Normal MPS Research Articles

Correction of Clinical Manifestations of Canine Mucopolysaccharidosis I with Neonatal Retroviral Vector Gene Therapy

Mucopolysaccharidosis I (MPS I) (Hurler syndrome) is due to deficient alpha-L-iduronidase (IDUA) activity and is the most common of the MPS disorders. Neonatal MPS I dogs were injected intravenously (IV) with a gamma retroviral vector containing a complete long-terminal repeat (LTR) and an internal human alpha(1)-antitrypsin (hAAT) promoter upstream of the canine IDUA complementary DNA (cDNA). This resulted in stable serum IDUA activity of 366 +/- 344 units (U)/ml (28-fold normal) for up to 1.8 years, which likely derived primarily from secretion of IDUA by transduced liver cells. Retroviral vector (RV)-treated dogs had >18% of normal IDUA activity in organs and had decreased severity and/or incidence of hernias, chest deformities, joint disease, facial dysmorphia, corneal clouding, valvular heart disease, and aortic dilatation as compared with untreated MPS I dogs. The marked reduction that was observed in lysosomal storage in the brain of RV-treated dogs may have been due in part to expression from the LTR of the vector in cells in the brain. This possibility will be explored in future studies, because the potential for insertional mutagenesis has raised concerns about using vectors with an intact LTR. If proven safe, this gene therapy technique may be utilized in treating children with Hurler syndrome.

Impact of Body Mass Index on Cardiac Mortality in Patients With Known or Suspected Coronary Artery Disease Undergoing Myocardial Perfusion Single-Photon Emission Computed Tomography

The purpose of this study was to assess the relationship between body mass index (BMI) and the prognostic value of myocardial perfusion single-photon emission computed tomography (MPS). The prognostic value of MPS in the obese has not been evaluated. We studied 4,720 patients with and 10,019 patients without known coronary artery disease (CAD) who underwent rest Tl-201/stress Tc-99m sestamibi MPS, including 5,233 gated MPS studies and followed up (mean 2.7 to 3.2 years). Patients were categorized as normal weight (BMI 18.5 to 24.9 kg/m2), overweight (BMI 25.0 to 29.9 kg/m2), or obese (BMI > or =30.0 kg/m2). Unadjusted annual rates of cardiac death (CD) rose versus stress MPS abnormalities in all weight groups (p < 0.001). Obese or overweight patients with or without known CAD who had normal MPS were at low CD risk (<1%/year), similar to normal weight patients. In CAD, obese and overweight patients with abnormal MPS had lower rates of CD compared with normal weight patients (p < 0.01). In patients with low ejection fraction (EF) by gated MPS, those with normal weight had highest CD rate (p = 0.001). Multivariable models revealed that BMI was not a predictor of CD in suspected CAD patients (hazard ratio [HR] 0.99; 95% confidence interval [CI] 0.95 to 1.02) but was an independent inverse predictor of CD in known CAD patients (HR 0.95; 95% CI 0.92 to 0.98), especially in women, adenosine stress, low EF, or abnormal perfusion. Normal MPS was associated with low risk of CD in patients of all weight categories. In patients with known CAD undergoing MPS, obese and overweight patients were at lower risk of CD over three years than normal weight patients.

Detection of hunter syndrome (mucopolysaccharidosis type II) in Taiwanese: Biochemical and linkage studies of the iduronate-2-sulfatase gene defects in MPS II patients and carriers

Background Hunter syndrome (mucopolysaccharidosis type II) is an X-linked recessive lysosomal storage disease caused by a defect of the iduronate-2-sulfatase (IDS) gene. The result is impaired IDS enzyme function. Methods To characterize the biochemical and molecular defects in IDS-deficient patients and their families, we measured IDS enzyme activity by fluorimetric enzyme assay and identified the IDS gene mutations in 14 unrelated Taiwanese patients with varying clinical phenotypes. In addition, haplotype analysis was also performed. Results Three novel (IVS2 + 1G > C, 1055del12, and G489D) and 7 previously reported (N63K, P228L, K347E, R468Q, R468W, I485R, and 1241delAG) mutations were found. Together R468Q and R468W account for 42.8% mutations found in our patients. Haplotype analysis using IDS flanking markers DXS1113 and DXS1123 revealed that the unrelated R468Q alleles were independent in origin whereas the unrelated R468W alleles are probably of the same origin. The R468Q mutation in patient 1150 and I485R mutation in patient 710 occurred de novo in male meioses. Once the mutation in a family was identified, restriction analysis was also performed for rapid diagnosis of female carriers in 8 families. Leukocyte IDS measurement revealed significantly wide range of IDS activity in normal controls and MPS II carriers (19.2∼70.6 vs. 8.4∼26.6 nmol/h/mg cell protein). The average leukocyte IDS activity of normal controls ( n = 43) was 43.9 ± 13.3 nmol/h/mg protein, whereas patients with MPS II ( n = 14) had < 5% of mean normal IDS activity (0.9 ± 0.6 nmol/h/mg protein), and carriers ( n = 13) had a mean activity of 17.5 (± 5.7) nmol/h/mg protein. The mean leukocyte IDS activity in female carriers was less than a half of the normal level. Conclusion Due to a small overlapping range of normal and carriers, the level of enzyme activity cannot be used alone for carrier detection.

Prognostic value of myocardial perfusion SPECT versus exercise electrocardiography in patients with ST-segment depression on resting electrocardiography

The value of exercise-induced ST-segment depression for the prognostic evaluation of patients with 1 mm of ST depression or greater on the resting electrocardiogram is controversial. Patients who underwent exercise myocardial perfusion single photon emission computed tomography (MPS) and had resting ST depression of 1 mm or greater with a nondiagnostic exercise electrocardiographic response (n = 1122) were followed up for 3.4 +/- 2.3 years. Those with paced rhythm, pre-excitation, left bundle branch block, or myocardial revascularization within the first 60 days after MPS were excluded. Additional exercise-induced ST-segment depression was considered significant if > or = 2 mm MPS was scored semiquantitatively by use of a 20-segment model of the left ventricle; the percentage of myocardium involved with stress defects (% myo) was derived by normalizing to the maximal possible score of 80. Hard events were defined as nonfatal myocardial infarction or cardiac death. A Cox analysis was used to determine independent predictors of hard events among clinical, exercise, and nuclear variables. Hard event rates increased as a function of % myo for either patients with exercise-induced ST depression (1.4%/y for normal MPS vs 4.1%/y for % myo >10%, P < .03) or those without it (0.7%/y for normal MPS vs 3.0%/y for % myo >10%, P = .0001). Age, diabetes mellitus, shortness of breath as the presenting symptom, and % myo were independent predictors of hard events. Exercise-induced ST depression was predictive of hard events only when it was 3 mm or greater. The presence and extent of perfusion defects, reflected in the % myo, had incremental prognostic value over clinical variables and also over all degrees of exercise-induced ST depression. Although MPS effectively risk-stratifies patients with resting ST depression of 1 mm or greater, the prognostic value of exercise-induced ST depression is limited in these patients, with a small added risk when severe (> or = 3 mm).

The use of nuclear cardiology in clinical decision making

Extensive data exist to support the role of myocardial perfusion single-photon emission computed tomography (MPS) in risk stratification. Normal MPS studies usually are associated with very low risk, and patient risk increase significantly as a function of MPS results. Ventricular function measurements from gated single-photon emission computed tomography further augment risk stratification, particularly with respect to identifying patients at risk of cardiac death. Ancillary findings are prognostically important, particularly in the setting of normal or near-normal MPS results. Recent data suggest that MPS results can identify which patients will benefit from revascularization versus medical therapy and have expanded the understanding of how stress MPS is helpful in the identification of risk, enhanced the means of identifying risk, and improved its use as a means to identify optimal posttest treatment.

Development of an assay for the detection of mucopolysaccharidosis type VI patients using dried blood-spots

Background Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disorder (LSD), which is caused by a deficiency in the enzyme N-acetylgalactosamine 4-sulfatase (4-sulfatase). MPS VI is characterized by severe skeletal abnormalities, somatic tissue pathology and early death. Treatment possibilities include bone marrow transplantation (BMT) and enzyme replacement therapy (ERT; currently in phase III clinical trial). Early diagnosis of MPS VI will allow treatment before the onset of irreversible pathology. Methods Sensitive immune assays have been developed to detect 4-sulfatase protein and activity in normal control and MPS VI blood-spots. Results Dried blood-spots from MPS VI patients contained no detectable 4-sulfatase protein and activity, compared to 3.5–21 μg/L of 4-sulfatase protein and 291–1298 nmol/min/L of activity for normal human controls. In this evaluation study, the assay was sensitive and 100% specific, allowing reliable detection of individuals with MPS VI. Conclusions The assays reported here have the potential to detect MPS VI patients using dried blood-spots.

Dispersal of Therapeutic Donor Cells throughout the Brain of Mice with Lysosomal Storage Disease Occurs Following in Utero Transplantation of Fetal-Derived Neuronal Stem Cells.

Mucopolysaccharidosis Type VII (MPS VII) is one of many lysosomal storage diseases and is caused by a deficiency of beta-glucuronidase (GUS). Progressive accumulation of undegraded glycosaminoglycan (GAG) intermediates occurs in the lysosome diminishing intellect, mobility, organ function, and life span. Previously, the efficacy of enzyme replacement therapy (ERT), bone marrow transplantation, and gene therapy have been tested in MPS VII mice. GUS supplied to serum by ERT or released into extracellular spaces from transplanted (Tx) normal or gene transduced cells is taken up by receptor mediated endocytosis and transported to the lysosome. All these treatments reduce lysosomal storage in visceral organs, but poorly deliver sufficient enzyme to the central nervous system (CNS). We hypothesized that in utero intrathecal transplantation of primary neuronal stem cells (NSC) would remedy the GUS deficiency of MPS VII CNS. We isolated NSC from the cortical ventricular zone (CVZ) of E14 GUS+ eGFP+ fetuses. CVZ NSC incubated with EGF and bFGF formed neurospheres in 3–5 days. E14 MPS VII GUS- recipients were Tx intrathecally with NSC and examined for engraftment by histological staining for GUS and eGFP expressing cells. Successful engraftment occurred in 13 of 23 (56.5%) MPS VII recipients. Three animals Tx with 5–25,000 (K) had GUS+ cells in the rostral migratory stream (RMS). Two of seven animals Tx with 50-80K had donor cells in the RMS and five had donor cells in olfactory bulb, cerebrum (cortical layers, corpus callosum and striatum), midbrain, hippocampus (including dentate gyrus), and the cerebellum (Purkinje layers, peduncle, and lobular regions). This demonstrates “complete” dispersal. One mouse Tx with 90K had donor cells lining the lateral ventricle. Another Tx with 160K had donor cells lining ventricular spaces and throughout the cerebrum away from the RMS. An animal Tx with 180K NSC had “complete” donor engraftment like those above. Histological staining for GUS+ donor cells in mice with “complete” engraftment showed levels of staining greater than untreated heterozygous controls, both in numbers of cells positive and in intensity of staining. Biochemistry of tissue sections from these animals averaged 59.5 ±9.2% (mean ±SE) of normal (+/+) activity. The oldest animal detected with “complete” engraftment thus far was 7 months old at sacrifice, demonstrating long-term durability of engraftment. Using anti-eGFP, beta-tubulin III (neurons), and GFAP (astrocytes) antibodies, we confirmed the “complete” engraftment was due to widespread dispersal and integration of donor derived cells and not to diffusion of GUS enzyme from a few engrafted cells. The lineage markers confirmed normal differentiation of donor CVZ NSC. Further evaluations are underway to measure cognitive function in treated animals by the Repeated Acquisition and Performance Chamber (RAPC). Comparison of untreated MPS VII adult mice to normal controls by the RAPC indicates significant deficiencies in learning and memory in untreated MPS VII mice and confirms our ability to measure functional changes in treated animals. In summary, GUS+ eGFP+ fetal NSC primary isolates engraft brain of MPS VII fetal recipients and restore GUS activity ≥ phenotypically normal MPS VII heterozygotes.

Relationship between stress-induced myocardial ischemia and atherosclerosis measured by coronary calcium tomography

We assessed the relationship between stress-induced myocardial ischemia on myocardial perfusion single-photon emission computed tomography (MPS) and magnitude of coronary artery calcification (CAC) by X-ray tomography in patients undergoing both tests. There has been little evaluation regarding the relationship between CAC and inducible ischemia or parameters that might modify this relationship. A total of 1,195 patients without known coronary disease, 51% asymptomatic, underwent stress MPS and CAC tomography within 7.2 +/- 44.8 days. The frequency of ischemia by MPS was compared to the magnitude of CAC abnormality. Among 76 patients with ischemic MPS, the CAC scores were >0 in 95%, >or=100 in 88%, and >or=400 in 68%. Of 1,119 normal MPS patients, CAC scores were >0, >or=100, and >or=400 in 78%, 56%, and 31%, respectively. The frequency of ischemic MPS was <2% with CAC scores <100 and increased progressively with CAC >or=100 (p for trend <0.0001). Patients with symptoms with CAC scores >or=400 had increased likelihood of MPS ischemia versus those without symptoms (p = 0.025). Absolute rather than percentile CAC score was the most potent predictor of MPS ischemia by multivariable analysis. Importantly, 56% of patients with normal MPS had CAC scores >or=100. Ischemic MPS is associated with a high likelihood of subclinical atherosclerosis by CAC, but is rarely seen for CAC scores <100. In most patients, low CAC scores appear to obviate the need for subsequent noninvasive testing. Normal MPS patients, however, frequently have extensive atherosclerosis by CAC criteria. These findings imply a potential role for applying CAC screening after MPS among patients manifesting normal MPS.

Stress myocardial perfusion single-photon emission computed tomography is clinically effective and cost effective in risk stratification of patients with a high likelihood of coronary artery disease (CAD) but no known CAD

ObjectivesWe sought to evaluate the prognostic and cost implications of stress myocardial perfusion single-photon emission computed tomography (SPECT), or MPS, in patients with a high pretest likelihood (>0.85) of coronary artery disease (CAD) with no previous CAD. BackgroundSparse data are available regarding the prognostic performance characteristics of MPS in this patient group. MethodsWe followed up 1,270 consecutive patients with no previous revascularization or myocardial infarction (MI), with a pre–exercise tolerance test (ETT) likelihood of CAD ≥0.85, who underwent exercise or adenosine stress MPS (follow-up 94.4% complete; 2.2 ± 1.2 years; 60 hard events [5.9%, 2.6%/year]). Risk adjustment of survival data was done using Cox proportional hazards analysis. Costs per reclassification of risk were calculated using assumed costs and threshold analyses. ResultsIn patients treated medically after MPS, normal MPS had a low risk of cardiac death and hard events (0.6% and 1.3% per year, respectively). With increasing extent and severity of MPS defects, the risk of both cardiac death and hard events increased significantly (p < 0.05). Cox models indicated that the addition of MPS data resulted in incremental prognostic value over pre-MPS data (chi-square increase 48 to 87, p < 0.0001). Compared with strategies of initial referral to ETT in patients able to exercise, initial referral to MPS appeared to be a more cost-effective strategy. Similarly, compared with a strategy of direct referral to catheterization in patients with a high likelihood of CAD, initial referral to MPS is a cost-saving approach. ConclusionsIn patients with a high likelihood of CAD but without known CAD, stress MPS yields incremental value and achieves risk stratification in a cost-effective manner. The current results support a strategy of initial stress imaging in this patient cohort, as a reasonable alternative to direct referral to catheterization or initial ETT.

Transient ischemic dilation ratio of the left ventricle is a significant predictor of future cardiac events in patients with otherwise normal myocardial perfusion SPECT

ObjectivesThis study evaluated the prognostic value of transient ischemic dilation (TID) of the left ventricle (LV) in patients with normal stress myocardial perfusion single photon emission computed tomography (MPS). BackgroundThe prognostic value of TID in patients with an otherwise normal MPS has not been defined. MethodsWe identified 1,560 patients who had normal stress MPS (436 vasodilator and 1,124 exercise stress), and no rest LV enlargement (Population 1) and followed up for 2.30 ± 0.67 years for hard events (HE) (cardiac death or myocardial infarction) and soft events (SE) (revascularization). Prediction of first HE or SE (total events [TE]) was evaluated by multivariable Cox analysis, which was also applied to a broader group of 2,037 patients (including patients with minimal defects (Population 2). ResultsIn Population 1, there were 13 HE, 36 SE, and 42 TE. Patients in the highest TID quartile (TID ≥1.21) had a higher TE rate than others, regardless of stress type. By multivariable analysis, highest TID quartile was predictive of TE (p = 0.008). Other independent predictors of TE were age, typical angina, and diabetes. In Population 2, TID was also predictive of TE. ConclusionsAn entirely normal stress MPS study does not always imply an excellent prognosis. In patients with otherwise normal MPS, TID is an independent and incremental prognostic marker of TE even after significant clinical variables—age, typical angina, and diabetes—are accounted for. When TID is present, caution in making low-risk prognostic statements may be warranted, especially in patients with typical angina, the elderly, and diabetics. Our findings also appear to apply to the broader population of “normal” MPS, which included patients with minimal perfusion defects.

Determinants of risk and its temporal variation in patients with normal stress myocardial perfusion scans: What is the warranty period of a normal scan?

ObjectivesThe aim of this study was to determine the predictors of risk and the temporal characteristics of risk associated with normal myocardial perfusion single photon emission computed tomography (MPS). BackgroundNo empiric data exist regarding predictors of risk after normal MPS and their temporal characteristics. MethodsFollow-up (mean: 665 ± 200 days, 96% complete) of 7,376 consecutive patients with normal exercise or adenosine MPS identified 78 hard events (HE) (45 cardiac deaths, 33 non-fatal myocardial infarction; 1.1% cumulative HE rate, 0.6%/year). Cox proportional hazards analysis was used to identify predictors of HE. Parametric survival analysis was used to model predicted time to HE. ResultsThe HE rates were greater in patients with versus without previous coronary artery disease (CAD). The Cox proportional hazards model identified pharmacologic stress, known CAD, diabetes mellitus (DM), male gender, and increasing age, with interactions between stress type and previous CAD (lower risk in patients without previous CAD undergoing exercise stress vs. all others) and between DM and gender (higher risk in DM females vs. all others) as the model most predictive of HE. The highest risk subgroups had a maximal event rate of 1.4% to 1.8%/year. Parametric survival models revealed that in patients without previous CAD the level of risk was uniform with time, but in patients with known CAD, risk increased with time (e.g., risk in the first year was less than in the second year, hence, a dynamic temporal component of risk was present). ConclusionsMultiple clinical factors add incremental prognostic value in patients with normal MPS, affecting their risk and its temporal pattern, and may alter the appropriate timing of repeat testing, hence establishing the existence of a “warranty” period for normal MPS studies.

Arginine metabolic pathways involved in the modulation of tumor-induced angiogenesis by macrophages

Neovascularization, an essential step for tumor progression and metastasis development, can be modulated by the presence of macrophages (Mps) in the tumor microenvironment. The ability of Mps to regulate the angiogenicity of the LMM3 tumor cell line was studied. Peritoneal Mps from LMM3 tumor-bearing mice (TMps) potentiate in vivo LMM3 angiogenicity. These results were confirmed by CD31 immunoblotting assays. The activity of TMps depended on nitric oxide synthase (NOS) and arginase (A) activity. By immunoblotting we evidenced that AI and AII isoforms were up-regulated in TMps while the inducible and neuronal NOS isoforms were highly expressed in normal Mps. TMps might positively modulate tumor growth by stimulating angiogenic cascade mainly through polyamine synthesis.

Molecular diagnostic tests for ascertainment of genotype at the mucopolysaccharidosis type VII locus in dogs

Abstract Objective To develop a molecular diagnostic test to ascertain genotype of the mucopolysaccharidosis type VII (MPS VII) locus. Sample Population Blood samples from 45 mixed-breed (German Shepherd Dog X Beagle) dogs that were phenotypically normal or affected with MPS VII. Procedure The canine ß-glucuronidase gene (exon 3) was amplified by polymerase chain reaction (PCR), using 2 pairs of primers to determine the genotype of the MPS VII locus by 2 independent methods. For the first method, PCR products were used for heteroduplex analysis, using conformation-sensitive gel electrophoresis. In the second method, an allele-specific restriction site was created by use of a mismatch primer in PCR. The amplified DNA fragment was digested with a restriction enzyme (Eag I) to enable identification of the wild-type and mutant alleles. Results Conformation-sensitive gel electrophoresis resulted in a single DNA band representing homoduplex when the sample contained a wild-type or MPS VII allele, but 2 bands representing hetero- and homoduplexes when both alleles were in the sample. Restriction digestion of the DNA fragment obtained by use of a mismatch primer was cleaved only when the template was a wild-type allele. Thus, samples from phenotypically normal carrier dogs that contained wild-type and MPS VII alleles were partially digested by the enzyme. Conclusions The diagnostic test used 2 strategies for independently ascertaining the wild-type or mutant MPS VII alleles in dogs. Thus, test results can distinguish phenotypically normal MPS Vll-carrier dogs from homozygous normal dogs. (Am J Vet Res 1998;59:1092-1095)

Enzyme replacement therapy in Mucopolysaccharidosis VI: evidence for immune responses and altered efficacy of treatment in animal models

Enzyme replacement therapy (ERT) can potentially result in an immunological response to the introduced protein. The immunological response by Mucopolysaccharidosis type VI (MPS VI) cats to recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) ERT has been investigated. Plasma antibody titres to rh4S were detected in untreated MPS VI and normal control cats, but the antibody titres to rh4S were higher in ERT treated MPS VI cats. The reactivity by cats to rh4S did not appear to be just due to species cross reactivity, as plasma antibodies from normal control, MPS VI and MPS VI ERT cats reacted equally with feline and human 4-sulfatase. Normal control and MPS VI human plasma also had antibody titres to rh4S. Plasma antibodies to rh4S, from an ERT treated cat, could be temporarily removed from circulation by enzyme infusion, confirming specificity for rh4S and indicating a possible window for ERT in the absence of antibody. In enzyme distribution studies with 3H-rh4S, evidence of altered targeting, and enzyme inactivation and degradation were observed in high compared to low titre rats. In high titre rats, the observed loss of 3H-label from vacuolar organelles of the liver may represent either degradation of antibody bound 3H-rh4S for reutilisation within the liver, or antigen presentation. The development of high titre antibody may have a detrimental effect on the efficacy of ERT.