葡萄糖神经酰胺合成酶与肿瘤多药耐药

多药耐药(multidrug resistence,MDR)是肿瘤化疗失败的主要原因,多年来医学界一直致力于多药耐药机制的研究。神经酰胺信号系统在细胞凋亡中的作用逐渐引起人们的重视,神经酰胺是细胞凋亡过程中的第二信使,葡萄糖神经酰胺合成酶(glucosylceramide synthase,GCS)可以催化神经酰胺糖基化,使其转变为无细胞毒性的葡萄糖神经酰胺,进而促进鞘糖脂的合成,大量研究表明细胞内GCS水平的增高与MDR表型有关。     

Glycosphingolipid studies of visceral tissues and brain from type 1 Gaucher disease variants

Glucosylceramide and glucosylsphingosine isolated from spleen, liver and brain were quantitated and characterized in two unrelated patients with Gaucher disease, neither of whom had clinical or neuropathologic evidence of neuronal involvement. Visceral glucosylceramide accumulation did not differ in the two patients. Hepatic glucosylsphingosine content was 2-fold greater in a young severely affected 3-year-old American Black patient compared to that in a 56-year-old Ashkenazi Jewish patient. In contrast, significant differences in glycosphingolipid content and composition were observed in the brains of these two cases. Cerebral and cerebellar cortical glucosylceramide accumulated to a greater extent (3-fold) in the severely affected 3-year-old patient compared to that in the older case. The compositions of the acyl and sphingosyl base residues of glucosylceramide in the cerebral and cerebellar cortices from the Ashkenazi Jewish patient were similar to those in normal individuals. In comparison, the gray matter glucosylceramide in the severely affected patient had increased percentages of stearic acid (18:0) and eicosasphingenine (d20:1), suggesting that the accumulated substrate was derived from the brain ganglioside pool. Glucosylsphingosine was found in large amounts only in cerebral and cerebellar cortices from the severely affected patient. The glycolipid content and composition in this patient was similar to that found in the Norrbottnian (Type 3) form of Gaucher disease. The differences in glucosylceramide acyl and sphingosyl base composition in gray matter from the severely affected patient and that in the Ashkenazi Jewish patient suggested that the accumulated substrates were metabolized differently by the residual enzymes in each case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucosylceramide accumulation is not confined to the lysosome in fibroblasts from patients with Gaucher disease

Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme β-glucosidase leading to the accumulation of glucosylceramide (GC) in lysosomes of affected cells. In order to determine the effect of GC accumulation on intracellular lipid content in fibroblasts from patients with GD, we measured individual species of ceramide, di- and trihexosylceramide, sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol using electrospray ionisation-tandem mass spectrometry. The different subspecies of each lipid class correlated with each other and were summed to give total lipid concentrations. In addition to GC, we also noted secondary elevations in other lipids, especially in type 2 GD. Sub-cellular fractionation showed that GC was not confined to the lysosome but increased throughout the cell. The sequelae of extra-lysosomal accumulation may have implications in the pathogenic mechanisms of GD by interaction with biochemical and metabolic pathways located outside the lysosome. The elevation of ceramide in confluent type 2 GD fibroblasts redistributed from its primary site of accumulation in the lysosome to the endosomal region at four-weeks post-confluence. The accumulation of lipids in the endosome and lysosome suggests both impaired trafficking of lipids and reduced capacity of the lysosome to degrade lipids.     

The D409H variant in GBA1: Challenges in predicting the Gaucher phenotype in the newborn screening era

Gaucher disease (GD) is an autosomal recessive disorder resulting from glucocerebrosidase deficiency due to pathologic variants in GBA1. While clinically heterogeneous, GD encompasses three types, non-neuronopathic (GD1), acute neuronopathic (GD2), and chronic neuronopathic (GD3). Newborn screening (NBS), which has made remarkable inroads in detecting certain diseases before detrimental health consequences and fatality ensues, is now being piloted for GD in several states and countries. Early on, clinical features of GD2 can overlap with GD3; hence, predicting outcome is challenging. As NBS for GD becomes more available, the increased detection of GD in neonates is inevitable. As a result, health care providers and families will be faced with uncertainty with respect to clinical management. Since more severe GBA1 variants are generally associated with neuronopathic GD, there has been an increased dependence on genotypic information. We present an infant detected by NBS with genotype D409H(p.Asp448His)/RecNciI (p.Leu483Pro; p.Ala495Pro;p.Val499=). To assist in genetic counseling, we performed a retrospective review of other patients in our cohort carrying D409H and reviewed the literature. The study illustrates the challenges faced in counseling for infants with neuronopathic GD, even with known GBA1 variants, and the tough management decisions that can ensue from detection in newborns.     

Glucocerebrosidase genotype of Gaucher patients in The Netherlands: Limitations in prognostic value

Gaucher disease is a recessively inherited lysosomal storage disorder that is caused by a deficiency in glucocerebrosidase activity. The clinical expression is markedly heterogeneous with respect to age of onset, progression, severity, and neurological involvement. The relative incidence of glucocerebrosidase (GC) mutations has been studied extensively for Jewish but not for non-Jewish Caucasian patient populations. The present survey on mutant GC genotypes prevalent in Gaucher disease in The Netherlands was taken of 72 patients from different genetic backgrounds. This number is more than half the total number of affected Gaucher patients to be expected on the basis of the incidence of the disorder in this country. Analysis of nine GC mutations led to the identification of 74% of the mutant GC alleles in patients from 44 unrelated Dutch families (i.e., families that have lived in The Netherlands for at least several generations) and of 44% of the mutant GC alleles in patients from nine unrelated families that recently immigrated from both European and non-European countries. The N370S (cDNA 1226G) GC mutation proved to occur most frequently (41%) in the unrelated Dutch patients and less frequently (6%) in the unrelated immigrant patients and was always associated with the nonneuronopathic (Type 1) form of the disease. Apart from the association of the N370S mutation with Type 1 Gaucher disease, the prognostic value of GC genotyping was limited, since a particular GC genotype did not correlate closely to a specific clinical course, or to a specific relative responsiveness to enzyme-supplementation therapy. Hum Mutat 10:348–358, 1997. © 1997 Wiley-Liss, Inc.     

Gaucher disease: studies of phenotype, molecular diagnosis and treatment

This report summarizes the results on 39 patients with Gaucher disease who have been genotyped, evaluated, and/or followed at this center. Mutation analysis for 4 common mutations; N370S, L444P, 84gg and IVS2 (+1), was performed for all patients. Mutation analysis identified both mutant alleles in 69% and at least one mutant allele in 90% of all chromosomes. This study group of 39 patients included 32 type 1, four type 2 and three type 3 patients. We include the details of the clinical course of two patients with Gaucher disease treated with enzyme replacement therapy (ERT). One patient with chronic neuronopathic Gaucher disease has been treated with enzyme replacement therapy (ERT) at a dose of 60 U/kg every 2 weeks since 2.5 years of age and has shown no progression of neurologic involvement. A second patient with non-neuronopathic Gaucher disease has demonstrated an unusually delayed response to ERT. No clinical response was noted following 17 months of treatment at 60 U/kg every 2 weeks. Only after the dose was increased to 60 U/kg every week was a clinical response evident. Response to treatment at 15 U/kg every 2 weeks was variable in the four type 1 patients treated at the lower dose. In two of these patients with identical genotypes, one patient demonstrated a positive clinical response to low dose treatment while the other patient did not.     

Inhibition of UDP‐glucosylceramide synthase in mice prevents Gaucher disease‐associated B‐cell malignancy

Clonal B‐cell proliferation is a frequent manifestation of Gaucher disease – a sphingolipidosis associated with a high risk of multiple myeloma and non‐Hodgkin lymphoma. Gaucher disease is caused by genetic deficiency of acid β‐glucosidase, the natural substrates of which (β‐d ‐glucosylceramide and β‐d ‐glucosylsphingosine) accumulate, principally in macrophages. Mice with inducible deficiency of β‐glucosidase [Gba(tm1Karl/tm1Karl)Tg(MX1‐cre)1Cgn/0] serve as an authentic model of human Gaucher disease; we have recently reported clonal B‐cell proliferation accompanied by monoclonal serum paraproteins and cognate tumours in these animals. To explore the relationship between B‐cell malignancy and the biochemical defect, we treated Gaucher mice with eliglustat tartrate (GENZ 112638), a potent and selective inhibitor of the first committed step in glycosphingolipid biosynthesis. Twenty‐two Gaucher mice received 300 mg/kg of GENZ 112638 daily for 3–10 months from 6 weeks of age. Plasma concentrations of β‐d ‐glucosylceramide and the unacylated glycosphingolipid, β‐d ‐glucosylsphingosine, declined. After administration of GENZ 112638 to Gaucher mice for 3–10 months, serum paraproteins were not detected and there was a striking reduction in the malignant lymphoproliferation: neither lymphomas nor plasmacytomas were found in animals that had received the investigational agent. In contrast, 14 out of 60 Gaucher mice without GENZ 112638 treatment developed these tumours; monoclonal paraproteins were detected in plasma from 18 of the 44 age‐matched mice with Gaucher disease that had not received GENZ 112638. Long‐term inhibition of glycosphingolipid biosynthesis suppresses the development of spontaneous B‐cell lymphoma and myeloma in Gaucher mice. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Increased cerebroside concentration in plasma and erythrocytes in Gaucher disease: Significant differences between Type I and Type III

A method was developed for the determination of cerebrosides in 1 ml of plasma or 1 ml of packed erythrocytes. At least 90% of the cerebroside fraction consisted of glucosylceramide. In the erythrocytes, nothing but glucosylceramide was identified. The method was applied to plasma samples from 25 controls, 34 Gaucher Type III obligate carriers, 16 Gaucher Type III patients, 7 Gaucher Type I patients and 7 patients with myelogenous or lymphatic leukemia, as well as to erythrocyte samples from 20 controls, 6 Gaucher Type III obligate carriers, 16 Gaucher Type III patients and 6 Gaucher Type I patients. The concentration of plasma cerebroside was 11.4±4.2 (S.D.) in controls, 11.8 ± 2.6 in Gaucher Type III carriers, 30.4 ± 7.7 in Gaucher Type III patients and 21.8 ± 6.7 μmol/l in Gaucher Type I patients. The Gaucher patients had Significantly increased (p <0.001) plasma cerebroside values, while the plasma cerebroside concentration of the leukemic patients was only slightly increased, 14.7 ± 5.7 μmol/l. In the packed erythrocyte pellet the corresponding values were: Controls 2.9 ± 0.7, Gaucher Type III carriers 2.9 ± 0.7, Gaucher Type III patients 9.2 ± 2.4 and Gaucher Type I patients 6.5 ± 2.7 μmol/l. The phospholipid concentration was the same in all the three Gaucher groups and was not significantly different from that in the controls.     

A novel alteration in metaxin 1, F202L,is associated with N370S in Gaucher disease

The gene for glucocerebrosidase (GBA), the enzyme deficient in Gaucher disease, is located in a gene-rich region on 1q21. Metaxin 1(MTX1) is a convergently transcribed gene contiguous to the 3 end of the GBA pseudogene. A single nucleotide alteration in MTX1, 628TC, resulting in the amino acid change F202L, was identified in patients with Gaucher disease in association with the common N370S mutation in GBA. The polymorphism was also present on 4.6% of 152 control alleles, but could have functional consequences that have a modifying role in Gaucher disease.     

Gaucher disease in Spanish patients: Analysis of eight mutations

Gaucher disease is particularly prevalent among Ashkenazi Jews; thus most studies have been reported on this ethnic group. We present the first data on Spanish patients with Gaucher disease and provide one of the first reports on a fairly well defined, large, non-Jewish population. Eight mutations were analyzed in 35 patients, with different clinical subtypes, by restriction enzyme digestion or allelespecific oligonucleotide (ASO) hybridization, after PCR amplification of genomic DNA. Analysis of the eight mutations allowed identification of 77.2% of the disease alleles, N370S and L444P alone accounting for 70%. Mutation N370S, carried by 31 alleles (44.3%), appeared to be the most prevalent in the Spanish population. The frequency of this mutation and of the N370S/N370S genotype is closer to those described for Ashkenazi Jews than to the frequencies found in other non-Jewish populations. Mutation L444P, the second most abundant mutation, occurred in 25.7% of the disease alleles. Four alleles carrying mutation D409H (5.7%) were detected in patients of different clinical expression and one RecNciI allele in a type I patient. Mutations 84GG, IVS2 + l, R463C, and RecTL were also screened but were not found in any of our patients. © 1995 Wiley-Liss, Inc.     

Revisiting the diagnosis of Gaucher disease in a family with multiple GBA1 variants

Our ability to identify different variants in GBA1, the gene mutated in the lysosomal storage disorder Gaucher disease (GD), has greatly improved. We describe a multigenerational family with type 1 GD initially evaluated over three decades ago. Re-evaluating both the genotype and phenotype, we determined that one family member with genotype N370S/T369M (p.N409S/p.T408M), was likely erroneously diagnosed with GD. This case substantiates that GBA1 variant T369M, while mildly reducing glucocerebrosidase activity, does not result in GD. The observation has clinical relevance as cases with this genotype will increasingly be ascertained through screening programs in newborns and in movement disorder clinics.     

Type 1 Gaucher disease: null and hypomorphic novel chitotriosidase mutations-implications for diagnosis and therapeutic monitoring

Human plasma chitotriosidase (Chito) is a useful diagnostic and therapeutic biomarker for Type 1 Gaucher disease (GD). However, approximately 40% of Caucasians are heterozygous or homozygous for a common null mutation, c.1049_1072dup24 (dup24) in the chitotriosidase gene (chitinase 1, CHIT1), that complicates interpretation for heterozygotes and precludes use for null homozygotes. 320 Type 1 GD patients were screened for CHIT1 genotype and plasma Chito enzyme levels; 37% were heterozygous and 4% were homozygous for the CHIT1 dup24 allele. Four patients who had no or very low plasma Chito activities had wild-type (wt)/dup24 or wt/wt CHIT1 genotypes, suggesting the presence of other mutations. Sequencing their CHIT1 genes revealed three novel mutations: p.E74K (E74K), p.G102S (G102S), and a complex exon 10 lesion (c.[1060G>A; 1155G>A; 1156+5_1156+8delGTAA], p.[G354R; L385L; missplicing], designated "complex E/I-10"). The G102S mutation was common in Type 1 GD patients and controls ( approximately 30% of alleles). In contrast, the E74K mutation was rare, present only in three Type 1 GD patients ( approximately 1% of alleles), all of Ashkenazi Jewish (AJ) descent, but it was not found in normal controls. The complex E/I-10 mutation occurred in two Caribbean Hispanic/African Type 1 GD patients and was present in 0 to 6% of alleles among normal controls from different populations. In vitro expression demonstrated that the E74K and G102S alleles had approximately 51% and approximately 23% of wild-type Chito catalytic efficiency, respectively. Expression of the G354R allele alone or with the L385L silent substitution did not produce detectable Chito activity or protein. RNA studies indicated that the complex E/I-10 allele also caused missplicing. Recognition of these mutations, particularly G102S, will facilitate the use and interpretation of plasma Chito activities for disease diagnosis, estimating disease severity, and monitoring therapeutic efficacy in GD.     

Novel mutations in type 2 Gaucher disease in Chinese and their functional characterization by heterologous expression

We investigated 10 unrelated Chinese patients with type 2 Gaucher disease and performed ex vivo expression for the novel mutations to characterize their functional defects. These patients were diagnosed by enzymatic assays and clinicopathologic features over the past five years in a national centre in China. Genomic DNA was sequenced by a two-stage PCR approach for mutations in the functional GBA gene. Novel mutations were expressed with baculovirus-transfected Sf21 cells. Six novel mutations were found (in traditional nomenclature): P122L, Y363C, N382K, L383R, L385P, and M416V. Review of reported mutations indicated clustering of type 2 mutations in three regions of the GBA gene. Expression of novel mutations revealed that the enzyme defect could arise from one of two mechanisms: loss of catalytic activity (Y363C and M416V) or enzyme instability (P122L and N382K).     

Altered level of plasma exosomes in patients with Gaucher disease

Mutations in the glucocerebrosidase gene (GBA) cause Gaucher disease (GD), the lysosomal storage disorder (LSD), and are the most common genetic risk factor of Parkinson's disease (PD). Lysosome functionality plays a critical role for secretion of extracellular vesicles (EVs) and their content. Here we compared EVs from the blood plasma of 8 GD patients and 8 controls in terms of amounts, size distribution, and composition of their protein cargo. EVs were isolated via sequential centrifugation and characterized by сryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). The presence of exosomal markers HSP70 and tetrasponins were analyzed by Western blot and flow cytometry. Protein profiling was performed by mass-spectrometry (shotgun analysis).Here, for the first time we reported an increased size and altered morphology in exosomes derived from blood plasma of GD patients. An increased size of plasma exosomes from GD patients compared to controls was demonstrated by cryo-EM and DLS (р<0.0001, p < 0.001, respectively) and confirmed by mode size detected by NTA (p < 0.02). Cryo-EM demonstrated an increased number of double and multilayer vesicles in plasma EVs from GD patients. We found that the EVs were enriched with the surface exosomal markers (CD9, СD63, CD81) and an exosome-associated protein HSP70 in case of the patients with the disease. Proteomic profiling of exosomal proteins did not reveal any proteins associated with PD pathogenesis. Thus, we showed that lysosomal dysfunction in GD patients lead to a striking alteration of plasma exosomes in size and morphology.     

Movement and mood disorder in two brothers with Gaucher disease

Gaucher disease (GD) is a lysosomal storage disorder with a wide spectrum of phenotypic presentations. We report the case histories of two adult brothers with GD who developed both parkinsonism and psychiatric symptoms. Direct sequencing and real-time polymerase chain reaction were used to establish that the patients were homozygous for mutation L444P. While parkinsonism has been described previously in GD, these patients had atypical features, including a complicated mood disorder. The comorbidity of GD and a mood disorder is a new finding, as psychiatric manifestations of GD have been described rarely. The etiology of the mental illness could be related to the processes contributing to the development of parkinsonism.     

Delineating pathological pathways in a chemically induced mouse model of Gaucher disease

Great interest has been shown in understanding the pathology of Gaucher disease (GD) due to the recently discovered genetic relationship with Parkinson's disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid β‐glucosidase (GCase) using the irreversible inhibitor conduritol B‐epoxide (CBE) is particularly attractive, although few systematic studies examining the effect of CBE on the development of symptoms associated with neurological forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both the levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBE‐treated mice and a genetic GD mouse model, the Gba^flox/flox;nestin‐Cre mouse, with 120 of the 144 genes up‐regulated in CBE‐treated mice also up‐regulated in Gba^flox/flox;nestin‐Cre mice. We also demonstrate that various aspects of neuropathology and some behavioural abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for time‐consuming crossing of mice. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Gaucher disease: mutation and polymorphism spectrum in the glucocerebrosidase gene (GBA)

Gaucher disease (GD) is an autosomal recessive disorder caused by the deficiency of glucocerebrosidase, a lysosomal enzyme that catalyses the hydrolysis of the glycolipid glucocerebroside to ceramide and glucose. Lysosomal storage of the substrate in cells of the reticuloendothelial system leads to multisystemic manifestations, including involvement of the liver, spleen, bone marrow, lungs, and nervous system. Patients with GD have highly variable presentations and symptoms that, in many cases, do not correlate well with specific genotypes. Almost 300 unique mutations have been reported in the glucocerebrosidase gene (GBA), with a distribution that spans the gene. These include 203 missense mutations, 18 nonsense mutations, 36 small insertions or deletions that lead to either frameshifts or in-frame alterations, 14 splice junction mutations, and 13 complex alleles carrying two or more mutations in cis. Recombination events with a highly homologous pseudogene downstream of the GBA locus also have been identified, resulting from gene conversion, fusion, or duplication. In this review we discuss the spectrum of GBA mutations and their distribution in the patient population, evolutionary conservation, clinical presentations, and how they may affect the structure and function of glucocerebrosidase.     

Correlating liver stiffness with disease severity scoring system (DS3) values in Gaucher disease type 1 (GD1) patients

Gaucher disease (GD) is an autosomal-recessive lysosomal storage disease caused by a deficiency of the enzyme, glucocerebrocidase, resulting in accumulation of lipid-laden storage cells in multiple organs such as bone marrow, liver, spleen, and lungs. Type 1 Gaucher disease is the most common form of this condition in which the brain and spinal cord (the central nervous system) are not affected. The Gaucher disease severity scoring system (GD-DS3) is typically used to assess disease severity accounting for skeletal, hematologic, and visceral disease. In addition to being time consuming for the clinician to calculate the scores, some of the assessments are subjective and may falsely increase or decrease disease severity. The purpose of this study was to determine if there is a correlation between liver stiffness values obtained from MR elastography (MRE) and the GD-DS3 score.An IRB approved, HIPAA compliant retrospective study was performed. All patients with type 1 GD imaged with MRE between 2011 and 2016 were included in this study. Clinical and imaging data was collected. Two pediatric radiologists analyzed MR images from abdomen and thigh studies independently to determine bone marrow involvement using a semi-quantitative scoring system with one reviewer analyzing a subset of studies to determine inter-observer reliability. The collected data was used to calculate a GD-DS3 score for all patients. GD-DS3 scores were compared with liver MRE stiffness values.Clinical MRE scores were plotted against GD-DS3 severity scores for 31 patients (15 males, 16 females; median age 27 years, age range: 4–67 years). The median GD-DS3 score was 4 (range: 1–10.1) and median MRE value was 2.43 kPa (range: 1.30–5.20 kPa). A significant positive correlation was found between MRE and GD-DS3 scores; Pearson's correlation coefficient value of r = 0.47, p < 0.001 for all scores, r = 0.68, p < 0.001 for complete scores and r = 0.46, p < 0.07 for incomplete scores. The inter-observer variation of bone marrow burden showed only fair agreement with a Kappa coefficient of 0.26.There is a significant positive correlation between increasing liver stiffness and increasing composite GD-DS3 scores. This supports the use of MRE, a non-invasive reproducible quantitative test, as both an additional assessment and independent marker for monitoring disease severity and progression in GD.     

Type I Gaucher disease due to homozygosity for the 259T mutation in a Bedouin patient

A 26-year-old Bedouin with moderate thrombocytopenia and enlarged spleen and liver was diagnosed as having type I Gaucher disease based on the presence of Gaucher cells in the bone marrow biopsy and enzymatic determination of glucocerebrosidase activity. Molecular analysis excluded 10 common mutations in the glucocerebrosidase gene. Homozygosity for the C → T mutation in nucleotide 259 of the cDNA (1763 genomic) was detected by digestion with restriction enzyme StyI after an amplification of a portion of exon 3 by mismatched primers. This is the first known case of homozygosity for this mutation. The fact that it produces a very mild phenotype, confirms a previous suggestion that 259T can be classified as a “mild” mutation. Association of the 259T mutation with the “Pv 1.1 +” haplotype is consistent with a common origin of the mutated alleles. Am. J. Med. Genet. 72:77–78, 1997. © 1997 Wiley-Liss, Inc.     

Pulmonary involvement in type 1 Gaucher disease: functional and exercise findings in patients with and without clinical interstitial lung disease

Pulmonary disease is a well-known complication of Type 1 Gaucher disease (GD), although its incidence is not well established and its severity varies. The purpose of this study was to determine the frequency and extent of pulmonary involvement in patients with GD. Pulmonary involvement was assessed by history, physical examination and chest radiograph in 150 consecutive patients with Type 1 GD presenting at a specialized center for genetic diseases. Five patients were noted to have clinical evidence of pulmonary involvement. Full pulmonary function tests were performed in these five patients and in an additional 13 patients randomly selected from the remaining 145. Many of the 18 patients also underwent radionuclide body imaging with 67 Gallium citrate and 111Indium-tagged leucocyte scans, as well as incremental cardiorespiratory exercise tests. Lung biopsies were available in two patients with lung disease, and a second examination of lung tissue was performed in one of these two patients post-mortem. Clinical lung disease was detected in five patients. All five had dyspnea, diffuse infiltrates, restrictive impairment and low single breath CO diffusing capacity (DLCOSB). Two of these patients underwent exercise testing and showed abnormalities consistent with lung disease (ventilatory limitation, excessive ventilation and increased dead space) as well as decreased VO2 max. and anaerobic threshold (AT). In contrast, in the other 13 patients, physical examination, chest radiographs and pulmonary function were normal (except for a low DLCOSB in one patient). Responses on exercise testing (performed in six of the 13 patients) were consistent with a circulatory impairment (decreased VO2 max. and AT). Our study found that <5% of patients with Type 1 GD have clinical interstitial lung disease. In addition, we found that some patients, without evident lung involvement, may experience limitations in physical exertion and are easily fatigued; this is attributable to impaired circulation.