Ruhua Yang

The underrecognized progressive nature of N370S Gaucher disease and assessment of cancer risk in 403 patients

Mutations in GBA1 gene that encodes lysosomal glucocerebrosidase result in Type 1 Gaucher Disease (GD), the commonest lysosomal storage disorder; the most prevalent disease mutation is N370S. We investigated the heterogeneity and natural course of N370S GD in 403 patients. Demographic, clinical, and genetic characteristics of GD at presentation were examined in a cross‐sectional study. In addition, the relative risk (RR) of cancer in patients compared with age‐, sex‐, and ethnic‐group adjusted national rates of cancer was determined. Of the 403 patients, 54% of patients were homozygous (N370S/N370S) and 46% were compound heterozygous for the N370S mutation (N370S/other). The majority of N370S/N370S patients displayed a phenotype characterized by late onset, predominantly skeletal disease, whereas the majority of N370S/other patients displayed early onset, predominantly visceral/hematologic disease, P < 0.0001. There was a striking increase in lifetime risk of multiple myeloma in the entire cohort (RR 25, 95% CI 9.17–54.40), mostly confined to N370S homozygous patients. The risk of other hematologic malignancies (RR 3.45, 95% CI 1.49–6.79), and overall cancer risk (RR 1.80, 95% CI 1.32–2.40) was increased. Homozygous N370S GD leads to adult‐onset progressive skeletal disease with relative sparing of the viscera, a strikingly high risk of multiple myeloma, and an increased risk of other cancers. High incidence of gammopathy suggests an important role of the adaptive immune system in the development of GD. Adult patients with GD should be monitored for skeletal disease and cancers including multiple myeloma. Am. J. Hematol., 2009.

Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation

Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human βGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, βGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human βGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders.

Glucocerebrosidase 2 gene deletion rescues type 1 Gaucher disease.

Significance Type 1 Gaucher disease (GD1) is a rare autosomal recessive disorder caused by inherited mutations in the glucocerebrosidase (GBA1) gene. This disease results in a marked accumulation of glycosphingolipid substrates, causing visceromegaly, cytopenia, and osteopenia. Here, we have rescued this clinical phenotype in GD1 mice by genetically deleting Gba2, a gene encoding a downstream extralysosomal enzyme, GBA2. We also report that sphingosine production in GD1 patients may contribute to the low-turnover bone loss. Our data suggest that GBA2 and sphingosine are potential targets for pharmacological intervention in GD1 patients. The inherited deficiency of the lysosomal glucocerebrosidase (GBA) due to mutations in the GBA gene results in Gaucher disease (GD). A vast majority of patients present with nonneuronopathic, type 1 GD (GD1). GBA deficiency causes the accumulation of two key sphingolipids, glucosylceramide (GL-1) and glucosylsphingosine (LysoGL-1), classically noted within the lysosomes of mononuclear phagocytes. How metabolites of GL-1 or LysoGL-1 produced by extralysosomal glucocerebrosidase GBA2 contribute to the GD1 pathophysiology is not known. We recently recapitulated hepatosplenomegaly, cytopenia, hypercytokinemia, and the bone-formation defect of human GD1 through conditional deletion of Gba in Mx1–Cre+:GD1 mice. Here we show that the deletion of Gba2 significantly rescues the GD1 clinical phenotype, despite enhanced elevations in GL-1 and LysoGL-1. Most notably, the reduced bone volume and bone formation rate are normalized. These results suggest that metabolism of GL-1 or LysoGL-1 into downstream bioactive lipids is a major contributor to the bone-formation defect. Direct testing revealed a strong inhibition of osteoblast viability by nanomolar concentrations of sphingosine, but not of ceramide. These findings are consistent with toxicity of high circulating sphingosine levels in GD1 patients, which decline upon enzyme-replacement therapy; serum ceramide levels remain unchanged. Together, complementary results from mice and humans affected with GD1 not only pinpoint sphingosine as being an osteoblast toxin, but also set forth Gba2 as a viable therapeutic target for the development of inhibitors to ameliorate certain disabling consequences of GD1.

Glucosylsphingosine is a key biomarker of Gaucher disease.

Gaucher disease (GD) involves the accumulation of glucosylceramide (GL1) and its deacylated lysolipid, glucosylsphingosine (lyso‐GL1) which is implicated in mediating immune dysregulation and skeletal disease. The aim of our study was to assess plasma Lyso‐GL1 as a biomarker of GD and its response to therapy.

Gaucher disease gene GBA functions in immune regulation

Inherited deficiency of acid β-glucosidase (GCase) due to biallelic mutations in the GBA (glucosidase, β, acid) gene causes the classic manifestations of Gaucher disease (GD) involving the viscera, the skeleton, and the lungs. Clinical observations point to immune defects in GD beyond the accumulation of activated macrophages engorged with lysosomal glucosylceramide. Here, we show a plethora of immune cell aberrations in mice in which the GBA gene is deleted conditionally in hematopoietic stem cells (HSCs). The thymus exhibited the earliest and most striking alterations reminiscent of impaired T-cell maturation, aberrant B-cell recruitment, enhanced antigen presentation, and impaired egress of mature thymocytes. These changes correlated strongly with disease severity. In contrast to the profound defects in the thymus, there were only limited cellular defects in peripheral lymphoid organs, mainly restricted to mice with severe disease. The cellular changes in GCase deficiency were accompanied by elevated T-helper (Th)1 and Th2 cytokines that also tracked with disease severity. Finally, the proliferation of GCase-deficient HSCs was inhibited significantly by both GL1 and Lyso-GL1, suggesting that the “supply” of early thymic progenitors from bone marrow may, in fact, be reduced in GBA deficiency. The results not only point to a fundamental role for GBA in immune regulation but also suggest that GBA mutations in GD may cause widespread immune dysregulation through the accumulation of substrates.

Genome-wide association study of N370S homozygous Gaucher disease reveals the candidacy of CLN8 gene as a genetic modifier contributing to extreme phenotypic variation†

Mutations in GBA1 gene result in defective acid β‐glucosidase and the complex phenotype of Gaucher disease (GD) related to the accumulation of glucosylceramide‐laden macrophages. The phenotype is highly variable even among patients harboring identical GBA1 mutations. We hypothesize that modifier gene(s) underlie phenotypic diversity in GD and performed a GWAS study in Ashkenazi Jewish patients with type 1 GD (GD1), homozygous for N370S mutation. Patients were assigned to mild, moderate, or severe disease categories using composite disease severity scoring systems. Whole‐genome genotyping for >500,000 SNPs was performed to search for association signals using OQLS algorithm in 139 eligible patients. Several SNPs in linkage disequilibrium within the CLN8 gene locus were associated with the GD1 severity: SNP rs11986414 was associated with GD1 severity at P value 1.26 × 10−6. Compared to mild disease, risk allele A at rs11986414 conferred an odds ratio of 3.72 for moderate/severe disease. Loss of function mutations in CLN8 causes neuronal ceroid‐lipofuscinosis, but our results indicate that its increased expression may protect against severe GD1. In cultured skin fibroblasts, the relative expression of CLN8 was higher in mild GD compared to severely affected patients, in whom CLN8 risk alleles were overrepresented. In an in vitro cell model of GD, CLN8 expression was increased, which was further enhanced in the presence of bioactive substrate, glucosylsphingosine. Taken together, CLN8 is a candidate modifier gene for GD1 that may function as a protective sphingolipid sensor and/or in glycosphingolipid trafficking. Future studies should explore the role of CLN8 in pathophysiology of GD. Am. J. Hematol., 2012.

Correlation of MRI-Based Bone Marrow Burden Score with Genotype and Spleen Status in Gaucher's Disease

OBJECTIVE The purpose of this study was to correlate skeletal pathologic findings quantified by MRI-based bone marrow burden score with genotype and spleen status and other clinical parameters, including liver size and duration of enzyme replacement therapy, in patients with Gauchers disease. MATERIALS AND METHODS Two radiologists retrospectively reviewed MR images of 47 patients with Gauchers disease and determined bone marrow burden scores by consensus on the basis of previously published criteria. The bone marrow burden scores were correlated with genotype, liver volume, spleen status, age, and cumulative duration of enzyme replacement therapy. RESULTS Subjects with compound heterozygous N370S alleles had significantly higher overall, lumbar spinal, and femoral bone marrow burden scores than did N370S homozygotes. There was a significant positive correlation between an enlarged or surgically absent spleen and bone marrow burden score. There were no significant associations between bone marrow burden score and liver volume, age, cumulative duration of enzyme replacement therapy, or cumulative duration of untreated disease. Femoral and lumbar spinal bone marrow burden scores had a weak but significant positive correlation across all patients. CONCLUSION Skeletal pathologic findings in Gauchers disease encapsulated as bone marrow burden score correlate significantly with the number of copies of the N370S allele, which has an ameliorative effect on bone marrow disease. Splenectomy or splenomegaly is associated with greater risk of bone marrow disease. Femoral and lumbar spinal bone marrow burden scores, although only weakly correlated, independently illustrated both the protective role of the N370S allele and the unfavourable implication of splenectomy. This finding suggests that axial and appendicular bone marrow burdens are related but distinct and justifies multiple-compartment evaluation in Gauchers disease.

Validating glycoprotein non-metastatic melanoma B (gpNMB, osteoactivin), a new biomarker of Gaucher disease

In the spleens of Gaucher disease mice and patients, there is a striking elevation of expression of glycoprotein non-Metastatic Melanoma B (gpNMB). We conducted a study in a large cohort of patients with Gaucher disease to assess the utility of serum levels of soluble fragment of gpNMB as a biomarker of disease activity. There was >15-fold elevation of gpNMB in sera of untreated patients with Gaucher disease. gpNMB levels correlated with overall disease severity as well as the severity of individual organ compartments: liver, spleen, bone and hematological disease. Imiglucerase enzyme replacement therapy resulted in significant reduction of gpNMB. Serum levels of gpNMB were highly correlated with accumulation of bioactive lipid substrate of Gaucher disease, glucosylsphingosine as well as established biomarkers, chitotriosidase and chemokine, CCL18. Our results suggest utility of gpNMB as a biomarker of Gaucher disease to monitor individual patients and cohorts of patients for disease progression or response to therapy. Investigation of gpNMB in Gaucher disease pathophysiology is likely to illuminate our understanding disease mechanisms.