La maladie de Gaucher est caractérisée par une accumulation de glucosylcéramides dans les lysosomes, liée à une diminution de l’activité de la béta-glucocérébrosidase acide. Cette baisse d’activité dépend de la quantité d’enzyme mutante mature qui parvient aux lysosomes et elle est modulée par l’importance de la dégradation de chaque variant par la voie ERAD du réticulum endoplasmique. L’ERAD est un processus complexe qui régule l’identification des protéines mal repliées dans le réticulum endoplasmique, leurs tentatives de repliage, leur sortie du réticulum endoplasmique vers le cytoplasme, leur poly-ubiquitination et leur dégradation dans le protéasome. Toutes ces étapes mettent en jeu un grand nombre de protéines, dont certaines sont des protéines chaperonnes calcium-dépendantes du réticulum tandis que d’autres participent à la poly-ubiquitination. Les taux de calcium et de cholestérol du réticulum endoplasmique ainsi que d’autres facteurs non encore identifiés déterminent le degré d’ERAD des différents variants mutants de la béta-glucocérébrosidase acide. Pour les auteurs, ce processus joue un rôle important dans le développement de la maladie de Parkinson, et d’autres maladies, chez les porteurs et les patients qui présentent une maladie de Gaucher. Les variants mutants de la béta-glucocérébrosidase subissent une poly-ubiquitination à laquelle participent les ligases E3: celles-ci sont alors détournées de leur activité sur d’autres substrats cellulaires, ce qui annule des processus cellulaires normaux.

OB

Le paradigme de la maladie de Gaucher : du réticulum endoplasmique aux comorbidités

Production woes weigh on Genzyme parent

Sanofi fails to put end to drug supply disruptions

September 27, 2011|By Robert Weisman, Globe Staff

 French drug maker Sanofi SA had hoped Genzyme Corp.’s high-profile production problems would be quickly put to rest when it acquired the Cambridge company.

 But five months after Sanofi completed its $20.1 billion takeover, supply disruptions for a pair of drugs to treat rare genetic disorders continue to dog Genzyme’s new owner.

 Earlier this month, Sanofi’s Genzyme division told health care providers it would reduce shipments for the next four months of its best-selling Gaucher disease drug, Cerezyme, which had recently returned to full production. Last month, the company said it was delaying shipments of its Fabry disease drug, Fabrazyme, which already had been rationed.

 Cerezyme patients in the United States whose normal biweekly doses had been restored in January, were temporarily back to once-a-month regimens. Fabrazyme patients faced a one-month delay in receiving already reduced dosages.

 “Because we have low inventory, we’ve said that any disruption in our manufacturing will be felt by the [patient] community,’’ said Genzyme spokeswoman Lori Gorski.

 The recent moves came after a rocky summer. Sanofi disclosed in July that it would not make a milestone payment to Genzyme investors because the company failed to meet manufacturing targets for the two drugs, made at the Allston Landing plant in Boston.

 Around the same time, the French company gave Bill Aitchison, previously head of manufacturing for its Sanofi Pasteur vaccines business, oversight for all of Genzyme’s biologics manufacturing operations. Aitchison replaced Scott Canute, who left the company.

 Sanofi’s ability to pull Genzyme out of the production morass it has been stuck in for the past two years will be key to the success of its buyout. The state of Genzyme’s manufacturing recovery was an issue hanging over the merger talks last year, with Sanofi leaders cautioning progress might be slower than their Genzyme counterparts were suggesting.

 The potential milestone payment, designed to bridge the parties’ differences over how much Genzyme was worth, put those competing views to the test. Thus far, Sanofi’s skepticism has been borne out. But as Genzyme’s new owner, Sanofi is now saddled with the responsibility to fix the problems at what had been the largest biotechnology company in Massachusetts.

 Sanofi can reap substantial benefits from Genzyme if it gets drug-making operations back on track, said Jonathan P. Gertler, senior partner at the Boston consulting firm Back Bay Life Science Advisors. He said Sanofi, with its global reach, can bring more resources to bear on ending the supply constraints than Genzyme could have as a stand-alone company.

 “The manufacturing issue looms large in everyone’s mind,’’ Gertler said. “It’s an absolutely critical issue for Sanofi. It’s critical for them to get the buy-in from the former Genzyme employees. It’s critical for Wall Street. And it’s critical for the long-term success of what are now the Sanofi products.’’

 Genzyme’s Gorski said the latest setbacks were aggravated by the low inventory at Allston Landing, where drugs go out to patients as soon as they are ready. The plant continues to produce Cerezyme and Fabrazyme as the company awaits approval from the Food and Drug Administration to open a new facility in Framingham next year.

 Allston Landing, on the banks of the Charles River, was the site of a virus found in a bioreactor in the summer of 2009. That forced Genzyme to temporarily suspend production and ration doses of the enzyme replacement therapies to thousands of patients.

 The drugs are produced by growing genetically modified Chinese hamster ovary cells in giant bioreactors. The conditions they treat, Gaucher and Fabry diseases, cause waste to build up in the body, swelling organs. The drugs, taken intravenously about every other week, cost up to $300,000 a year per patient.

 While it keeps rebuilding its inventory, Genzyme recently has experienced smaller than expected Cerezyme “productivity’’ - meaning the amount of a drug in the production process that is ready to ship. “You expect variability in biologics manufacturing,’’ Gorski said. “But we didn’t have the buffer of inventory.’’

 Genzyme has also had to make adjustments while implementing new procedures agreed to in a so-called consent decree with the FDA last year. The plant was placed under federal oversight for seven years as it worked to correct quality control problems.

 “We understand better what the implications of the consent decree are,’’ Gorski said. “It’s a new environment for that [Allston Landing] facility.’’ At the same time, she said, “We remain on target with the new Framingham plant, which is pivotal to both products.’’

 Patients, however, are growing restless. Some who suffer from Gaucher disease have switched to Vpriv, a rival drug produced by Irish drug maker Shire PLC, which has based its human genetic therapies division in Lexington. Some Fabry disease patients, who have been reduced to one dose or a half dose a month from their normal two a month for the past two years, might be open to switching to an alternative treatment - if there was one on the market.

 Genzyme has “not been able to move as fast as the patient community would have liked,’’ said Jack Johnson, executive director of the Fabry Support and Information Group, based in Concordia, Mo. “It has created a loss of confidence, and we’ve told them so.’’

 While many Fabry patients have been able to tolerate the reduced dosages, some have experienced pain, gastrointestinal problems, or other conditions, Johnson said.

 Members of the Fabry group were in Washington, D.C., last week to discuss their predicament with FDA officials. Johnson said they are urging the agency to fast-track alternative Fabry disease treatments being developed by Shire and New Jersey-based Amicus Therapeutics Inc.

But they are also urging the agency to speed approval of Genzyme’s plant in Framingham, where Fabrazyme will be made.

“The rationing has gone on since 2009,’’ Johnson said. “Patients want this to come to a conclusion as soon as possible.’’

Robert Weisman can be reached at weisman@globe.com.

One consultant said Sanofi can reap substantial benefits from Genzyme if it gets the drug-making operations on track. Its an absolutely critical issue for Sanofi, said Jonathan P. Gertler.

http://articles.boston.com/2011-09-27/business/30209007_1_genzyme-cerezyme-sanofi-pasteur

Genzyme parts de marché s'effondre

Article classé dans la catégorie : "NOUVELLES des LABORATOIRES".

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Ghislaine SURREL

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Ongoing supply problems for Genzyme’s Cerezyme

Article | 19 September 2011

US biotech Genzyme, now a subsidiary of French drug major Sanofi (Euronext: SAN), says it will have only limited supplies of its Gaucher disease drug Cerezyme (imiglucerase, injection) available for the next four months starting October.

In a letter to US health care providers, published by the National Gaucher Foundation, Genzyme said the shortage was caused by "a temporary decrease in Cerezyme yields," coupled with "changes to our product release processes and procedures".

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The company has had problems with supplies of Cerezyme, as well as its Fabry disease drug Fabrazyme (agalsidase beta) for patients worldwide, as a result of earlier manufacturing issues since June 2009 (The Pharma Letters passim). The Genzyme problems have benefited Ireland-headquartered Shire, as it has resulted in switching to the company’s Replagal (agalsidase alfa) from Fabrazyme and to Vpriv (velaglucerase alfa) from Cerezyme.

Adjustments to individual treatment plans

Changes to Cerezyme availability will be felt globally, the letter stated, adding that delays in shipments will likely affect patients and some adjustment to individual treatment plans may be necessary. For the USA from October through January, Genzyme expects to provide:

• one full dose (equivalent to the currently prescribed dose) per month for patients aged 19 years and older currently treated with Cerezyme; and
• two full doses per month for patients aged 18 years and younger and for Type 2/3 patients currently treated with Cerezyme.

Genzyme will confirm shipping availability each month. Given current improvements to productivity and progress with the firm’s manufacturing recovery, Genzyme currently anticipates an improving Cerezyme supply outlook from February 2012 forward.

In order to support the current level of patient demand, Cerezyme is made available as it is produced, which does not allow the build-up of inventory, the company explained. “Operating with little or no inventory means changes like these in our manufacturing plans can impact supply. Over the past four years there has been significant investment in a new manufacturing facility to produce Fabrazyme in Framingham, Massachusetts. We are in the process of plant validation and currently anticipate completing regulatory approval processes that will allow us to ship product in the first part of 2012. This is a critical step as it will allow us to improve Cerezyme production at the Allston facility which is producing both Cerezyme and Fabrazyme today,” said Genzyme’s letter.

http://www.thepharmaletter.com/file/107413/ongoing-supply-problems-for-genzymes-cerezyme.html?utm_source=2009_11_06-Pharma+Clean&utm_campaign=d261c6d402-RSS_EMAIL_CAMPAIGN&utm_medium=email

Bisphosphonates and Atypical Femoral Shaft Fractures

N Engl J Med 2011; 365:377July 28, 2011

Article

To the Editor:

Schilcher et al. (May 5 issue)1 report the findings of a cohort analysis that examined the risk of atypical femoral fractures with bisphosphonate use. They found a statistically significant increase in such fractures, with an absolute risk of 5 per 10,000 patient-years, similar to that reported in other studies.2 They conclude that their results are reassuring for patients taking bisphosphonates, since “the magnitude of the absolute risk [is] small.” This level of absolute risk appears identical to the absolute risks reported for hormone-replacement therapy (HRT) by the Women's Health Initiative investigators,3 yet those investigators described the risks of HRT as “substantial,” even though many of those increased risks appeared more likely to be due to chance rather than the intervention when subsequent analyses were undertaken or appropriate statistics were applied.4 This report from the Women's Health Initiative led to substantial reduction in the use of HRT (up to 50% worldwide), even though the results indicated neither harm nor benefit for more than 99% of participants. But Schilcher et al. say that they find the data on the risk of bisphosphonate use to be reassuring. Am I missing something?

John C. Stevenson, F.R.C.P.
Royal Brompton Hospital, London, United Kingdom
j.stevenson@imperial.ac.uk

Dr. Stevenson reports receiving research grants from Eli Lilly, Janssen-Cilag, Novo Nordisk, Organon-Schering-Plough, Schering, Shire, Solvay, and Wyeth; serving on the advisory boards of Novo Nordisk, Procter & Gamble, and Pfizer-Wyeth; and receiving consulting fees from AstraZeneca, Bayer-Schering, Novo Nordisk, Orion, Procter & Gamble, Servier, Solvay, Theramex, and Pfizer-Wyeth.

No other potential conflict of interest relevant to this letter was reported.

4 References

Author/Editor Response

Any judgment about the magnitude of a risk must be seen in relation to other risks and benefits. Women with osteoporosis run a high risk of fracture, which is substantially reduced by bisphosphonate therapy.1 The numbers needed to treat with bisphosphonates for 3 years are 91 for hip fractures and 14 for radiologic vertebral fractures.1 Without consideration of duration of use, we found that the number needed to harm given 3 years of treatment was 667 — that is, the benefits with the therapy outweigh the risks. The absolute risk of stress (atypical) fracture in our study tended to be higher with a longer duration of bisphosphonate use. With more than 2 years of treatment, the difference in absolute risk as compared with no treatment was 8 per 10,000 women per year of treatment. This estimate corresponds to a number needed to harm of 417 for a 3-year treatment period. Thus, theoretically, for each stress fracture caused, at least 30 vertebral and about 5 hip fractures will be prevented. This is reassuring. However, without a proper indication, the benefit–risk ratio with bisphosphonate use may not be advantageous.

Jörg Schilcher, M.D.
Linköping University, Linköping, Sweden

Karl Michaëlsson, M.D., Ph.D.
Uppsala University, Uppsala, Sweden

Per Aspenberg, M.D., Ph.D.
Linköping University, Linköping, Sweden
per.aspenberg@liu.se

Since publication of their article, the authors report no further potential conflict of interest.

1 Reference

http://www.nejm.org/doi/full/10.1056/NEJMc1106551?query=TOC&

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Vous trouverez des articles traitant du même sujet dans la catégorie "A propos de la maladie de Gaucher" 

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Ichtyose congénitale dans la maladie de Gaucher de type II sévère avec mutation nulle homozygote
Voici l’observation d’un nouveau-né présentant une maladie de Gaucher de type II dont le phénotype était inhabituellement sévère avec une ichtyose congénitale, une hépatosplénomégalie , une hypotonie musculaire, des myoclonies et une insuffisance respiratoire. L’étude de la peau en microscopie électronique a montré au niveau de la couche cornée la présence de structures lamellaires considérées comme typiques de la maladie de Gaucher de type II. Le bébé est mort d’insuffisance respiratoire à 1 mois sans avoir fait de progrès neurologique. L’analyse moléculaire a permis d’identifier une mutation homozygote nulle jamais signalée auparavant du gène de la beta-glucocérébrosidase : c.1505G A. La maladie de Gaucher de type II (forme neuronopathique aiguë) est une maladie néo-natale rare de mauvais pronostic. La mutation homozygote décrite ici est responsable de la destruction du site donneur d’épissage entre les exons 10 et 11 à l’origine d’une suppression fonctionnelle de toute l’activité enzymatique : cette mutation entraîne donc la forme clinique la plus sévère de la maladie de Gaucher de type II. La microscopie électronique est utile au diagnostic précoce cat elle permet de montrer les reliquats caractéristiques du trouble métabolique de la barrière lipidique épidermique ; la beta-cérébroglucosidase est essentielle au fonctionnement de la barrière épidermique.

Congenital Ichthyosis in Severe Type II Gaucher Disease with a Homozygous Null Mutation
[27-04-2011]

Sabine Haverkaemper^a, Thorsten Marquardt^c, Ingrid Hausser^d, Katharina Timme^a, Thomas Kuehn^a, Christoph Hertzberg^b, Rainer Rossi^a

Departments of
^aPediatrics and
^bPediatric Neurology and Social Care, Klinikum Neukoelln, Berlin,
^cDepartment of Pediatrics, University Hospital, Muenster, and
^dDepartment of Dermatology, Electron Microscopy Laboratory, University Hospital, Heidelberg, Germany

Abstract

This paper describes a neonate with type II Gaucher disease. The phenotype was unusually severe with congenital ichthyosis, hepatosplenomegaly, muscular hypotonia, myoclonus and respiratory failure. Electron microscopy of the skin revealed lamellar body contents in the stratum corneum interstices, appearances considered to be typical of type II Gaucher disease. The baby died from respiratory failure 1 month postpartum having made no neurological progress. Molecular analysis identified a previously not reported homozygous null mutation, c.1505G→A of the β-glucocerebrosidase gene.

2109298_CongenitalIchthyosisinSev

Article classé dans la catégorie : "Examens biologiques, IRM,...".

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Maladie de Gaucher de type 1 : anomalie de l’adhésion plaquettaire et risque de saignement muqueux

Les patients qui présentent une maladie de Gaucher de type 1 peuvent avoir une tendance clinique significative au saignement, sans rapport avec le niveau de leur taux de plaquettes. Pour vérifier le rôle joué par les plaquettes une étude israélienne a inclus 48 patients avec une maladie de Gaucher de type 1, 52 porteurs (parents d’enfants avec une maladie de Gaucher) et 19 témoins bien portants. Chez les patients l’adhésion des plaquettes était significativement plus faible que chez les témoins ou chez les porteurs. L’adhésion plaquettaire n’était pas modifiée par la prise d’une enzymothérapie substitutive spécifique de la maladie, mais elle était améliorée après splénectomie. Cette diminution de l’adhésion était liée à un défaut plaquettaire intrinsèque. Un saignement muqueux était signalé chez 17 patients (35,4%) et était associé à une anomalie de l’adhésion (odd ratios : 5,73). Chez 5 patients il existait une baisse de l’agrégation plaquettaire et tous présentaient une diminution de l’adhésion plaquettaire. Le défaut d’agrégation plaquettaire n’était pas associé avec des saignements muqueux. Une anomalie de l’adhésion plaquettaire constitue une thrombocytopathie majeure chez les patients qui présentent une maladie de Gaucher de type 1 et peut expliquer en partie la tendance augmentée au saignement.   Platelet adhesion defect in type I Gaucher Disease is associated with a risk of mucosal bleeding [27-04-2011] Galia Spectre 1, Batia Roth 1, Galia Ronen 3, Dror Rosengarten 4, Deborah Elstein 4, Ari Zimran 4, David Varon 1, Shoshana Revel‐Vilk 2 1 Coagulation Unit 2 Paediatric Haematology/Oncology Department, Hadassah‐Hebrew University Medical Centre, Jerusalem, Israel 3 Radiology Department, Sourasky Medical Centre, Tel Aviv 4 Gaucher Clinic, Shaare Zedek Medical Centre, Jerusalem, Israel *Correspondence:G. Spectre, Coagulation Unit, Hadassah‐Hebrew University Medical Centre, Jerusalem 91120, Israel. E‐mail: galias@hadassah.org.il Summary Patients with type I Gaucher Disease (GD) may have a clinically significant bleeding tendency that is disproportionate to their platelet count. We hypothesized that impaired platelet adhesion might contribute to bleeding tendency. Adult patients with type I GD with platelet counts ≥130 × 109/l and haematocrit ≥30% (n = 48), obligatory carriers (n = 52), and healthy controls (n  = 19) were studied. Platelet adhesion, using the IMPACT‐R (Cone and Plate(let) Analyser), and platelet aggregation were determined. Type I GD patients had significantly lower platelet adhesion [surface coverage %, median (interquartile range)] 4·6 (3·2–7·5), compared to controls, 8·7 (7·6–10·3), or carriers, 8·1 (6·5–9·4; P = 0·001). Platelet adhesion was not affected by the use of disease‐specific enzyme replacement therapy but was improved in patients after splenectomy, 7·2 (5·8–9·3). Mixing tests showed that the reduced adhesion was an intrinsic platelet defect. Mucosal bleeding was reported in 17 (35·4%) patients and was associated with abnormal adhesion [P = 0·037, with an Odds Ratio (95% confidence interval) of 5·73 (1·1–29·6)]. Five patients (22%) had reduced platelet aggregation, all of whom had reduced platelet adhesion. Platelet aggregation defect was not associated with mucosal bleeding. In conclusion, platelet adhesion defect is a major thrombocytopathy in type I GD patients and can explain part of the increased tendency to bleeding. A bleeding tendency is a prominent feature of type I Gaucher disease (GD). The main bleeding symptom of these patients is mucocutaneous, which manifests as epistaxis, gingival bleeding, easy bruising, and/or heavy menstrual bleeding (Granovsky‐Grisaru , 1995; Larsen , 2003; Zimran , 2005). Increased bleeding tendency in GD is commonly attributed to thrombocytopenia secondary to hypersplenism and/or to bone marrow infiltration. Reduction in the activity of various coagulation factors, in particular low factor XI levels, and increased fibrinolysis, have also been described, mainly in unsplenectomized patients (Hollak , 1997; Katz , 1999; Deghady , 2006; Giona , 2006). A high gene frequency of both factor XI deficiency and type I GD in Ashkenazi Jews can explain the relatively common concurrence of both genetic disorders (Seligsohn , 1976; Berrebi , 1992). However, it has been considered that some GD patients have a clinically significant bleeding tendency that is not proportional to their platelet counts or coagulation abnormalities. Thus, an additional cause for bleeding may be an abnormal platelet function (Kelsey , 1994; Gillis , 1999; Giona , 2006). Abnormal platelet aggregation has been described in seven patients with type I GD in a series of 32 patients (22%) from our clinic (Gillis , 1999) and in six of 15 patients (40%) in another study (Giona , 2006). Adhesion of platelets to the injured vessel wall, the initial step of the haemostatic response, is crucial for normal platelet function. To date, this important aspect of platelet response has not been studied in GD. We hypothesized that impaired platelet adhesion might contribute to the bleeding diathesis observed in patients with GD. In the present study we tested platelet adhesion under arterial shear conditions in patients with type I GD, obligatory carriers of the disease, and healthy volunteers. We used the IMPACT‐R [Cone and Plate(let) Analyser] technique (Shenkman , 2000). In addition, mixing studies were performed in order to evaluate the differential role of platelets, plasma, and red blood cells (RBCs) in platelet adhesion in GD. Materials and methods Patients and controls Type I GD adult patients followed at the Gaucher Clinic, Shaare Zedek Medical Centre were eligible for this study. Diagnosis of GD was made by demonstration of decreased glucocerebrosidase activity in leucocytes (Beutler & Kuhl, 1970) and by mutation analysis at the DNA level (Beutler , 1992). In a few patients bone marrow findings of Gaucher cells in bone marrow, with a combination of two mutated alleles were acceptable in the absence of an enzymatic diagnosis. Lower platelet counts and/or haematocrit levels are known to be associated with abnormal adhesion using the IMPACT‐R system (Varon , 1997; Kenet , 1998), therefore we included only patients with platelet counts ≥130 × 109/l and haematocrit ≥30%. Data were recorded from the clinic medical files of all patients, including year of birth, ethnic origin, mutation analysis, history of bleeding episodes, history of splenectomy, and history of medical treatment, i.e. enzyme replacement therapy (ERT) or substrate reduction therapy (SRT). Disease severity was calculated by the severity score index (SSI; Zimran , 1989). Obligatory carriers, parents of children with GD coming to the clinic at the time of study period, and a group of hospital personnel as healthy controls were also enrolled in the study. All study participants signed an informed consent form. The study was approved by the local ethics (Helsinki) committee. Blood counts and platelet adhesion tests Samples for complete blood count were collected in EDTA tubes. Platelet adhesion under flow conditions was studied using the IMPACT‐R (Cone and Plate (let) Analyser; DiaMed, Cressier, Switzerland; Shenkman , 2000). Blood samples for the IMPACT‐R test were collected in citrate tubes containing 0·38% sodium citrate, and were analysed within 3 h after blood draw. Citrated whole blood (130 μl) were placed on polystyrene plates (Nunc, Roskilde, Denmark) and subjected to a shear rate of (1800/s) for 2 min using a rotating teflon cone. The wells were then thoroughly washed with tap water, stained with May–Gruenwald stain (Sigma, Rehovot, Israel). The image of adhered platelets was analysed with an inverted light microscope (Olympus, Tokyo, Japan) connected to an image analysis system (Galai, Migdal Haemek, Israel). Two parameters of platelet adhesion were evaluated: percent surface coverage (SC, %) which is the percentage of total area covered by platelets (single platelets and clusters/aggregates) and the average size (AS, μm2) of the polystyrene bound platelet clusters/aggregates. The samples were analysed in duplicates, and the higher platelet adhesion result was used for statistical analysis. The normal values of the IMPACT‐R test were previously specified as SC: ≥7% and AS: ≥23 μm2, based on the 5th percentile of log transformed data from 98 adult controls (Revel‐Vilk , 2009). Preparation of platelet rich plasma, platelet poor plasma, and red blood cells Platelet rich plasma (PRP) was prepared by centrifugation of anti‐coagulated whole blood 180 g for 12 min at room temperature. Red blood cells were isolated by further centrifugation of the remaining layer of whole blood at 1200 g for 5 min. The supernatant consisted of platelet poor plasma (PPP). Mixing studies Mixing studies were performed in nine pairs of patients and healthy volunteers, matched for blood type. Normal RBC (adjusted to the haematocrit of 40%) were reconstituted with patient’s PRP, incubated for 5 min with gentle rotation (10 rpm) and then analysed by the IMPACT‐R test. In the same manner, normal PRP was reconstituted with the patients’ RBC, and patients’ PPP was added to normal whole blood. In all experiments, autologous reconstitution of the healthy volunteer’s blood served as a control. Platelet aggregometry Platelet aggregation was measured by a routine platelet aggregometer (PACKS‐4, Helena Laboratories, Beaumont, TX, USA) using the following agonists (obtained from Diamed, Switzerland): ADP (10 μmol/l), epinephrine (10 μmol/l), and collagen (5 μg/ml). Platelet aggregation was considered normal if maximal aggregation amplitude was >60%. Statistical analysis Characteristics of study subjects are presented as nominal data. The percentages are presented with Fisher’s exact 95% confidence interval (CI). Nominal variables are presented as median (interquartile range) or mean (95% CI), where applicable. Differences in age, platelet count, haematocrit, and SC/AS measurements between the different groups were assessed using the parametric and non‐parametric tests for normally and non‐normally distributed data, respectively. Adjustment of P values to correct for possible significance resulting from performance of multiple tests on the same data (Bonferroni like correction‐Hommel adjusted p; Wright, 1992) was performed with WINPEPI (PEPI‐for‐Windows, Version 2.8, March 2007). Differences for mixing tests were evaluated by a paired T‐test. To compare between GD patients with and without mucosal bleeding a logistic regression model was used. The following variables were considered: platelet count, haematocrit, SC/AS measurement, platelet aggregation test, SSI, and ERT. Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS), version 14 for Windows. A P value <0·05 was considered significant. Results Forty‐eight adult patients with type I GD, 52 obligatory carriers, and 19 controls were enrolled to this study. The clinical characteristics of the GD patients are presented in Table I. I  Characteristics of patients with type I Gaucher disease.   No % Total patients 48   Male 23 48 Ashkenazi Jewish origin 47 98 Mutations  N370S homozygous 23 48  Compound heterozygous (N370S/other) 25 52 Severity Score Index (SSI)  Mild (1–10) 31 65  Moderate (11–25) 16 33  Severe (26–30) 1 2  Splenectomy (total or partial) 17 35  Medical therapy 36 75 Table 1 Effect of gaucher disease on platelet adhesion Patients with type I GD had a significantly lower SC as compared to controls and obligatory carriers (Table II). The platelet counts of GD patients were not significantly different from controls; however they were significantly lower as compared to obligatory carriers. No differences in AS measurements were found between patients, obligatory carries and controls.   II  Laboratory characteristics of patients and obligatory carriers and healthy volunteers.     Patients Controls Carriers P‐value* P‐value† P‐value‡ Number (M:F) 48 (23:25) 19 (13:6) 52 (26:26) –   – Platelet count, ×109/l 193 (153–252) 222 (200–245) 249 (217–302) 0·06 0·003 0·06 Haematocrit, % 40·7 (37·3–44·1) 43 (40·5–45·8) 41·1 (37·2–45) 0·18 0·52 0·31 Surface coverage, % 4·6 (3·2–7·5) 8·7 (7. 6–10·3) 8·1 (6·5–9·4) 0·002 0·002 0·36 Average size, μm2 24·1 (20·1–33·9) 28·0 (24·0–34·0) 27·0 (24·0–37·0) 0·15 0·07 0·86   Data presented as median (interquartile range). M, male; F, female.   *P value for Gaucher patients compared to control.   †P value for Gaucher patients compared to obligatory carriers.   ‡P value for controls compared to obligatory carriers. Table 2 Given that differences in platelet counts could potentially affect the difference observed in SC, 15 participants with platelet counts <160 × 109/l were excluded from the analysis in order to attain a median platelet count that was not significantly different between patients, obligatory carriers, and controls (P = 0·1). Also in this subgroups analysis, SC was significantly lower in patients, median (interquartile range), 5·9 (3·4–9), as compared to obligatory carriers, 8·1 (6·7–9·4) or controls, 8·8 (7·9–10·2; P = 0·001). Correlation was found between SC and platelet counts in patients with type I GD (correlation coefficient 0·34, P  = 0·017, Spearman correlation test). No correlation was found with age and the SSI. No correlation was found between SC and platelet count in controls or in obligatory carriers. Representative photographs of platelet adhesion in a patient and a healthy control are shown (Fig 1). Figure 1  Reduced platelet adhesion in Gaucher disease. A representative platelet adhesion picture (IMPACT‐R, Cone and platelet analyser) of a healthy control (right): Surface coverage (SC) 9% Average size (AS) 27 μm2, and a patient with Type I Gaucher disease (left) SC 3·3%, AS 23 μm2. Effect of ert and splenectomy on platelet adhesion ERT‐treated patients had similar platelet counts, SC, and AS of aggregates compared to untreated patients (Table III). Splenectomized patients on ERT had significantly higher platelet counts and SC as compared to patients on ERT only. Only one splenectomized patient did not receive ERT and was excluded from this analysis. III  Characteristics of patients with Gaucher type I with or without enzyme replacement therapy (ERT).   No ERT ERT only ERT & splenectomy* P‐value† P‐value‡ P‐value§ Number 11 20 16 – – – Age, years 39 (35–48) 40 (25–59) 48·5 (37–61) 0·82 0·16 0·12 Platelet count, ×109/l 154 (138–204) 169 (142–193) 262 (214–302) 0·99 0·002 <0·001 Haematocrit, % 40·6 (38·8–42·4) 39·7 (36·6–42·9) 41·3 (39·1–43·6) 0·44 0·89 0·58 Surface coverage, % 3·8 (1·9–7·5) 3·8 (2·4–5·3) 7·2 (5·8–9·3) 0·99 0·09 0·002 Average size, μm2 20·0 (17·1–25·1) 25·6 (22·5–35·8) 24·9 (22·3–31·7) 0·21 0·16 0·79 Mucosal bleeding 6 (54%) 7 (35%) 4 (25%) 0·29 0·15 0·7 Data presented as median (interquartile range). ERT, enzyme replacement therapy.   *One splenectomized patient who did not receive ERT was excluded from the analysis.   †P value for patients without therapy compared to those who received ERT.   ‡P value for patients without therapy compared to those who received ERT and underwent splenectomy.   §P value for patients who received ERT compared to who received ERT and underwent splenectomy. Table 3 Although a trend of higher platelet count was found in splenectomized patients compared to controls (Fig 2A), the SC of those patients tended to be lower compared to controls (Fig 2B).   Figure 2  (A) Platelet counts in patients with Gaucher disease and controls. A box plot for the platelet count (×109/l) in untreated patients (no Rx, n = 11), patients receiving only ERT (ERT = 20), splenectomized patients also receiving ERT (ERT and splenectomy, n = 16), and controls (n = 19). A trend for higher platelet count in splenectomized patients on ERT compared to controls (P  = 0·066). (B) Surface coverage in patients with Gaucher disease and controls. A box plot curve for the surface coverage (% IMPACT‐R) in untreated patients (no Rx, n = 11), patients receiving only ERT (ERT n = 20), splenectomized patients receiving also ERT (ERT and splenectomy, n = 16) and controls (n = 19). A trend for lower surface coverage in splenectomized patients on ERT as compared to controls (P = 0·071). No correlation was found between platelet counts and SC during analysis of the different treatment groups, i.e. untreated with intact spleen, ERT with intact spleen, and ERT with splenectomy. Bleeding history in patients with gaucher disease Twenty‐eight patients reported a previous history of bleeding episodes (58·3%, 95% CI 43·2%‐72·4%). Eleven patients (22·9%) reported only easy bruising. Mucosal bleeding was reported 17 patients (35·4%), including epistaxis, menorrhagia, gingival bleeding, rectal bleeding, and prolonged bleeding after cuts or surgery. A bleeding history was not obtained in one patient. A history of mucosal bleeding was not associated with the SSI, ERT, platelet counts, haematocrit level, and/or SC/AS measurements. To improve the diagnostic value of the IMPACT‐R test as a potential predicative test for mucosal bleeding, the results of both the SC and the AS measurements were used to define an abnormal test. An abnormal IMPACT‐R test, (defined as an abnormal SC < 7% and/or AS < 23 μm2) was found in 32 patients (66·7%, 95% CI 51·6–79·6%) and was associated with history of mucosal bleeding. An abnormal IMPACT‐R test was found in 15/17 (88·2%, 95% CI 63·6–98·5%) of patients with mucosal bleeding as compared to 17/30 (56·6%, 95% CI 37·4–74·5%) of patients without mucosal bleeding (P = 0·037). The Odds Ratio (95% CI) for mucosal bleeding in GD patients with an abnormal IMPACT‐R test was 5·73 (1·1–29·6). Mixing studies in patients and controls Platelet adhesion in the IMPACT‐R system is influenced by platelets, RBCs, and plasma components (Shenkman , 2000; Peerschke , 2007). To test the differential role of each component in the observed reduced platelet adhesion in GD, we performed mixing studies in nine pairs of patients and controls. Patient PRP mixed with control RBC resulted in a lower SC compared to control PRP mixed with control RBC, mean (95% CI), 3·5% (1·5–5·5%) and 8·5% (7·2–9·8%), respectively (P < 0·001; Fig 3).   Figure 3  Mixing studies in patients and controls. RBC, red blood cell; PRP, platelet rich plasma; PPP, platelet poor plasma; WB, whole blood; C, control; P, patient; NS, not significant. Surface coverage (%) of reconstituted samples of patients and controls (n  = 9) as described in material and methods. Surface coverage was significantly reduced when RBC‐C were reconstituted with PRP‐P. No change in SC was observed when patient PPP or RBC was reconstituted with control blood. Mixing of control PPP with control whole blood diluted the sample and reduced platelet adhesion, mean SC (95% CI), 4·54% (3·34–5·74%). However, mixing of patient PPP with control whole blood did not reduce platelet adhesion further: mean SC (95% CI), 4·29% (2·85–5·72%). Mixing of patient RBC with control PRP did not change platelet adhesion (for the whole group of patients, n = 8), mean SC (95% CI), 5·98% (3·7–8·26%; Fig 3). However, a splenectomized patients’ RBC mixed with normal PRP reduced SC as compared to autologous mixing of control RBC and control PRP (n = 5, P = 0·04). This phenomenon did not occur in the patients with intact spleens (n = 3, P = 0·4). Platelet aggregation Platelet aggregation in response to epinephrine, ADP, collagen and ristocetin was tested in 22 patients. Platelet counts, SC/AS measurements, and rate of abnormal IMPACT‐R test were not different in patients who were tested or not for platelet aggregation. Isolated abnormal platelet aggregation in response to epinephrine was observed in three (13%) patients (Kambayashi , 1996). Abnormal platelet aggregation in response to ADP and/or collagen was observed in five (22%) patients, all of whom had an abnormal IMPACT‐R test [positive predictive value of 100% (95% CI 89–100%)]. Aggregation in response to ristocetin was normal in all patients. Platelet aggregation defect alone was not associated with mucosal bleeding (P = 0·32). Discussion This study was designed to assess the platelet adhesion in patients with type I GD. Platelet adhesion was found to be lower in patients with GD compared to obligatory carriers and controls. Platelet adhesion was not affected by the use of ERT and was improved after splenectomy. A platelet adhesion defect, measured by the IMPACT‐R system, occurred in two‐thirds of GD patients and was associated with history of mucosal bleeding. The IMPACT‐R system measures both the adhesion of platelets to the polystyrene surface (SC), as well as the aggregation of circulating platelets around adherent platelets associated with the release reaction (AS). The significantly lower SC in GD patients compared to controls suggests that a platelet adhesion defect is a major thrombocytopathy in patients with GD. This thrombocytopathy might be missed when testing platelet function only with the platelet aggregometer. Indeed, abnormal platelet aggregation was found in only one‐fifth of the patients in this study and others (Gillis , 1999; Giona , 2006). The mechanism of reduced platelet adhesion in patients with type I GD is not clear. Based on mixing tests, reduced adhesion is an intrinsic platelets defect and not affected by RBCs or plasma of these patients. The increased plasma levels of glucocerebroside in patients with GD may affect platelet activation (Nilsson , 1982; Aerts & Hollak, 1997; Gousset , 2002). However, this mechanism could possibly explain platelet abnormalities only in untreated patients. Alternatively, extracts of platelets were found to be rich in an aggregated, activated form of the glucocerebrosidase (Yatziv , 1974), thus lack of the enzyme in GD might cause increased glucocerebroside in the platelets and affect platelet function. In our study, patients receiving ERT did not have higher platelet counts or platelet adhesion compared to untreated patients. This is in contrast to previous studies where platelet counts and platelet aggregation were shown to be improved after starting ERT (Hollak , 1997; Gillis , 1999; Giona , 2006). By excluding patients with low platelet counts, we might have influenced the chance to find differences in platelet counts and platelet adhesion between ERT‐treated and untreated patients. In contrast, splenectomy was associated both with improved platelet counts and platelet adhesion as measured by the IMPACT‐R system. The improved platelet adhesion found after splenectomy might be directly related to the higher platelet counts. Alternatively, splenomegaly has been suggested to cause chronic platelet activation that might lead to platelet exhaustion with reduced function (Humphries & Hess, 1994; Hollak , 1997). The study inclusion criterion of a platelet count >130 × 109/l has resulted in a relatively high percentage of splenectomized patients (35%) in the era of disease‐specific ERT. The trend to lower SC in splenectomized GD patients compared to controls, in the presence of a tendency for higher platelet counts suggests that the platelet adhesion defect we observed in GD patients is a true pathological finding and is not only a reflection of the low platelet counts. The observation of a lower SC in a subgroup analysis of patients with platelet counts >160 × 109/l, supports a similar conclusion. Preliminary data from mixing studies indicate that RBC from GD patients with intact spleens may reduce the SC of normal platelets, as compared to RBC from splenectomized patients. These preliminary results suggest that the better platelet adhesion profile of splenectomized patients could also result from elimination of a yet unknown effect of splenomegaly on RBC physiology under high shear stress. Further research is needed to study the mechanism of the platelet adhesion defect in GD patients. Of interest is the association of abnormal platelet function measured by the IMPACT‐R system with history of mucocutaneous bleeding. No similar association was reported with the more commonly performed platelet aggregation test. Studying whole blood platelet adhesion under high shear rate may be a more relevant parameter for correlation with bleeding symptoms. In previous studies, the IMPACT‐R method was shown to be effective in the assessment of platelet function in thrombocytopenic patients (Kenet , 1998) and in patients undergoing cardiac surgery (Gerrah , 2004). Recently, the IMPACT‐R method was shown to be a useful screening tool for bleeding disorders in children evaluated for bleeding tendency (Revel‐Vilk , 2009). The diagnosis of bleeding risk is clinically important in preparing patients before surgery or delivery. The current recommendations in patients with GD include performing a complete history of bleeding tendency (including easy bruising and recurrent gum or nose bleeds, and heavy menstrual bleeds), a complete blood count, coagulation factors assays if the prothrombin time and/or partial thromboplastin time are abnormal, platelet aggregation tests, and von Willebrand factor levels and activity (Ioscovich , 2005; Zimran , 2005). Platelet adhesion studies are currently used in the Gaucher Clinic, Shaare Zedek Medical Centre, as part of the routine check of the haemostatic system before surgical procedures or delivery. In conclusion, platelet adhesion defect is commonly found in type I GD patients and can explain some of the increased bleeding tendency in these patients. Testing platelet adhesion should be considered before surgery, delivery or dental procedures in order to assess the need for platelet transfusions, antifibrinolytic agents or desmopressin (DDAVP) in the individual patient. The first two authors contributed equally to the paper. <p><img src="http://www.gnmhealthcare.com/clients/images/11/04/11/BJH_8613_f1.gif" border="0"/></p> <p><b>Figure 1</b>  Reduced platelet adhesion in Gaucher disease. A representative platelet adhesion picture (IMPACT‐R, Cone and platelet analyser) of a healthy control (right): Surface coverage (SC) 9% Average size (AS) 27 μm<sup>2</sup>, and a patient with Type I Gaucher disease (left) SC 3·3%, AS 23 μm<sup>2</sup>.</p> <p><img src="http://www.gnmhealthcare.com/clients/images/11/04/11/BJH_8613_f2.gif" border="0"/></p> <p><b>Figure 2</b>  (A) Platelet counts in patients with Gaucher disease and controls. A box plot for the platelet count (×10<sup>9</sup>/l) in untreated patients (no Rx, <i>n</i> = 11), patients receiving only ERT (ERT = 20), splenectomized patients also receiving ERT (ERT and splenectomy, <i>n</i> = 16), and controls (<i>n</i> = 19). A trend for higher platelet count in splenectomized patients on ERT compared to controls (<i>P</i> = 0·066). (B) Surface coverage in patients with Gaucher disease and controls. A box plot curve for the surface coverage (% IMPACT‐R) in untreated patients (no Rx, <i>n</i> = 11), patients receiving only ERT (ERT <i>n</i> = 20), splenectomized patients receiving also ERT (ERT and splenectomy, <i>n</i> = 16) and controls (<i>n</i> = 19). A trend for lower surface coverage in splenectomized patients on ERT as compared to controls (<i>P</i> = 0·071).</p> <p><img src="http://www.gnmhealthcare.com/clients/images/11/04/11/BJH_8613_f3.gif" border="0"/></p> <p><b>Figure 3</b>  Mixing studies in patients and controls. RBC, red blood cell; PRP, platelet rich plasma; PPP, platelet poor plasma; WB, whole blood; C, control; P, patient; NS, not significant. Surface coverage (%) of reconstituted samples of patients and controls (<i>n</i> = 9) as described in material and methods. Surface coverage was significantly reduced when RBC‐C were reconstituted with PRP‐P. No change in SC was observed when patient PPP or RBC was reconstituted with control blood.</p> <p><b>I</b>  Characteristics of patients with type I Gaucher disease. </p> <table frame="box" border="1" bordercolor="#999999"> <tr> <th></th> <th>No</th> <th>%</th> </tr> <tr> <td>Total patients</td> <td>48</td> <td> </td> </tr> <tr> <td>Male</td> <td>23</td> <td>48</td> </tr> <tr> <td>Ashkenazi Jewish origin</td> <td>47</td> <td>98</td> </tr> <tr> <td colspan="3">Mutations</td> </tr> <tr> <td> N370S homozygous</td> <td>23</td> <td>48</td> </tr> <tr> <td> Compound heterozygous (N370S/other)</td> <td>25</td> <td>52</td> </tr> <tr> <td colspan="3">Severity Score Index (SSI)</td> </tr> <tr> <td> Mild (1–10)</td> <td>31</td> <td>65</td> </tr> <tr> <td> Moderate (11–25)</td> <td>16</td> <td>33</td> </tr> <tr> <td> Severe (26–30)</td> <td>1</td> <td>2</td> </tr> <tr> <td> Splenectomy (total or partial)</td> <td>17</td> <td>35</td> </tr> <tr> <td> Medical therapy</td> <td>36</td> <td>75</td> </tr> </table> <p><b>II</b>  Laboratory characteristics of patients and obligatory carriers and healthy volunteers. </p> <table frame="box" border="1" bordercolor="#999999"> <tr> <th></th> <th>Patients</th> <th>Controls</th> <th>Carriers</th> <th> <i>P</i>‐value*</th> <th> <i>P</i>‐value†</th> <th> <i>P</i>‐value‡</th> </tr> <tr> <td>Number (M:F)</td> <td>48 (23:25)</td> <td>19 (13:6)</td> <td>52 (26:26)</td> <td>–</td> <td> </td> <td>–</td> </tr> <tr> <td>Platelet count, ×10<sup>9</sup>/l</td> <td>193 (153–252)</td> <td>222 (200–245)</td> <td>249 (217–302)</td> <td>0·06</td> <td>0·003</td> <td>0·06</td> </tr> <tr> <td>Haematocrit, %</td> <td>40·7 (37·3–44·1)</td> <td>43 (40·5–45·8)</td> <td>41·1 (37·2–45)</td> <td>0·18</td> <td>0·52</td> <td>0·31</td> </tr> <tr> <td>Surface coverage, %</td> <td>4·6 (3·2–7·5)</td> <td>8·7 (7. 6–10·3)</td> <td>8·1 (6·5–9·4)</td> <td>0·002</td> <td>0·002</td> <td>0·36</td> </tr> <tr> <td>Average size, μm<sup>2</sup> </td> <td>24·1 (20·1–33·9)</td> <td>28·0 (24·0–34·0)</td> <td>27·0 (24·0–37·0)</td> <td>0·15</td> <td>0·07</td> <td>0·86</td> </tr> </table> <p>Data presented as median (interquartile range). M, male; F, female.</p> <p>*<i>P</i> value for Gaucher patients compared to control.</p> <p>†<i>P</i> value for Gaucher patients compared to obligatory carriers.</p> <p>‡<i>P</i> value for controls compared to obligatory carriers.</p> <p><b>III</b>  Characteristics of patients with Gaucher type I with or without enzyme replacement therapy (ERT). </p> <table frame="box" border="1" bordercolor="#999999"> <tr> <th></th> <th>No ERT</th> <th>ERT only</th> <th>ERT & splenectomy*</th> <th> <i>P</i>‐value†</th> <th> <i>P</i>‐value‡</th> <th> <i>P</i>‐value§</th> </tr> <tr> <td>Number</td> <td>11</td> <td>20</td> <td>16</td> <td>–</td> <td>–</td> <td>–</td> </tr> <tr> <td>Age, years</td> <td>39 (35–48)</td> <td>40 (25–59)</td> <td>48·5 (37–61)</td> <td>0·82</td> <td>0·16</td> <td>0·12</td> </tr> <tr> <td>Platelet count, ×10<sup>9</sup>/l</td> <td>154 (138–204)</td> <td>169 (142–193)</td> <td>262 (214–302)</td> <td>0·99</td> <td>0·002</td> <td><0·001</td> </tr> <tr> <td>Haematocrit, %</td> <td>40·6 (38·8–42·4)</td> <td>39·7 (36·6–42·9)</td> <td>41·3 (39·1–43·6)</td> <td>0·44</td> <td>0·89</td> <td>0·58</td> </tr> <tr> <td>Surface coverage, %</td> <td>3·8 (1·9–7·5)</td> <td>3·8 (2·4–5·3)</td> <td>7·2 (5·8–9·3)</td> <td>0·99</td> <td>0·09</td> <td>0·002</td> </tr> <tr> <td>Average size, μm<sup>2</sup> </td> <td>20·0 (17·1–25·1)</td> <td>25·6 (22·5–35·8)</td> <td>24·9 (22·3–31·7)</td> <td>0·21</td> <td>0·16</td> <td>0·79</td> </tr> <tr> <td>Mucosal bleeding</td> <td>6 (54%)</td> <td>7 (35%)</td> <td>4 (25%)</td> <td>0·29</td> <td>0·15</td> <td>0·7</td> </tr> </table> <p>Data presented as median (interquartile range). 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Ghislaine SURREL

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D-dimer assay in Egyptian patients with Gaucher disease: correlation with bone and lung involvement

[13-05-2011]

Sherif, Eman Ma; Tantawy, Azza AGa; Adly, Amira AMa; Kader, Hossam Ab; Ismail, Eman ARc

aDepartment of Pediatrics, Egypt, bDepartment of Radiology, Egypt, cDepartment of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Received 20 August, 2010, Revised 17 October, 2010, Accepted 27 October, 2010

Correspondence to Eman A.R. Ismail, MD, 5 Nageb Mahfoz Street, Agouza, 12654 Giza, Egypt Fax: +202 3337 5435; e-mail: eman.ismail_70@yahoo.com

Abstract

Gaucher disease is the most frequent lysosomal storage disorder. Bone and lung involvement are two major causes of morbidity in this disease. D-dimer is a reliable indicator of active microvascular thrombosis, even in patients without overt hypercoagulation. This study aimed to assess D-dimer levels in Gaucher disease, correlating this marker to clinical characteristics and radiological parameters to investigate its role as a potential predictor for the occurrence and severity of skeletal and pulmonary manifestations. The study population consisted of 56 Egyptian patients with Gaucher disease, 36 had type 1 Gaucher disease (64.3%) and 20 had type 3 Gaucher disease (35.7%). Thirty healthy individuals were enrolled as a control group. D-dimer levels were significantly higher in all patients with Gaucher disease compared with controls (P < 0.001). Patients with type 3 showed significantly higher D-dimer concentrations compared with type 1 (P < 0.001). Pulmonary involvement was present in a significant proportion among type 3 Gaucher patients (P < 0.05), whereas bone changes were present in a higher percentage in type 1 compared with type 3 Gaucher patients. D-dimers were significantly higher in patients with abnormal MRI findings of the long bones and in those with ground glass appearance on high-resolution computerized tomography of the chest compared with patients with normal radiology (P < 0.001). Splenectomized patients displayed significantly higher D-dimer levels compared with nonsplenectomized patients (P < 0.001). Our results suggest that D-dimer is significantly elevated in Gaucher disease, particularly type 3, and may be considered as a potential marker of risk prediction of bone and lung involvement that could be used to monitor treatment response.

Introduction

The lysosomal storage diseases have a cumulative incidence of 1 in 5000 live births [1]. Gaucher disease predominates among this group, having a frequency of 1 in 40 000 in the United States [2,3]. Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme acid [beta]-glucosidase [glucocerebrosidase (GBA)], which is responsible for hydrolysis of glucocerebroside [glucosylceramide (GLC)] [4]. Mutations in the GBA gene result in Gaucher disease and many of these mutations are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein [5]. Absent or reduced enzymatic activity leads to accumulation of GLC in various cells of the macrophage–monocyte system (Gaucher cells) [6,7]. Classically, the pathophysiology of Gaucher disease has been attributed to the amount, location and rate of accumulation of the stored material [8,9]. The manifestations of Gaucher disease occurring due to accumulation of Gaucher cells in three main anatomical compartments, namely the osseous skeleton, the bone marrow and visceral organs [4,10].

As with most genetic diseases, the signs and symptoms of Gaucher disease present along a continuum, ranging from the lethal neonatal form to the asymptomatic form [11]. Currently, Gaucher disease is classified into three clinical forms: nonneuropathic (type 1), acute neuropathic (type 2) and chronic neuropathic (type 3) [3,10]. Patients with type 1 disease may present at any age with hepatosplenomegaly, anemia, thrombocytopenia, often skeletal involvement (such as avascular necrosis of the large joints or pathological fractures) or lung disease. Age and mode of presentation as well as the eventual clinical course are highly variable, yet phenotypic heterogeneity can be attributed only in part to specific mutations [9,12].

Bone disease usually designates the advanced stages of Gaucher disease, but susceptibility to fractures and avascular necrosis can be the first sign of Gaucher disease in otherwise asymptomatic patients [13]. The skeletal aspects of the disease have a much greater impact on patients' quality of life than the hematological and visceral aspects. Moreover, skeletal manifestations are commonly seen in patients with normal hematology [14]. Therefore, it is important for physicians not to focus only on hematological and visceral complications on the expense of skeletal involvement when assessing the response to enzyme replacement therapy (ERT) [15,16]. Nearly, all patients with Gaucher disease have radiological evidence of skeletal involvement including Erlenmeyer flask deformity, osteopenia, osteosclerosis, osteonecrosis, fractures and bone marrow infiltration. Skeletal involvement follows three basic processes: focal disease (irreversible lesions such as osteonecrosis and osteosclerosis), local disease (reversible abnormalities adjacent to heavily involved marrow such as cortical thinning and long bone deformity) and generalized osteopenia [17]. For more accurate assessment of bone disease in adults and children, it is desirable to conduct MRI studies at centers with radiologists experienced in evaluating patients with Gaucher disease [18].

Symptomatic lung involvement may be common at presentation, and may progress over the course of the disease to pulmonary hypertension in some patients, particularly in type 3 Gaucher disease [13]. Chest radiographs may demonstrate reticulonodular changes, and on high-resolution computerized tomography (HRCT), a range of abnormal patterns including widespread ground glass opacification may be present [19,20]. 

Many factors may trigger or aggravate symptoms and signs of the disease including environmental or acquired conditions, such as viral infections or pregnancy. In addition, other concurrent genetic defects such as partial deficiency of coagulation factors aggravate the tendency to bleeding and may contribute to the variability of clinical manifestations in Gaucher patients. These modifiers may be helpful in predicting risk of developing bone and/or lung disease [3,10,21]. A logical theory to explain the incidence of avascular bone necrosis and pulmonary hypertension in Gaucher disease was an inherited predilection for hypercoagulability [9,22]. Gaucher disease has been the playground to develop new therapeutic interventions such as ERT and substrate-reduction therapy. The availability of these costly therapies has stimulated research regarding suitable biomarkers to monitor onset and progression of disease, as well as the efficacy of therapeutic intervention [23].

D-dimer assays are ordered, along with laboratory tests that help to rule out, diagnose and monitor conditions associated with hypercoagulability [24,25]. The possibility of correlation between avascular bone necrosis and pulmonary hypertension in Gaucher patients with D-dimer levels was considered because it would implicate involvement of microthrombosis, without expecting a quantitative correlation with hypercoagulability per se [9]. However, there have been few studies that evaluated the impact of this reliable indicator of coagulation on the development and severity of skeletal and pulmonary manifestations in Gaucher disease.

Therefore, this study aimed to assess D-dimer levels in Egyptian patients with Gaucher disease as a potential predictor for the occurrence and severity of bone and lung involvement correlating this marker to clinical characteristics and radiological parameters.

Patients and methods

This cross-sectional study was conducted on 56 consecutive patients with Gaucher disease attending the Hematology/Oncology Clinic in Pediatric Hospital, Ain Shams University, from June 2009 to May 2010. They were 33 men and 23 women, with a male to female ratio of 1.4: 1. The mean age at diagnosis in the study population as a whole was 6.0 ± 3.9 years (range, 2.5–13 years). Control samples (peripheral blood) were obtained from 30 healthy children and adolescents with no history of inborn errors of metabolism in their first-degree and second-degree relatives. Twelve were men and 18 were women (male to female ratio, 1: 1.5). The mean age of the control population was 7.4 ± 4.24 years (range, 3–15 years). A written consent was obtained from the guardian of each case and control before participation in the study. The study was approved from the local ethical committee.

All patients were subjected to full medical history with special emphasis on bone manifestations (pains or fractures), hematological manifestations (pallor, bleeding and history of blood transfusion), pulmonary manifestations (cough, dyspnea and recurrent chest infections) and neurological symptoms (squint, convulsions, bulber manifestations, trismus and manifestations suggestive of cerebellar affection). Thorough clinical examination was performed laying stress on anthropometric measures, abdominal examination with assessment of hepatic and splenic size and full neurological examination including cranial nerves, motor and sensory systems. Radiological investigations included plain radiograph on long bones, chest radiograph, abdominal ultrasonography to assess the volume of liver and spleen in cubic centimeter (calculated as multiples of normal sizes predicted for body weight) [15] and MRI on pelvis and femur, as well as HRCT on both lung fields.

Laboratory investigations for Gaucher patients included complete blood count, examination of peripheral blood-stained smears, bone marrow aspiration and examination of stained smears and coagulation profile, as well as liver and renal function tests. GBA enzyme activity was assessed in peripheral blood leukocytes and plasma chitotriosidase was measured at diagnosis and for follow-up of the response of ERT as a sensitive indicator of dose effects. Molecular analysis of the acid GBA gene was performed for 18 patients.

Diagnostic criteria

Diagnosis of Gaucher disease was based on the presence of hepatosplenomegaly, bone involvement, hematological manifestations and developmental delay with or without neurological manifestations in the form of occulomotor abnormalities and/or convulsions. This was confirmed by laboratory findings of Gaucher cells in bone marrow aspirate and biopsy, low GBA activity [4] and high plasma level of chitotriosidase activity [26,27].

The clinical and laboratory characteristics of the studied Gaucher patients are summarized in Table 1. The studied patients were divided into two groups: 

Table 1 Clinicopathological characteristics of the studied Gaucher patients

Group I included 36 patients with type 1 Gaucher disease (64.3%). They were 20 men and 16 women with a male to female ratio of 1.3: 1. Their ages at the time of the study ranged from 2.5 to 18 years with a mean of 6.6 ± 5.7 years. Type 1 patients presented initially with hepatosplenomegaly, bony manifestations, anemia and thrombocytopenia. At time of the study, 19 of 36 type 1 patients had anemia and 15 had thrombocytopenia as well as organomegaly. The duration of treatment ranged from 1 to 8 years with a mean of 2.9 ± 2.4 years. They were under ERT in a dose ranging from 60 to 90 U/kg per 2 weeks with a mean of 66 ± 13.4 U/kg per 2 weeks.

 Group II included 20 patients with type 3 Gaucher disease (35.7%). They were 12 men and eight women (male to female ratio, 1.5: 1). Their ages at the time of the study ranged from 2.5 to 15 years with a mean of 7.8 ± 4.4 years. Type 3 patients presented with neurological manifestations in the form of ophthalmoplegia, neck stiffness, dysphagia, convulsions and trismus, in addition to visceral and hematological manifestations. The duration of treatment ranged from 1.5 to 8 years with a mean of 6.6 ± 5.7 years. They were under ERT in a dose ranging from 60 to 120 U/kg per 2 weeks with a mean of 84 ± 20.8 U/kg per 2 weeks.

 None of the studied patients had advanced liver disease, active inflammatory conditions or were on anticoagulant therapy at time of study. Fourteen patients (35%) were splenectomized prior to the onset of ERT. All patients were under regular ERT [28,29] for a period of 1–8 years starting from January 1999. ERT was first given using the placenta-derived aglucerase (Ceredase; Genzyme Corporation, Cambridge, Massachusetts, USA) at low dose regimen (15 U/kg per 2 weeks). The recombinant enzyme imiglucerase (Cerezyme; Genzyme Corporation) was applied in March 1999 in a dose of 20–30 U/kg per month, divided into four equal weekly doses. In July 1999, Cerezyme was administered in a dose of 60 U/kg per 2 weeks. Six patients with type 3 received high doses of 120 U/kg per 2 weeks, one of them had progressive neurological disease and the others suffered severe pulmonary involvement.

 Blood sampling and assay of d-dimer

 As part for routine work-up for Gaucher disease, peripheral blood samples were collected on 3.2% buffered sodium citrate at a ratio of nine parts blood to one part anticoagulant (1: 10 ratio) and centrifuged at 1600 g for 15 min. D-dimer concentrations were determined in platelet-poor plasma using Tina-quant assay (Roche Diagnostics, Mannheim, Germany) on a Hitachi 917 analyzer (Roche Diagnostics). Tina-quant D-dimer is an immunoturbidimetric assay in which a beam of monochromatic light is passed through the suspension of latex microparticles coated by covalent bonding with monoclonal antibodies specific for D-dimer. The wavelength of the light is greater than the diameter of the latex microparticles and thus the solution of latex microparticles only slightly absorbs the light. In the presence of D-dimer, the particles aggregate and turbidity increases. The increase in scattered light is proportional to the amount of D-dimer in the test sample. The latex particles are coated with a monoclonal antibody that reacts with fibrin D-dimer or fragment D of fibrin. The antibody has no cross-reactivity with fibrinogen. This allows quantitative determination of D-dimer in human plasma. Normal limit of the assay is 500 µg/l. The test has a lower detectable limit of 40 µg/l [25,30–32].

 Statistical analysis

 The processing of data was computed using statistical package for social science (SPSS), version 14 IBM compatible PC (SPSS Inc., Chicago, Illinois, USA). Data were described in the form of number and percentage, and range and mean ± SD. In order to compare quantitative variables between two groups, both the Student's t-test and the nonparametric Mann–Whitney U-test were applied. A [chi]2-test was used to compare qualitative variables. A P-value of 0.05 or less and 0.01 or less was considered significant and highly significant, respectively, in all analyses.

Results

 Clinical, hematological and radiological features of gaucher patients

 Type 1 Gaucher disease was found in 64.3% of patients, and type 3 in 35.7%. Molecular analysis of the acid GBA gene in 18 patients revealed homozygosity for L444P in 11 patients (all were type 3), whereas six type 1 Gaucher patients showed mutation of N370S and only one patient with type 1 Gaucher disease displayed the uncommon homozygous D409H mutation.

 As shown in Table 1, age and sex did not differ significantly between patients in both groups, although positive family history for Gaucher disease was significantly present in type 3 compared with type 1 Gaucher patients (P < 0.05). Underweight was frequently observed in type 3 patients, whereas short stature was frequently observed in type 1 (P < 0.001). Mean hepatic and splenic volumes reported in multiples of normal size predicted for body weight in type 1 Gaucher disease were significantly higher when compared with those of type 3 patients (P < 0.001). White blood cell count, hemoglobin levels and platelet counts did not show any significant difference between both study groups of Gaucher disease (P > 0.05). Type 3 Gaucher patients received significantly higher doses of ERT compared with type 1 (P < 0.001).

 As regards radiological findings (Table 1), in type 1 Gaucher disease, 21 patients (58%) had Erlenmeyer flask-shaped deformity in radiograph of long bones, as well as bone marrow expansion with cortical thinning in MRI. Moreover, 13 patients (36.1%) had ground glass appearance on HRCT chest. In type 3 Gaucher disease, nine patients (45%) had Erlenmeyer flask-shaped deformity in radiograph and bone marrow expansion with cortical thinning in MRI (Fig. 1). Fourteen patients (70%) had ground glass appearance in HRCT chest (eight of them had interlobular thickening). Pulmonary involvement shown by HRCT results (Figs 2 and 3) was present in a significantly higher percentage among type 3 Gaucher patients compared with type 1 (P < 0.05). Although bone changes represented by MRI findings were present in a higher percentage in type 1 compared with type 3 Gaucher disease, yet the difference did not reach statistical significance (P > 0.05). It was noticed that the studied Gaucher patients under ERT in a dosage of 60 U/kg per 2 weeks suffered from clinically and radiologically evident pulmonary symptoms confirmed by HRCT findings. However, when these patients received higher doses of ERT ranging from 90 to 120 U/kg per 2 weeks, a remarkable improvement in their clinical manifestations was observed. The frequency and severity of chest infections that required hospitalization were markedly decreased. There was decreased dyspnoea, clubbing and cyanosis in some patients, although on radiology, lung pathology was not normalized. Others showed improved respiratory compliance, with a significant improvement of the radiological findings.

 D-dimer levels in control population and gaucher patients

 D-dimer was negative (<500 µg/l) in controls with a range of 77.8–410 µg/l and a mean value of 215.7 ± 32.5 µg/l. All patients with Gaucher disease had significantly elevated D-dimer levels, with a range of 510–4329 µg/l and a mean of 2018 ± 368 µg/l at time of the study (P < 0.001). Patients with type 1 and type 3 Gaucher disease displayed significantly high D-dimer concentrations than controls when compared separately (P < 0.001) (Fig. 4). Moreover, D-dimer levels were significantly higher in type 3 patients compared with type 1 (mean, 1863.5 ± 414 vs. 830 ± 275.2 µg/l; P < 0.001) (Table 1 and Fig. 4).

 D-dimer analysis results in relation to clinical and radiological data

 Analysis of D-dimer in relation to clinical and radiological data of Gaucher patients (Table 2) revealed significantly higher levels in patients with abnormal MRI findings of long bones compared with those with normal MRI study (mean, 1563.6 ± 357 vs. 810.7 ± 250 µg/l; P < 0.001). In addition, Gaucher patients with pulmonary involvement on HRCT chest had higher D-dimer concentrations compared with those with normal radiological study (mean, 1980 ± 400 vs. 990 ± 205 µg/l; P < 0.001). Splenectomized patients displayed significantly higher D-dimer levels compared with nonsplenectomized patients (mean, 1670 ± 326 vs. 746 ± 187 µg/l; P < 0.001).

Table 2 Clinical and radiological features of the studied Gaucher patients according to D-dimer levels

Discussion

Studies of genotype–phenotype correlations in Gaucher disease, the most common sphingolipidosis, revealed significant genotypic heterogeneity among clinically similar patients and vastly different phenotypes among patients with the same mutations [11]. The incidence of the subacute neuronopathic (type 3) form is less than one in 100 000 patients. The distribution of type 3 Gaucher disease is panethnic, but this form predominates in some geographic regions such as northern Sweden [33,34]. In the present study, type 3 Gaucher disease was found in 35.7% of patients. This could be explained by the higher prevalence of the genotype L444P among this ethnic group as it was reported that N370S homozygotes generally present with a less severe phenotype, whereas L444P and D409H homozygosity confers neurologic involvement [2,4,35]. This is in contrast to Ashkenazi Jewish population in which the genotype N370S is predominant, resulting in higher prevalence of type 1 Gaucher disease with mild clinical manifestations [13]. Moreover, some genetic mutations are unique to individual groups or families. For instance, D409H/D409H causes calcification of the heart valves and occulomotor apraxia, but without visceral affection occurring uniquely in Jenin Arabs, Japanese and Spanish patients [36,37].

 Failure to thrive and underweight were prominent features in the studied patients, especially in type 3 Gaucher disease. In line with our results, Zimran et al. [38] noticed that poor weight gain and growth retardation in those patients occurred likely due to the excessive metabolic burden from accumulation of undegraded substrate and GLC in tissues, together with the chronic nature of the disease and anemia that accounts for the chronic fatigue reported by many patients. The low-grade, smoldering and subclinical internal inflammation in individuals with Gaucher disease is accompanied by an increased degree of erythrocyte and leukocyte adhesiveness/aggregation. These findings might have rheological consequences in terms of microcirculatory slow flow and tissue hypoxemia. An additional factor in type 3 Gaucher patients is pseudobulbar palsy resulting in dysphagia, regurgitation and recurrent aspiration pneumonia [39]. Similarly, Khalifa et al. [40] reported that patients with Gaucher disease were more susceptible to recurrent infection, which is a main cause of debilitation. The tendency toward infection in Gaucher disease patients was attributed to their postsplenectomy state. Furthermore, Orvisky et al. [8] noticed resumption of normal growth in children after receiving ERT.

As previously reported, the estimated life expectancy for patients with type 1 Gaucher disease was mildly decreased than the reference population being 9 years less [41]. Disease progression varies in type 1 Gaucher disease and survival may be normal depending on the severity of complications. On the contrary, evolution is rapid in type 2 Gaucher disease leading to death within the first 2 years of life, usually because of lung failure, whereas type 3 Gaucher disease patients often survive to the second or third decades of life [2,42]. In this study, the significantly higher doses of ERT received by patients with type 3 Gaucher disease compared with type 1 were in concordance to the report of Vellodi et al. [43], who stated that there is clear evidence that ERT improves systemic involvement in nonneuronopathic as well as neuronopathic Gaucher disease enhancing quality of life. In addition, ERT has reversed, stabilized or slowed the progression of neurological involvement in some patients and is considered the gold standard treatment for Gaucher disease type 1 and type 3 patients [27,43,44]. High dose provides a faster clinical response and should be considered for patients with more aggressive disease [45].

 Despite some general genotype–phenotype correlations, disease severity and clinical outcomes cannot be predicted on the basis of genotype [13,46]. Genetic modifiers may play an important role in determining the eventual Gaucher phenotype [39]. Identification of biochemical markers characteristic of pathology may be of use in predicting the progression of the disease state [23,34,47]. There are no predictive tests to ascertain patients at risk for bone and lung involvement in Gaucher disease, which are slow to respond to ERT [4,9]. As reported by Deghady et al. [48], bleeding tendency in Gaucher disease may not always be related to absolute platelet counts, but may be influenced by coagulation factor deficiencies or abnormal platelet function. Acquired coagulation factor deficiencies have been demonstrated in Gaucher disease. The mechanism involved is unclear, but may include low-grade disseminated intravascular coagulation or sequestration of coagulation factors by substrate-loaded cells [10].

 Quantitative D-dimer determination has become routine practice in patients evaluated for the presence of deep venous thrombosis or pulmonary emboli [25,49]. In this study, plasma fibrin D-dimer was assayed in Gaucher disease using a new quantitative latex test for cross-linked fibrin degradation products, involving a specific monoclonal antibody. This method has been proven to be rapid, sensitive and reproducible [30].

The relevance of the determination of D-dimer in Gaucher disease was evaluated and high plasma D-dimer levels were found among patients with Gaucher disease, especially type 3. In addition, a significant elevation was observed in those with bone changes and pulmonary involvement as reflected by radiological findings. In agreement with our results, Shitrit et al. [9] stated that the levels of elevated of D-dimers were related to the presence of pulmonary infiltration, by Gaucher cells, as well as bone marrow infiltration. They implied that microthrombi may be part of the pathogenesis for avascular necrosis as well as pulmonary hypertension in patients with Gaucher disease. The significant correlation of D-dimer levels with treatment may be an indirect marker of bone and/or lung in Gaucher disease [9]. Additionally, Boot et al. [33] and Maire et al. [34] reported that the elevation of the serum concentration of several serologic markers (e.g. D-dimer, CCL18/PARC, CD163) in persons with Gaucher disease is considered a possible surrogate indicator of disease burden that could be used in monitoring treatment response.

 Moreover, it was reported that a normal MRI study in Gaucher patients with elevated D-dimer levels does not rule out subsequent development of bony changes in those patients, especially if splenectomized. Therefore, serial follow-up is required and the rising titer of D-dimer may determine or indicate the proper timing of the next MRI study [20].

 Strikingly, higher D-dimer concentrations were found among splenectomized patients with Gaucher disease. These results were in line with those obtained by Ashkenazi et al. [50], who observed that patients with Gaucher disease usually start to suffer from severe bony manifestations and pulmonary hypertension after splenectomy. In a preliminary study, Tunaci et al. [51] reported that asplenia was strongly associated with life-threatening forms of pulmonary hypertension in Gaucher disease, as removal of the spleen, which is the primary reservoir of storage cells, promotes migration of the mononuclear phagocyte system toward other tissue macrophage pools (lung and bone). Recently, Mistry et al. [52] observed a higher risk of avascular necrosis was observed among patients who had previously undergone splenectomy.

 Bone involvement was evident clinically and radiologically in the studied patients with type 1 Gaucher disease while pulmonary involvement reflected by HRCT findings was significantly found among type 3 Gaucher disease compared to type 1. This finding could be explained by the significantly elevated D-dimer levels encountered in type 3 Gaucher patients compared to type 1 Gaucher disease which is a less severe form. In this context, El-Beshlawy et al. [46] reported that bone manifestations were the most common presenting symptoms in Egyptian patients with type 1 Gaucher disease as evidenced by MRI long bones and ERT was effective in ameliorating radiological manifestations of skeletal disease and achieving complete remission of bone pain but it required a long period of treatment. Moreover, Jmoudiak and Futerman [13] reported that lung involvement including interstitial lung disease and pulmonary hypertension was found in a small number of patients with type 1 Gaucher disease. Additionally, Miller et al. [53] reported a higher frequency of pulmonary involvement in type 3 Gaucher disease compared to type 1. On the other hand, few patients develop pulmonary hypertension on ERT [54]. Mistry et al. [22] also revealed a remarkable predisposition for pulmonary hypertension in type 1 Gaucher disease. Progression to severe, life-threatening pulmonary hypertension occurs in the presence of additional genetic factors (non-N370S GBA mutation, positive family history and angiotensin converting enzyme I gene polymorphism) and epigenetic modifiers (i.e. asplenia, female sex) [22]. Therefore, routine echocardiographic monitoring of all treated and untreated patients with type 1 Gaucher disease was recommended and consideration of treatment withdrawal was suggested if the TI gradient progresses to more than 30 mmHg [54].

 In the present analysis, the remarkable improvement occurred in pulmonary manifestations with higher doses of ERT confirmed the previous reports by Elstein et al. [54] and Ashkenazi et al. [50], who noticed that the lung and bone are less accessible compartments to ERT, so that a low-dose regimen may not be sufficient to overcome a threshold for Gaucher cell infiltration and the dose must be increased to allow an adequate enzyme concentration at the site of the pathology, especially if there are fibrotic or necrotic regions. Moreover, Goitein et al. [55] reported that in patients with Gaucher disease and symptomatic lung involvement, there is great heterogeneity in presentation and response to ERT. Clinically, some benefited significantly from ERT, but in contrast to the dramatic reduction in organomegaly, there was no normalization in pulmonary function or lung architecture. These findings emphasized the importance of optimal dose of ERT, avoidance of splenectomy and vigorous combination therapy with vasodilators, as appropriate for the best outcomes in Gaucher patients [16,29,43].

 In conclusion, elevated D-dimer levels in Gaucher patients can be used as a predictor for the development of skeletal and pulmonary complications and may suggest that the occurrence of these pathological changes is induced initially as microthrombi. Thereafter, it would be interesting to apply the fully quantitative automated D-dimer assay in a prospective manner to ‘borderline’ Gaucher patients with persistent dyspnea, bronchorrhea and recurrent pneumonopathy, to ascertain whether they have merely incipient pulmonary changes and follow-up them for the possibility of developing progressive pulmonary hypertension. Further studies are recommended to indicate whether a predictive cut-off level of D-dimer in Gaucher disease can be described.

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[30.] Schutgens REG, Haas FJLM, Ruven HJT, Spannagl M, Horn K, Biesma DH. No influence of heparin plasma and other (pre)analytic variables on D-dimer determinations. Clin Chem 2002; 48:1611–1613.

[31.] Weber T, Högler S, Auer J, Berent R, Lassnig E, Kvas E, et al. D-Dimer in acute aortic dissection. Chest 2003; 123:1375–1378.

[32.] Wieganda J, Kollerb M, Bingissera R. Does a negative D-dimer test rule out aortic dissection? Swiss Med Wkly 2007; 137:462.

[33.] Boot RG, Verhoek M, de Fost M, Hollak CE, Maas M, Bleijlevens B, et al. Marked elevation of the chemokine CCL18/PARC in Gaucher disease: a novel surrogate marker for assessing therapeutic intervention. Blood 2004; 103:33–39.

[34.] Maire I, Guffon N, Froissart R. Current development and usefulness of biomarkers for Gaucher disease follow up. Rev Med Intern 2007; 28(Suppl. 2):S187–S192.

[35.] Barranger J, Rice E. An overview of Gaucher disease. Gaucher Clin Perspect 1993; 1:1–5.

[36.] Tayebi N, Park J, Madike V, Sidransky E. Gene rearrangement on 1q21 introducing a duplication of the glucocerebrosidase pseudogene and a metaxin fusion gene. Hum Genet 2000; 107:400–403.

[37.] Beutler E. Gaucher disease: multiple lessons from a single gene disorder. Acta Paediatr Suppl 2006; 95:103–109.

[38.] Zimran A, Bashkin A, Elstein D, Rudensky B, Rotstein R, Rozenblat M, et al. Rheological determinants in patients with Gaucher disease and internal inflammation. Am J Hematol 2004; 75:190–194.

[39.] Mistry P. Phenotype variations in Gaucher disease. Rev Med Intern 2006; 27:S3–S6.

[40.] Khalifa A, Tantawy A, Monir E, Sadek A, Tiseer N. Immune dysfunction in patients with Gaucher disease: impact of disease severity and enzyme replacement therapy. Haematologica 2010; 95(suppl.2):37.

[41.] Weinreb NJ, Deegan P, Kacena KA, Mistry P, Pastores GM, Velentgas P, et al. Life expectancy in Gaucher disease type 1. Am J Hematol 2008; 83:896–900.

[42.] Grabowski GA. Phenotype, diagnosis, and treatment of Gaucher's disease. Lancet 2008; 372:1263–1271.

[43.] Vellodi A, Bebi B, de Villemeur TB, Collin-Histed T, Erikson A, Mengel E, et al. Management of neuronopathic Gaucher disease: a European consensus. J Inherit Metab Dis 2001; 24:319–327.

[44.] Cox TM, Aerts JM, Andria G, Beck M, Belmatoug N, Bembi B, et al, Advisory Council to the European Working Group on Gaucher disease. The role of the iminosugar N butyldeoxynojirimycin (miglustat) in the management of type I (nonneuronopathic) Gaucher disease: a position statement. J Inherit Metab Dis 2003; 26:513–526.

[45.] Altarescu G, Schiffmann R, Parker CC, Moore DF, Kreps C, Brady RO, Barton NW. Comparative efficacy of dose regimens in enzyme replacement therapy of type I Gaucher disease. Blood Cells Mol Dis 2000; 26:285–290.

[46.] El-Beshlawy A, Ragab L, Youssry I, Yakout K, El-Kiki H, Eid K, et al. Enzyme replacement therapy and bony changes in Egyptian pediatric Gaucher disease patients. J Inherit Metab Dis 2006; 29:92–98.

[47.] Whitfield PD, Nelson P, Sharp PC, Bindloss CA, Dean C, Ravenscroft EM, et al. Correlation among genotype, phenotype, and biochemical markers in Gaucher disease: implications for the prediction of disease severity. Mol Genet Metab 2002; 75:46–55.

[48.] Deghady A, Marzouk I, El-Shayeb A, Wali Y. Coagulation abnormalities in type 1 Gaucher disease in children. Pediatr Hematol Oncol 2006; 23:411–417.

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Imaging Findings in Pediatric Type 1 Gaucher Disease: What the Clinician Needs to Know
[13-05-2011]

Green, Brian A. MD^*; Alexander, Alan A.Z. MD^†; Hill, Phillip R. MD^*; Lowe, Lisa H. MD^‡

^*Department of Radiology, University of Missouri School of Medicine, 1 Hospital Drive, Columbia
^‡Department of Radiology, University of Missouri-Kansas City and Children's Mercy Hospital and Clinics, Kansas City, MO
^†Department of Radiology, University of Virginia, 1 Hospital Drive, Charlottesville, VA
The authors have no financial disclosures to report.
Reprints: Alan A.Z. Alexander, MD, Department of Radiology, University of Virginia, 1 Hospital Drive, Charlottesville, VA 22908 (e-mail: aaz.alexander@yahoo.com; e-mail: lhlowe@cmh.edu).

Abstract

This study presents visceral and skeletal imaging findings commonly observed in pediatric patients with type I Gaucher disease. Presented images show methods used for radiologic assessment of pediatric Gaucher patients, and imaging findings are discussed in the context of the underlying pathophysiology of the disease. Routine radiologic surveillance plays a central role in assessing Gaucher disease progression and response to treatment, but monitoring of pediatric patients presents specific challenges with regard to minimizing radiation exposure and interpreting extent of marrow involvement against the backdrop of normal growth-related changes in marrow composition. In addition to highlighting imaging findings in children with type I Gaucher disease, this manuscript discusses alternate modalities, which minimize radiation and may be just as accurate, if not better, than conventional methods exposing the child to radiation.

Keywords: gaucher disease; marrow; hepatosplenomegaly; imaging; enzyme replacement therapy

Table of contents

• Symptoms and management
• Imaging
• Acknowledgments
• References

Gaucher disease is an inherited autosomal recessive metabolic disorder resulting from mutations in the gene encoding [beta]-glucocerebrosidase, an enzyme in the glycosphingolipid degradation pathway.1 Glucocerebrosidase deficiency leads to massive accumulation of the insoluble lipid glucocerebroside in lysosomes of cells of monocyte/macrophage lineage, principally macrophages. Engorgement with glucocerebroside alters macrophage morphology producing the so-called “Gaucher cells” that are a hallmark of the disease (Fig. 1). Gaucher cells may occur anywhere where macrophages are found, but they are particularly abundant in tissues of the reticuloendothelial system.

Figure 1

Symptoms and management

Gaucher disease is a rare disorder, but is the most commonly occurring lysosomal storage disease.1 Gaucher disease has been divided into 2 major clinical subtypes based on whether the nervous system is affected. The non-neuronopathic form, designated as type 1, accounts for 90% of all cases.2 The extremely rare neuronopathic variants have been subdivided into acute (type 2) and subacute (type 3) forms. Type 2 manifests in early infancy and is characterized by rapidly progressive nervous system and brain degeneration with patients seldom surviving past 2 years of age.3 Type 3 has a more protracted course with neurological signs appearing in late infancy or childhood. Inidividuals reaching adolescence may survive into their third or forth decade.1,3

Non-neurological manifestations of Gaucher disease are due to progressive infiltration of organs by Gaucher cells. Anemia, thrombocytopenia, and hepatosplenomegaly arise in all 3 forms of the disease. Patients with type 1 disease commonly develop skeletal complications due to bone marrow infiltration. Macrophage activation concomitant with lipid deposition can cause chronic, low-grade inflammation or acute flare-ups with severe bone pain.4 Children with type 1 Gaucher disease are frequently growth restricted and experience delayed puberty.4,5

Treatment for type 1 disease is enzyme replacement therapy (ERT) using macrophage-targeted recombinant human glucoceribrosidase (Cerezyme, Genzyme Corporation, Cambridge, MA).6 Treatment goals include alleviating symptoms, reducing hepatosplenomegaly, stabilizing or reversing skeletal disease, and for children, restoring normal growth rates.2,6 Most type 1 patients have near normal life spans, particularly if ERT is started early.1

A misconception about type 1 is that it is an “adult” form of Gaucher disease; 66% of type 1 patients are diagnosed before the age of 20 years.6 Childhood onset of type 1 disease generally predicts a more rapid, severe course.6,7 Noting an underappreciation of the prevalence and severity of pediatric type 1 disease, the International Collaborative Gaucher Group has called for increased focus on recognizing and assessing non-neuronopathic Gaucher disease in children.7

Imaging

Although imaging is not generally used for diagnosis, radiology at initial presentation is useful in assessing initial severity and extent of disease in the skeleton. Furthermore, it plays an important role in monitoring disease progression and response to ERT. Careful monitoring of skeletal disease is essential for preventing irreversible complications and crippling disability.8 The following is a review of visceral and skeletal imaging findings typically encountered in children with type 1 Gaucher disease.

Spleen and liver imaging findings

Hepatosplenomegaly is one of the most common effects of Gaucher disease (Fig. 2A). In children, splenomegaly can be marked and often is accompanied by growth failure, cachexia, and hypermetabolism.5 Significant organ enlargement increases the likelihood of focal abnormalities identified with cross-sectional imaging, including Gaucher cell aggregations, vascular infarctions, and regions of fibrosis 5 (Fig. 2B).

Figure 2

Reductions in spleen and liver size are early, sensitive indicators of response to ERT.6 Therefore, obtaining accurate baseline measures of organ volumes is important for assessing treatment response and adjusting ERT dosage. Magnetic resonance imaging (MRI) and computed tomoghaphy (CT ) were generally considered to be superior to ultrasound (US) for making volume determinations, especially in older children, and have greater accuracy and increased sensitivity for detecting focal lesions (Fig. 3). However, recently developed 3-dimensional techniques available on contemporary US machines may offer an alternative method to assess organ volumes in younger children without need for sedation or ionizing radiation exposure. Many Gaucher specialists consider US measures to be sufficiently accurate for clinical decision making.9

Figure 3

International Collaborative Gaucher Group guidelines recommend volume determination for children at baseline, when ERT is begun, at time of dosage change, and every 12 to 24 months thereafter.6 Measured volumes can be compared with age-based or weight-based estimates of normal volumes to determine extent of enlargement.10,11

Skeletal imaging findings

Skeletal complications are the most painful and disabling aspect of type 1 disease, with the long bones and vertebrae being most commonly affected.6 Skeletal effects are not because of glucocerebroside deposition per se, but rather result from marrow packing and replacement by Gaucher cells. Effects related to marrow infiltration by Gaucher cells include vascular occlusion, ischemia, and cytokine-induced alterations in bone turnover rates.2,10 More than 80% of patients have some degree of skeletal involvement.12 Skeletal disease manifestations include bone pain related to inflammation and edema, trabecular resorption with fibrosis, generalized osteopenia, cortical thinning and bone remodeling, pathologic fractures, and joint deformity or collapse due to avascular necrosis. Joint replacement surgery may be necessary; however, arthroplasty failure rates are higher for Gaucher-diseased bones.13

Skeletal response to ERT is slow and may take years. Children respond more quickly than adults, possibly because of their higher bone turnover rates.14 Treatment goals in pediatric patients are to prevent pain and osteonecrosis and to restore bone mineral density by the second year of treatment.2

As substantial marrow infiltration can occur before changes become apparent on plain radiographs, MRI is the preferred modality for monitoring skeletal disease.6,13 T1-weighted sequences are most sensitive for detecting marrow infiltration (Fig. 4A). Mature, adipose-containing marrow is normally hyperintense on T1-weighted MR images, but Gaucher cell infiltration reduces marrow signal intensity to approximately that of muscle. T2-weighted imaging with fat-suppressed sequences facilitates detection of complications such as infarcts (Fig. 4B). Coronal T1 and T2-weighted scans of the femora should be performed at the same intervals and frequency as the visceral assessments described above. Sagittal echo train inversion-recovery (STIR ) images may be used as an alternative to T2-weighted images if desired.6

Figure 4

In very young children, growth-related changes in marrow composition complicate interpretation of marrow T1 images.6,13,14 As children mature, hypointense, hematopoietic red marrow is replaced by hyperintense fatty marrow in a centripetal pattern.13,14 Therefore, increase in signal intensity due to marrow maturation may be misread as a positive treatment response, and red marrow signal may obscure Gaucher cell infiltration in younger children. To circumvent this problem, some clinicians have suggested monitoring the distal regions of the lower extremities, that is, the tibiae and ankles.13 As marrow matures earliest in these regions, reduced T1 signal would indicate significant skeletal involvement.

Several semiquantitative scoring methods have been developed to assist in staging skeletal involvement and treatment response.12,13 A quantitative assessment of marrow involvement can be obtained using chemical shift MRI, which can directly measure absolute marrow fat content.13,14 However, this methodology has not yet been widely adopted into clinical practice in children (though it has in adults). The use of ^99mTc-sestamibi scintigraphy for assessment of skeletal involvement in Gaucher disease has also been discussed. Accumulation of sestamibi in the bone marrow reflects the degree of infiltration by Gaucher cells, and is not affected by physiologic age-related changes in bone marrow composition. Despite this, radiation exposure favors the use of MRI over sestamibi scintigraphy in children.8

With each sestamibi scan, the pediatric radiation exposure is at least 500 to 600 mrem, and upward of 10 mSv. According to the Alliance for Radiation Safety in Pediatric Imaging (Image Gently), <100 to 150 mSv is the acceptable cumulative low level dose during childhood years. Unique to children with radiation exposure, there is a longer time to manifest changes from exposure thereby increasing their cancer risk. Their tissues are also more radiosensitive, and the effective dose is higher for small cross sections in children when compared with adults (with similar CT parameters). Radiation dose is also cumulative over a lifetime and the risk estimate is based on a linear model; risks have been reported as high as 1:500 in the literature when exposed to low level radiation. For these reasons, we prefer use of US or MRI when possible. If CT is necessary, it is important to image only the area of interest, avoid multiphase scanning, and limit the radiation exposure by “child sizing” the CT technique.15

Dual-energy x-ray absorptiometry may also be used to monitor bone mineral density in children with Gaucher disease, as a persistently low bone mineral density in Gaucher patients on ERT may warrant consideration for bisphosphonate therapy.13

On the basis of The Gaucher disease Working Group recommended guidelines with regards to imaging, several of these aforementioned modalities may be used. The recommendations include imaging a substantial part of the bone marrow compartment (femur, pelvis, and spine) at baseline and at least every 2 years, ideally annually; T1-weighted and either T2-weighted or STIR sequences are recommended for routine evaluation, and STIR sequences are recommended to detect complications. Dual-energy x-ray absorptiometry is recommended to assess lumbar spine, proximal femur, and the entire body at baseline and annually thereafter.8

A number of Gaucher osseous effects are visible on conventional plain radiographs (Figs. 5, 6).16 Radiography is most useful for identifying causes of acute bone pain such as pathologic fractures or osteonecrosis. It is also the modality of choice for arthroplasty follow-up.12,13 Although not highly sensitive or specific, radiographs may be the primary method of evaluating the skeleton in locations with limited access to MRI. Infrequently, the diagnosis of Gaucher disease may be initially suspected by recognition of Erlenmeyer flask deformities of the long bone metaphyses (a classic radiography sign). Splenectomy is associated with the development of osteolytic lesions, osteonecrosis, and bone mineral density deficits. In addition, asymptomatic vertebral collapse may be noted, along with medial proximal humeral notching. Incidental radiographic findings may rarely provide the initial clues that a child has Gaucher disease, therefore it is essential to consider this in the differential when observing such findings.17

Figure 5

Figure 6

Acknowledgments

The authors thank Pamela S. Cooper, PhD, for editorial assistance in preparation of the manuscript, and Lei Shao, MD, for her assistance with the pathologic figure.

References

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Journal of Pediatric Hematology/Oncology 2011; 33(4): 301-5