Marie E. Grace

Pulmonary hypertension in type 1 Gaucher’s disease: genetic and epigenetic determinants of phenotype and response to therapy☆

Type 1 Gauchers disease (GD) is recognized for striking but unexplained phenotypic diversity. Rarely, severe pulmonary hypertension (PH) may occur in GD but its clinical spectrum, determinants or its response to enzyme replacement therapy (ERT)+/-vasodilators is not known. One hundred and thirty-four consecutive patients with Type 1 GD were screened to estimate right ventricular systolic pressure (RVSP) by Doppler echocardiography. Ninety-four patients were on ERT and 40 were untreated. Eight additional GD patients were studied that represented consecutive tertiary referrals with severe PH. Angiotensin converting enzyme (ACE) gene polymorphisms and acid beta-glucosidase gene (GBA) mutations were determined by DNA analysis. Mild, asymptomatic PH (RVSP>35<50 mmHg) was prevalent in Type 1 GD: 30% in untreated patients and 7.4% among patients receiving ERT (P<0.001). Splenectomy was strongly associated with severe, life-threatening PH: all patients with severe PH (RVSP 50-130 mmHg) were asplenic compared to only 31% of patients with RVSP<50 mmHg (Odds ratio [OR] 28.8, 95% CI 1.6-531.6, P<0.001). Other characteristics of patients presenting with severe PH were poor compliance to ERT (4/9 patients) or no ERT (5/9 patients), a family history of a sib with GD and PH (2/2 patients), an excess of ACE I allele (OR 2.3, 95% CI 1.1-4.9, P=0.034) and an excess of non-N370S GBA mutation (OR 6.0, 95% CI 1.1-33, P=0.003). Severe PH was ameliorated by ERT+/-vasodilators during 4.6+/-4.0 yr (range 1-12 yr) follow-up; three patients were initially considered for lung transplantation but improved such that they are no longer active transplant candidates. Our study reveals a remarkable predisposition for PH in type 1 GD. Progression to severe, life-threatening PH occurs in the presence of additional genetic factors (non-N370S GBA mutation, positive family history, and ACE I gene polymorphism) and epigenetic modifiers (i.e., asplenia and female sex). Splenectomy should be avoided and in high-risk patients, ERT+/-vasodilators/coumadin should be initiated.

Enzyme Augmentation in Moderate to Life- Threatening Gaucher Disease

ABSTRACT: Gaucher disease type 1 (GD type 1) is the most prevalent lysosomal storage disease and has its highest frequency in the Ashkenazi Jewish population. Deficiency of the enzyme, acid β-glucosidase, results in the deposition of glucocerebroside primarily in macrophages. The accumulation of such “Gaucher cells” leads to viscer-omegaly, hepatic and bone marrow dysfunction, hyper-splenism, and bony disease. Eleven GD type 1 patients, ages 4–52 y, with moderate to life-threatening manifestations, received 6–12 mo of enzyme augmentation with a macrophage-targeted acid β-ghicosidase preparation. Within 6 mo, substantial increases in Hb levels (mean = +30%) and platelet counts (mean = +39%) were observed. Hepatic and splenic volumes decreased by ∼20% (range = 3–35%) and ∼35% (20–52%), respectively. Hematotogic and hepatic volume improvements were similar in the splenectomized (n = 4) and nonsplenectomized (n = 7) patient groups. In this patient population, no major differences were observed in the hematologic and visceral improvements with enzyme doses of 30, 50, or 60 IU/kg administered every 2 wk. Normal levels of acid β-glucosi-dase activity were present in hepatic autopsy samples from one patient 11 d after enzyme infusion. In comparison, exogenous activity was absent from brain and lung specimens of the same patient High levels (∼10-fold normal) were present in bone marrow samples from two patients obtained at 1 and 11 d after infusions. These studies demonstrate biochemical and clinical improvements by targeted enzyme augmentation in GD type 1, even in far advanced, life-threatening involvement. These and previous studies indicate that earlier intervention in patients with more mild signs may be warranted to obviate the need to rescue extraordinarily ill patients with GD type 1.

Frequency of Unrecognized Fabry Disease Among Young European-American and African-American Men With First Ischemic Stroke

Background and Purpose— The cause of initial ischemic stroke in up to 30% of young patients remains unclear. Fabry disease, due to deficient α-galactosidase A (α-Gal A) activity, is a vascular endothelial glycosphingolipid storage disease typically presenting in childhood. With advancing age, patients develop renal, cardiac, and cerebrovascular disease and die prematurely. A European study suggested an increased prevalence of unrecognized Fabry disease in patients with cryptogenic stroke. We hypothesized that α-Gal A deficiency is a rare cause of initial early-onset ischemic stroke in men. Methods— The Stroke Prevention in Young Men Study enrolled >550 men (15 to 49 years) with first ischemic stroke in the Baltimore–Washington area in 2004 to 2007. Frozen plasma samples were assayed for α-Gal A activity, and DNA from patients with consistently low plasma α-Gal A activities were sequenced. Results— The study sample consisted of 558 men (42% African-American; median age 44 years). Stroke was cryptogenic in 154 men (40% African-American). In 10 patients with low plasma α-Gal A activities, DNA sequencing identified alterations in the α-Gal A gene in 2 patients. The polymorphism, D313Y, which results in low plasma enzyme activity, but near normal levels of cellular activity was seen in one European-American male. The Fabry disease-causing A143T mutation was seen in an African-American male with cryptogenic stroke (0.18% of all strokes: upper 95% CI=0.53%; 0.65% of cryptogenic strokes: upper 95% CI=1.92%). Conclusions— In this biracial population, unrecognized Fabry disease is a rare but treatable cause of initial ischemic stroke in young men.

Non-pseudogene-derived complex acid beta-glucosidase mutations causing mild type 1 and severe type 2 gaucher disease.

Gaucher disease is an autosomal recessive inborn error of glycosphingolipid metabolism caused by the deficient activity of the lysosomal hydrolase, acid beta-glucosidase. Three phenotypically distinct subtypes result from different acid beta-glucosidase mutations encoding enzymes with absent or low activity. A severe neonatal type 2 variant who presented with collodion skin, ichthyosis, and a rapid neurodegenerative course had two novel acid beta-glucosidase alleles: a complex, maternally derived allele, E326K+L444P, and a paternally inherited nonsense mutation, E233X. Because the only other non-pseudogene-derived complex allele, D140H+E326K, also had the E326K lesion and was reported in a mild type 1 patient with a D140H+E326K/K157Q genotype, these complex alleles and their individual mutations were expressed and characterized. Because the E233X mutation expressed no activity and the K157Q allele had approximately 1% normal specific activity based on cross-reacting immunologic material (CRIM SA) in the baculovirus system, the residual activity in both patients was primarily from their complex alleles. In the type 1 patient, the D140H+E326K allele was neuroprotective, encoding an enzyme with a catalytic efficiency similar to that of the N370S enzyme. In contrast, the E326K+L444P allele did not have sufficient activity to protect against the neurologic manifestations and, in combination with the inactive E233X lesion, resulted in the severe neonatal type 2 variant. Thus, characterization of these novel genotypes with non-pseudogene-derived complex mutations provided the pathogenic basis for their diverse phenotypes.

Identification and expression of acid beta-glucosidase mutations causing severe type 1 and neurologic type 2 Gaucher disease in non-Jewish patients.

Gaucher disease, the most prevalent lysosomal storage disease, occurs in three subtypes, all resulting from mutations in the acid beta-glucosidase gene. Molecular studies in five severely affected type 1 and two type 2 Gaucher disease patients of non-Jewish descent identified six new mutations: K74X, W179X, G195E, S271N, V352L, and a two-base deletion in exon 10 (1450del2). Two additional mutations identified in these patients (R48W and G202R) have been reported previously, but were not expressed or characterized. Heterologous expression in Sf 9 cells using the baculovirus system revealed that the missense mutations, R48W and V352L, had 14 and 7%, respectively, of the specific activity based on cross-reacting immunologic material expressed by the normal allele. In contrast, the G195E, G202R, and S271N mutant alleles were more severely compromised with only 1-2% of the normal expressed specific activity based on cross-reacting immunologic material. Structural distortion at the active site was probed by comparing the interaction of the mutant enzymes with active site-directed inhibitors (castanospermine, conduritol B epoxide and deoxynojirimycin). R48W, G202R, and S271N were normally inhibited, whereas the V352L and G195E mutant enzymes had significantly decreased binding affinity. These mutations further expand the genetic heterogeneity in the lesions causing Gaucher disease types 1 and 2, and further delineate genotype/phenotype correlations and functional domains within the acid beta-glucosidase gene.

Human acid β-glucosidase: Glycosylation is required for catalytic activity

Abstract The role of oligosaccharide modification in human acid β-glucosidase function was investigated. This lysosomal enzyme has five putative N-glycosylation sites, four of which are occupied. The unglycosylated human protein was stable when expressed in bacteria or in Spodoptera frugiperda cells in the presence of tunicamycin but lacked catalytic activity. Deglycosylation of purified acidβ-glucosidase from human placenta with N-Glycanase ™ under native conditions resulted in the removal of an accessible oligosaccharide chain from a single site with no effect on activity, whereas studies demonstrate that occupancy of at least one glycosylation site is required for the formation and maintenance of acid β-glucosidase in an active conformation.

Human acid beta-glucosidase: inhibition studies using glucose analogues and pH variation to characterize the normal and Gaucher disease glycon binding sites.

Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid beta-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., Kis much less than Kii) and had low apparent affinity for the enzyme (Kisapp = 20-500 mmol/l). beta-Glucose-1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-beta-glucoside hydrolysis in an uncompetitive pattern (Kiapp = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-beta-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5-imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with Kisapp(beta-glucose)/Kisapp approximately equal to 10(3)-10(5); an amine at C-2 did not alter Kisapp compared to beta-glucose. The variation of Kisapp with pH for the 5-imino- and 1-deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH2) of the diprotic (Vmaxapp and Vmaxapp/Kmapp) normal enzyme (pK1 = 4.7; pK2 = 6.7) with pH-independent Kisapp values of 2.9-9.0 mumol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that Kisapp(AJGD)/Kisapp(normal) = 4-17 for dNM and 1-deoxy-1-amino-beta-glucose; this ratio was approximately equal to 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid beta-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of beta-glucose and a residue with pKa = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid beta-glucosidase.

Expression of functional human acid beta-glucosidase in COS-1 and Spodoptera frugiperda cells.

A cDNA encoding human acid beta-glucosidase (N-acylsphingosyl-1-O-beta-D-glucoside: glucohydrolase, EC 3.2.1.45) expressed catalytically active enzyme in transfected COS-1 or infected Spodoptera frugiperda (Sf9) cells. The expression plasmid p91023(B) (p91023B/Glc) and a Baculovirus (AcMNPV/Glc) containing the cDNA were constructed and used with the respective cells. By immunoblotting a glycosylated, 63-kilodalton human acid-beta-glucosidase was detected in the transfected or infected cells. A 56-kilodalton human polypeptide was obtained after complete deglycosylation with N-Glycanase. The expressed human enzymes also had partial endoglycosidase H sensitivity. The human enzyme expressed at high levels in Sf9 cells and had normal immunologic properties. With the partially purified enzyme from Sf9 cells, intact function of active site was indicated by normal kcat and Kmapp or Kiapp values for alternative substrates or potent inhibitors, respectively. The expressed enzyme was also activated normally by the negatively charged lipid, taurocholate. The results of these studies indicate that the Baculovirus expression system could provide a convenient source of normal human enzyme for structure/function investigations. In addition, this expression system should prove useful for the identification and evaluation of putative etiologic point mutations in Gaucher disease variants with kinetically altered residual enzymes.

Synthesis and use of novel fluorescent glycosphingolipids for estimating β-glucosidase activity in vitro in the absence of detergents and subtyping Gaucher disease variants following administration into intact cells

Two novel fluorescent glycolipids, LRO-glucosylceramide (LRO-GC) and LRO-trihexosylceramide (LRO-THC) were synthesized and utilized for estimating activities of the lysosomal, acid beta-glucosidase in cell extracts and intact skin fibroblasts, derived from normal individuals and patients with Gaucher disease subtypes. The uniqueness of the glycolipids is the fact that a fluorescent probe (lissamine rhodamine) is linked in a sulfonylamide linkage to the sphingosyl residue of the sphingolipid. Thus, the product of enzymatic hydrolysis, lissamine rhodamine sulfonylamido sphingosine (LRO-ceramide) cannot be further hydrolyzed and remains a metabolic end product. A unique property of LRO-GC as a substrate for the lysosomal, acid beta-glucosidase in vitro was the observation that enzymatic hydrolysis occurs in the absence of detergents and that hydrolytic rates are, in fact, reduced in the presence of Triton X-100 and/or sodium taurocholate. Also, both glycolipids penetrated the membrane of intact fibroblasts in the absence of serum and were hydrolyzed in lysosomes of the intact cells. The rates of intracellular hydrolysis decreased with the severity of the Gaucher disease subtypes. Using LRO-THC as substrate, the intracellular ratio of LRO-ceramide to LRO-glucosylceramide was an indicator for the specific GD-subtype.

Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum

A rapid and simple turbulent flow liquid chromatography (TFC-LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC-LC-MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000nmol/L and for 7-OH MTX from 20 to 2000nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10nmol/L to 5×10(5)nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC-LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC-MS/MS assay.