general, patients displaying at least one L444P pathogenic mutation are at risk of developing neurologic impairment. Genotype/phenotype correlation is not completely accurate but genotype characterization provides prognostic considerations, as the residual enzyme activity level does not allow to predict the severity of the disease 8. In at-risk families, a pre-conceptional molecular testing and a prenatal diagnosis could be offer according to the wish of the family and taking into account the severity of the identified mutation in the index cases. Clinical criteria for individualized treatment in adults and children: Classification according to disease severity allows an individual fine-tuned therapeutic approach. The most important investigations that should be performed in every patient with GD form the base of such a classification. Some patient have very few symptoms which do not require specific treatment but only regular monitoring and others have a clear multisystemic disease with complications requiring high dose treatment. Before the advent of ERT, splenectomy was performed to treat thrombocytopenia and reduce liver volume in GD type 1. However, this procedure has shown to lead to more aggressive bone disease, pulmonary hypertension, liver disease, predisposition to malignancy, increased accumulation of Gaucher lipids in the brains of neuronopathic GD patients and is no more recommended 16,17. The disease severity classification and recommended dose therapy adapted to the severity of the disease are described in Table 3. Additional clinical criteria for severe involvement indicating high dose ERT therapy are summarized in Table 4. ERT is an effective and safe treatment for the nonneurological symptoms in the chronic neuronopathic form of GD type 3 but is unsuccessful in the treatment of the neurological symptoms which are prominent in the acute neuronopathic form of GD type 2. All recommendations for initial dosage should be individually adjusted based on clinical response and achievement of therapeutic goals. Well-described therapeutic goals are published in several guidelines 21,22. The recommended therapeutic management described is restricted to ERT in children. In adults however there is a place for the new oral therapy, eliglustat that can be given as a first-line medication in moderate and severe affected GD disease patients 23. A life-long, intravenous replacement of a human enzyme ß-glucocerebrosidase analogue produced by recombinant DNA technology has now been available for more than 20 years (imiglucerase) and for 5 years (velaglucerase) 24, 25,26,27,28,29. Enzyme Replacement Therapy (ERT) is given at regular intervals, once every 2 weeks. If this treatment is begun early enough, in a sufficiently high dose, it Tabel 3: Children (< 18 years) with Gaucher s disease type I : Criteria for clinical classification based on disease severity and recommended dosis for ERT Without clinical disease With clinical disease Mild Moderate Severe Haemoglobin (g/dl) 2gr/dl below lower limit for age > 3 gr/dl below lower limit for age Platelets (/mm 3 ) > < Liver size MN* (MRI or CT measures) < > 2.5 Spleen size MN* (MRI or CT measures) < > 15 Skeletal involvement symptoms - - (Chronic) bone pain, bone crisis (commonly misdiagnosed as osteomyelitis) Plain bone X ray Erlenmeyer flask deformity Asymptomatic areas of avascular necrosis Avascular necrosis, pathological fractures Bone MRI (> 4-6 years of age) Normal/slight decrease in signal intensity T1 Severe decrease in signal intensity T1 Severe decrease in signal intensity T1 Dual Energy X-Ray Absorptiometry (DXA) (>5 y-especially during puberty) Bone marrow fat fraction (Ff) Method : QCSI** > SD at lumbar spine L1 L4-1.5 > X >-2 SD at lumbar spine L1 L4 < - 2 SD at lumbar spine L1 L4 Ff > 23 % (normal population : 27% < Ff < 55%) Ff < 23% Ff < 23 % Growth Mild growth retardation <-1 SD/> - 2SD*** Severe growth retardation > -2SD*** Chitotriosidase (nmol/hr/ml) < > > Therapy Watchful waiting**** 60 U/kg***** 60 U/kg***** * ** *** **** ***** MN : Multiple of normal size (according to age) QCSI : Quantitative Chemical Shift Imaging. SD : Standard deviations from the Mid Parental Height Because reimbursement criteria in Belgium currently do not allow treatment in these cases. Because bone mass improvement by ERT is most effective in younger patients, suggesting a therapeutic window for best efficacy in paediatrics with high-dosage therapy in this critical period when peak bone mineral density is accrued 18 18

4 Tabel 4: Monitoring of clinical and radiologic parameters under ERT treatment Haematology Liver Bone Pulmonary Monitoring Rare complications and comorbidities usually leads to a significant improvement of hepatosplenomegaly, hematologic parameters and bone disease and various laboratory-biochemical changes. This results in a considerable improvement of patient s general condition/health status and quality of life. While this therapy is highly effective, such chronic treatment is a burden on the patient 23,24,25, 28,29,30,31,32. An oral therapy eliglustat which acts through substrate reduction (SRT) is available since 2016 in Belgium as an alternative treatment of adult patients affected by GD type 1. Testing for CYP2D6 pharmacogenetics is mandatory in order to determine patients who can be treated with this medication and the dose they can receive (slow versus fast metabolizers). Prescribers also need to be aware of its complex cytochrome P450 metabolism that complicates the concomitant use of some medications 22,33,34,35. A former SRT miglustat is no longer prescribed in Belgium because of its inferior efficacy and more frequent side effects (gastrointestinal, neurologic) 36,37. Other treatments are also effective in patients management: orthopaedic surgeryincluding protheses, adjunctive medications (pain, osteoporosis), physiotherapy, physical exercise, anti-epileptic treatment, psychologic support, heart surgeryvalve replacement, medication for pulmonary hypertension, endocrinology,... This list reminds us that GD is a severe multisystemic disease and patients should be treated in close collaboration with a reference center for inherited metabolic diseases and a multidisciplinary team. Conclusion What should be tested - Blood count: 1 x/3 months for 2 years - Danger : Hb level drops 1,5 g/dl and/or platelet count drops > 25% below initial value or below /mm3 when repeated twice at 2 weeks interval and/or clinically onset of bruising or bleeding - Every year : clinical evaluation, ultrasound - Every 2 3 years : elastography by fibroscan of liver and spleen or ARF1 (acoustic radiation force intensity); when abnormal : closer surveillance is indicated - X-ray examination repeated only when there is a clinical indication19 - Panoramic radiograph of the mandible in case of jawbone involvement - 99 mtc-methylene diphosphonate (99 mtc-mdp) bone scintigraphy, as an alternative to MRI, be used in the discrimination of osteomyelitis and AVN if performed 72 h after clinical onset. Scintigraphy can also be used for the investigation of occult fractures or the evaluation of loosening of hip joint prostheses, in which case 3-phase bone scintigraphy should be applied. - If treated for bone involvement: QCSI (after 6 months) and/or MRI (after 12 months) - Bone pathology: 1x/2 years (6 8 y) MRI or QCSI - Every year: BMD (if abnormal) - Every 3 4 years: extended skeletal survey in patients with bone manifestations at initial diagnosis 20 Only in case of pulmonary disease: interstitial lung disease; pulmonary hypertension - Once a year clinical evaluation, Doppler Echocardiography, lung X-ray, lungs high resolution CT - Adjuvant therapy in combination with ERT Every pulmonary involvement = severe disease - Polyclonal gammopathy; lymphoma; renal cell carcinoma; Gaucheroma (pseudotumor); iliopsoas bleeding; gall stones; fibromyalgia; amyloidosis, neuronopathic pain (small fibre disease), peripheral neuropathy, metabolic syndrome, Parkinson (mainly described in adults) In case of symptoms/clinical signs mimicking Gaucher disease (as chronic anaemia, thrombocytopenia, hypersplenism, retarded growth,...), paediatrician should be aware of excluding this rare disease as effective treatments are available for children. First symptoms or a moderate course of GD could not be obviously diagnosed at first as being linked to a GD by a non-aware practitioner. Furthermore, this rare lysosomal storage disease is probably underdiagnosed in case of moderate symptoms. Too mildly affected patients would probably never come to medical attention or be embarked for a long diagnostic odyssey 38. In case of a family history of GD or belonging to an at-risk group (as Askenazi Jewish genetic origin), children with suspect symptoms should be screened for Gaucher disease by a simple enzymatic test (on leukocytes or dried blood test) and further confirmed by GBA1 gene molecular sequencing if the enzyme activity is decreased. Although it has been recognised that a N370S genotype preclude a mild phenotype or a less severe disease, no one can extrapolate if the child with one or two N370S will remain asymptomatic lifelong. Identifying a child in a presymptomatic or mild state brings the advantage to put in place the right treatment early enough to avoid irreversible damage as bone complications, lung infiltration, chronic hypersplenism and poor growth/delayed puberty 38. Guidelines are available in the international litterature and updated Belgian expert opinion for diagnosis, treatment and monitoring of Gaucher disease is now available on the website of Metabolics, the belgian scientific association involved in Inborn Errors of Metabolism ( These guidelines can be of help in establishing the correct diagnosis at the appropriate time. REFERENCES: 1. Beutler E, Grabowski GA. Gaucher disease. In: Scriver C, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York: McGraw-Hill; 2001: Benko W, Ries M, Wiggs EA, Brady RO, Schiffmann R, et al. (2011) The Saccadic and Neurological Deficits in Type 3 Gaucher Disease. PLoS ONE 6(7):e22410.doi: / journal.pone Rolfs A et al. Epidemiologic data in M. Gaucher disease in the Caucasian population- usage of a simple ELISA-detection based PCR-format for the screening of the most frequent mutations. Biotechniques Goldblatt J. Type I Gaucher disease. J Med Genet 1988; 25(6): Zimran A, Gelbart T, Westwood B, Grabowski GA, Beutler E. High frequency of the Gaucher disease mutation at nucleotide 1226 among Ashkenazi Jews. Am J Hum Genet 1991; 49(4): Charrow J et al. The Gaucher registry-demographics and disease characteristics of 1698 patients with Gaucher disease. Arch Int Med 2000 Oct 9; 160: LaMarca ME, Scott CR, Sidransky E. Gaucher disease: mutation and polymorphism spectrum in the glucocerebrosidase gene (GBA). Hum Mutat 2008;29(5): GM Pastores and DA Hughes. Gaucher disease In Pagon RA, Adam MP, Ardinger HH, Wallace SE, Ameyima A, Bean LJH, Bird TD, Ledbetter N, Mefford HC, Smith RJH, Stephends K, Eds. GeneReviews (internet) Seattle (WA): University of Washington, Seattle; Jul 27 (updated 2015 Feb 26). 9. F Santamaria, G Parenti et al. Pulmonary manifestations of Gaucher disase: an increased risk for L444P homozygotes? Am J Respir Crit Care Med 1998; 157(3 PT1): P Mistry, S. Sirrs et al. Pulmonary hypertension in type 1 Gaucher s disease: genetic and epigenetic determinants of phenotype and response to therapy. Mol Genet Metab 2002; 77 (1-2): CE Hollak, S van Weely, M H van Oers, and J M Aerts. Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease. J Clin Invest. 1994; 93(3): Grace ME, Balwani M, Nazarenko I, Prakash-Cheng A, Desnick RJ. Type 1 Gaucher disease : null and hypomorphic novel chitotriosidase mutations-implications for diagnosis and theraupeutic monitoring. Human Mutat 2007;28(9) : Van Dussen L, Hendriks EJ, Groener JE, Boot RG, Hollak CE, Aerts JM. Value of plasma chitotriosidase to assess non-neuronopathic Gaucher disease severity and progression in the era of enzyme replacement therapy. J Inherit Metab Dis ; 37(6): Rolfs A, Giese A-K, Grittner U et al. Glucosylsphingosine is a Higly Sensitive and Specific Biomarker of Primary Diagnostic n Follow-up Monitoring in Gaucher Disease in a Non- Jewish, Caucasian Cohort of Gaucher Disease Patients. PLoS ONE 2013; 8(11): e9732. Doi: /journal.pone Murugesan V, Chuang WL, Liu J et al. Glucosylsphingosine is a key biomarker of Gaucher disease. Am J Hematol 2016; 91: P.R. Fleshner, A.H. Aufses Jr., G.A. Grabowski, R. Elias, A 27-year experience with splenectomy for Gaucher's disease, Am. J. Surg. 161 (1) (1991) O. Nilsson, L. Svennerholm, Accumulation of glucosylceramide and glucosylsphingosine (psychosine) in cerebrum and cerebellum in infantile and juvenile Gaucher disease, J. Neurochem. 39 (3) (1982) Andersson H et al. Eight-year clinical outcomes of long-term enzyme replacement therapy for 884 children with Gaucher disease type 1. Pediatrics 2008; 112:

5 19. G Marcucci, A Zimran, B Bembi et al Gaucher Disease and Bone Manifestations. Calcif Tissue Int 2014; 95: S vom Dahl, L Poll, M di Rocco et al. Evidence-based recommendations for monitoring bone disease and response to enzyme replacement therapy in Gaucher patients. Current medical research and opinions 2006; 22 (6): Kaplan P et al. Revised recommendations for the management of Gaucher disease in children.eur J Ped 2013; 172: Biegstraaten M, Cox TM, Belmatoug N, et al. Management goals for type 1 Gaucher disease: An expert consensus document from the European working group on Gaucher disease. Blood Cells Mol Dis Oct 24. pii: S (16) doi: /j. bcmd Mistry PK, Lukina E, Ben Turkia H, et al. Effect of Oral Eliglustat on Splenomegaly in Patients With Gaucher Disease Type 1. The ENGAGE Randomized Clinical Trial. JAMA 2015, 313 (7): Zimran A, Altanescu G, Elstein D. Screening for Gaucher disease: new challenges. Isr Med Assoc J 2014; 16 (11): Weinreb NJ, Charrow J, Andersson HC, et al. Effectiveness of enzyme replacement therapy in 1028 patients with type 1 Gaucher disease after 2 to 5 years of treatment: a report from the Gaucher Registry. Am J Med 2002;113(2): Elstein D, Zimran A. Review of the safety and efficacy of imiglucerase treatment of Gaucher disease. Biologics 2009;3: Pastores GM. Recombinant glucocerebrosidase (imiglucerase) as a therapy for Gaucher disease. BioDrugs 2010;24(1): Zimran A, Elstein D, Levy-Lahad E, et al. Replacement therapy with imiglucerase for type 1 Gaucher s disease. Lancet 1995;345: Grabowski GA, Barton NW, Pastores G, et al. Enzyme therapy in type 1 Gaucher disease: comparative efficacy of mannose-terminated glucocerebrosidase from natural and recombinant sources. Ann Intern Med 1995;122(9): Charrow J, Dulisse B, Grabowski GA, Weinreb NJ. The effect of enzyme replacement therapy on bone crisis and bone pain in patients with type 1 Gaucher disease. Clin Genet 2007;71: Wenstrup RJ, Kacena KA, Kaplan P, et al. Effect of enzyme replacement therapy with imiglucerase on BMD in type 1 Gaucher disease. J Bone Miner Res 2007;22: Altarescu G, Hill S, Wiggs E, et al. The efficacy of enzyme replacement therapy in patients with chronic neuronopathic Gaucher s disease. J Pediatr 2001;138(4): Elstein D, Abrahamov A, Zimran A. Ethical considerations for enzyme replacement therapy in neuronopathic Gaucher disease. Clin Genet 1998;54(3): Hollak CE, Hughes D, van Schaik IN, Schwierin B, Bembi B. Miglustat (Zavesca) in type 1 Gaucher disease: 5-year results of a post-authorisation safety surveillance programme. Pharmacoepidemiol Drug Saf 2009;18(9): Lukina E, Watman N, Avila Arreguin E, et al. Improvement in hematological, visceral, and skeletal manifestations of Gaucher disease type 1 with oral eliglustat tartrate (Genz ) treatment: two-year results of a phase 2 study. Blood 2010;116(20): McEachern KA, Fung J, Komarnitsky S, et al. A specific and potent inhibitor of glucosylceramide synthase for substrate reduction therapy of Gaucher disease. Mol Genet Metab 2007,91(3): Zimran A, Altarescu G, Philips M, et al. Phase 1/2 and extension study of velaglucerase alfa re- placement therapy in adults with type 1 Gaucher disease: 48-month experience. Blood 2010; 115(23): Elstein D, Altarescu G, Abrahamov A, Zimran A. Children with type 1 Gaucher disease: Changing profiles in the 21st century. Blood Cells Mol Dis Dec 19. pii: S (16)