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Gaucher disease, the most common lysosomal storage disease, is an autosomal recessive disorder. The primary biochemical defect is the deficiency of glucocerebrosidase, a lysosomal acid β-glucosidase, which results in the accumulation of glucosylceramide in lysosomes of reticuloendothelial cells (1).

The gene locus for glucocerebrosidase has been mapped to chromosome 1 in the q21 region (2). Several point mutations have been described, and the presence of two gene defects has been associated with the clinical expression of the disease (3). However, differences in the phenotype expression cannot be always explained by the amino acid changes or truncated proteins resulting from those gene defects (4).

Gaucher disease has several clinical subtypes. Type 1 Gaucher disease represents non-neuronopathic disease and it is characterized by splenomegaly, hepatomegaly, pancytopenia, and osteolytic and osteopenic degeneration of the skeleton. The clinical expression of the disease is variable and may manifest during the first years of life or remain undetected until the last decades of life (5). Type 2 is less common and is characterized by a rapidly progressive neurovisceral storage disorder and early death. Type 3 is less rapidly progressive and also involves a neurovisceral storage disorder and early death (6, 7).

Childhood-onset Gaucher disease has similar features, but there are a number of indirect manifestations of Gaucher disease in the affected child that also provide indicators of disease activity (8). One of these manifestations is growth retardation, which seems to be especially valuable in assessing severity as well as rate of progression of Gaucher disease in children. Nearly half of pediatric type 1 patients have anthropometric evidence of growth retardation, which correlates with disease severity as measured by other parameters (8, 9). In some children, growth retardation may be the sole overt manifestation of type 1 Gaucher disease (8). The cause of this poor growth is not yet elucidated. It has been reported that ERT accelerates growth in these patients (9). In a cohort of 15 Spanish patients with type 1 Gaucher disease who were receiving ERT, we have observed a significant increase in height z score. The estimated z score before treatment was −1.65, and after 13 ± 5.6 mo of ERT it increased to −1.25 (p < 0.05) (unpublished results).

IGF are recognized as important determinants of somatic growth (10). Recent research has proven that IGF have an anabolic effect even in adults (11). These peptides stimulate whole-body and muscle protein synthesis. IGF exist in the circulation complexed to IGFBP, which have been shown to increase the half-lives of IGF, and to modulate their bioactivities and bioavailabilities (12). Six high-affinity IGFBP have been classified; the predominant serum IGFBP is IGFBP-3, a glycosylated protein of 40–45 kD. IGFBP-3 is the only IGFBP that can circulate as part of a large 130–150 kD ternary complex in association with IGF and an ALS. The role of ALS has not been completely determined, but ALS may have a central role in regulating the bioavailability of IGF by stabilizing the IGF–IGFBP-3 complex (12, 13). These proteins, produced by liver, are GH-, age-, and nutritionally dependent (14). The major correlates of growth are changes in the concentrations of circulating IGFBP-3 and IGF-I. It has been proven that chronic diseases with liver involvement can cause IGF deficiency (15, 16).

The aim of this study was to asses the IGF axis in patients with childhood-onset Gaucher disease, before and after ERT, and its association with other clinical and analytical parameters.

METHODS

Twenty-two Spanish patients with Gaucher disease were studied. The diagnosis was established before 14 y of age in all patients. Biochemical and genetic studies were performed with the informed consent of all participants. Diagnosis of Gaucher disease was based on clinical features, appearance of Gaucher cells in samples of bones marrow, and low activity of acid β -glucosidase in leukocytes. Our experimental protocol was approved by the Committee on Human Experimentation at the University of Zaragoza, Zaragoza, Spain, and was in accordance with the Helsinki Declaration of 1975, as revised in 1983.

Clinical and analytical data were obtained from a questionnaire sent to pediatricians and hematologists who managed these patients with Gaucher disease in Spanish hospitals. The age-adjusted severity score index of Zimran et al.(17) was used to evaluate the clinical status. Enzymatic activity, genetic studies, and IGF determinations were done in a reference laboratory (the same for all the samples). Blood samples were obtained from patients after an overnight fast and stored in test tubes containing EDTA. Plasma was separated by low-speed centrifugation at 2000 g for 30 min at 4°C. The activity of glucocerebrosidase was measured on leukocytes with 4-methylumbelliferyl-β -d-glycopyranoside as substrate (18). Genomic DNA of all participants was isolated from white blood cells by standard methods (19, 20). The mismatched PCR method followed by digestion with Xho I was used to detect the N370S mutation (21); restriction analysis with Pvu II was used to detect the G377S mutation (22) and with Nci I and Msp I to analyze the L444P mutation (23). Major glucocerebrosidase gene rearrangements were determined with the procedure described by Beutler and Gelbart (24).

Baseline determinations in plasma samples of total IGF-I, free IGF-I, and IGFBP-3 were performed in 19 patients before starting ERT at 13.8 ± 11.2 y of age by immunoradiometric assay (IRMA) using the DSL-2800 ACTIVE Non-Extraction IGF-I Coated-Tube IRMA Kit, the DSL-9400 ACTIVE Free IGF-I Coated-Tube IRMA Kit, and the DSL-6600 ACTIVE IGF Binding Protein-3 Coated-Tube IRMA Kit, respectively (all from Diagnostic Systems Laboratories, Inc., Webster, TX, U.S.A.). The Spearman test was performed to establish the association with other clinical and analytical parameters. In a group of 13 patients receiving ERT, total IGF-I, free IGF-I, and IGFBP-3 were also measured during the first 2 y of treatment. The Wilcoxon test was performed for the statistical analysis. Total IGF-I and IGFBP-3 were expressed as SD score (SDS) using total IGF-I and IGFBP-3 standardized normal values by age and sex provided by Diagnostic Systems Laboratories and free IGF-I normal values provided by J. Argente and V. Barrios (“Niño Jesús” Hospital, Madrid, Spain). We have expressed the results in SDS because these proteins are age dependent.

RESULTS

The mean age at diagnosis of patients was 7.25 y (range, 0.3–14 y), with a male/female ratio of 12/10. The clinical and analytical data are summarized in Tables 1 and 2. Splenomegaly was present in all patients at diagnosis. In 18.1% of patients, the spleen was removed. Fifty percent of patients had Hb levels <11 g/dL, 45.4% had low platelet count (<100 × 109/ L) and 18.1% had low leukocyte count (<4.0 × 109/L). High acid phosphatase levels were observed in 100% of cases, and, in 63.6%, mild biochemical hepatic dysfunction was observed as a result of the alterations of liver transaminases. All of the studied patients on ERT experienced normalization of liver transaminases levels. Albumin, alkaline phosphatase, and bilirubin levels did not alter in the studied patients. The test for acid glucosidase showed a marked decrease in enzymatic activity in all patients. The two most common glucocerebrosidase mutations were N370S and the L444P, which accounted for 43.18% and 20.45% of all mutations, respectively (Table 3).

Table 1 Incidence of hepatomegaly, splenomegaly, splenectomy, and bone disease in the Gaucher disease study population (n = 22)
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Table 2 Analytical data for the Gaucher disease study population
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Table 3 Allelic frequency of the patients
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Total IGF-I, free IGF-I, and IGFBP-3 were determined in 19 patients before ERT. We found a significant association between total IGF-I SDS and the age-adjusted severity score index in this group before ERT (r = −0.62, p < 0.05). In the same group, total IGF-I and IGFBP-3 SDS correlated negatively with the presence of L444P mutation (r = −0.53 and −0.5, respectively, p < 0.05). Height SDS correlated with total IGF-I and IGFBP-3 SDS (r = 0.84 and 0.78, respectively, p < 0.05).

Changes in IGF axis during ERT during the first years of treatment were also studied. Total IGF-I SDS increased from −1.8 ± 0.8 to −0.8 ± 1.4 (p = 0.005) and free IGF-I SDS increased from −1.2 ± 1 to 1.1 ± 2.1 (p = 0.011) at 12 ± 6.8 mo of ERT (n = 10). IGFBP-3 showed the same response as it increased from −1.3 ± 0.6 to −0.2 ± 1.2 SDS (p = 0.012) at 12 ± 4.5 mo of ERT (n = 8) (Table 4). During the second year, total IGF-I SDS increased from −1.3 ± 0.7 to −0.5 ± 0.8 (p = 0.012) (n = 8). No significant changes were found during the second year or ERT for free IGF-I and IGFBP-3.

Table 4 Changes in IGF axis during ERT
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DISCUSSION

Type 1 Gaucher disease is characterized by splenomegaly, hepatomegaly, pancytopenia, and osteolytic and osteopenic degeneration of the skeleton (1). These are the classical features of Gaucher disease, but in some children growth retardation may be the sole manifestation of the disease. In these children, growth retardation has a valuable role in assessing severity and progression of Gaucher disease (8). Kaplan et al.(9) found a significant incidence of severe retardation of linear growth in untreated patients during the two first decades of life. They observed the following three patterns of growth: (1) in 28%, there was normal growth compared with both the general population and the children's parents; (2) in 22%, the growth pattern was normal for the general population but was low in comparison with the family; and (3) in 50%, the growth pattern was abnormal compared with both the general population and the child's family. Recently, Kauli et al.(25) has proven that growth retardation in childhood and delay of puberty are more frequent with severe disease. In 52.6% of a group of patients followed since early childhood, a deceleration of growth was observed at 3–5 y of age. Height SDS decreased from 0.34 at age 0–3 y to −1.93 (p < 0.01) at age 7–10 y, and was more pronounced with severe disease. With ERT, growth velocity increased dramatically at all ages in most children whose growth velocity was below normal (9).

The cause of this poor growth is not yet understood. It has been proven that chronic diseases with liver involvement are associated with growth retardation because of IGF deficiency (16, 17). Kaplan et al.(9) found a significant association between the z scores for height before treatment and liver enlargement (r = 0.57).

Mandel et al.(12), when examining the changes in IGF-I, IGFBP-3, GHBP, erythrocyte IGF-I receptors, and the growth rate in prepubertal GHD children during GH therapy, showed that the major correlates of growth are changes in the concentrations of circulating IGFBP-3 and IGF-I. IGFBP-3 acts synergistically with IGF-I in promotion of growth in GH deficiency, as well as normal children. In patients with type 1 Gaucher disease, we demonstrated that these two parameters, total IGF-I and IGFBP-3, show the best correlation with children's height before treatment (r = 0.84 and 0.78, respectively). IGF-I and IGFBP-3 are significantly reduced as the child's height is more affected. Total IGF-I has a significant negative correlation with the severity of the disease using Zimran's age-adjusted severity score index (r = −0.62). Both total IGF-I and IGFBP-3 correlate negatively with the presence of L444P mutation (r = −0.53 and −0.5, respectively). The presence of this mutation in type 1 Gaucher disease has been related with poor outcome (26). In this study, we found that the IGF axis is affected before treatment. This could be a consequence of liver involvement that could affect the synthesis of proteins, including IGF-I and IGFBP-3. The reduced levels of IGF-I and IGFBP-3 may reflect a GH insensitivity state, as has been shown in other chronic diseases.

The main consequence of IGF-I deficiency in childhood is growth retardation. However, recent research has proven that IGF have an anabolic effect even in adults. These peptides stimulate whole-body and muscle protein synthesis. The reduced IGF-I concentrations are associated with reduced muscle mass and strength (12). In GHD adults, it has been proven that the increase in IGF-I levels correlates with the changes in body composition (27) and serum lipid profile. Bengtsson et al.(27) demonstrated that quality of life, determined by a specific questionnaire, improved after 6 mo of GH therapy in those patients. These results could also be expected in Gaucher patients receiving ERT, because IGF-I is normalized.

When we analyzed the IGF axis response during ERT, we found that ERT results in significant increase of free IGF-I and IGFBP-3 during the first year of treatment and total IGF-I during the first 2 y of treatment. This could be explained by the normalization of liver function that would normalize the GH insensitivity state in the first months of ERT. There is a significant increase in IGF-I and IGFBP-3 levels that results in adequate IGF-I bioavailability.

In conclusion, we consider growth retardation in pediatric patients with Gaucher disease to be related to the alterations in the IGF axis. ERT results in significant increase of IGF during the first years of treatment, and further studies are necessary to clarify long-term response.