Mechanistic Insights into the Chaperoning of Human Lysosomal-Galactosidase Activity: Highly Functionalized Aminocyclopentanes and C-5a-Substituted Derivatives of 4-epi-Isofagomine.

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid ß-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent ß-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of ß-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

Morquio-B disease: Clinical and genetic characteristics of a distinct GLB1-related dysostosis multiplex.

BACKGROUND: Morquio-B disease (MBD) is a distinct GLB1-related dysostosis multiplex involving the trabecular parts of long bones and spine, presenting a mild phenocopy of GALNS-related Morquio-A disease. METHODS: We analyzed 63 (n = 62 published) cases with MBD to describe their clinical, biochemical and genetic features. RESULTS: Forty-one of 51 cases with informative clinical data had pure MBD including progressive growth impairment, kyphoscoliosis, coxa/genua valga, joint laxity, platyspondyly, odontoid hypoplasia. Ten of 51 had MBD plus neuronopathic manifestations including intellectual/developmental/speech delay, spasticity, ataxia dystonia. Corneal clouding, cardiac valve pathology, hepatosplenomegaly, spinal cord compression were infrequent and atlantooccipital dislocation, cardiomyopathy and cherry red spot were never reported. Urinary glycosaminoglycan and oligosaccharide excretion was consistently abnormal. Keratan sulphate-derived oligosaccharides were only detected using LC-MS/MS-based methods. Residual ß-galactosidase activities measured against synthetic substrates were 0%-17%.Among 28 GLB1 variants, W273 L (34/94 alleles) and T500A (11/94 alleles) occurred most frequently. W273L was invariably associated with pure MBD. Pure MBD also was reported in a case homozygous for R201H, and in the majority of cases carrying the T500A variant. Homozygous Y333C and G438E were associated with MBD plus neuronopathic manifestations. T82M, R201H, and H281Y, observed in seven alleles, previously have been found sensitive to experimental chaperones. CONCLUSION: Data provide a basis for future systematic collection of clinical, biochemical, morphologic, and genetic data of this ultra-rare condition.

The Clinical and Molecular Spectrum of GM1 Gangliosidosis.

OBJECTIVE: To evaluate the clinical presentation of patients with GM1 gangliosidosis and to determine whether specific clinical or biochemical signs could lead to a prompt diagnosis. STUDY DESIGN: We retrospectively analyzed clinical, biochemical, and genetic data of 22 patients with GM1 gangliosidosis from 5 metabolic centers in Germany and Austria. RESULTS: Eight patients were classified as infantile, 11 as late-infantile, and 3 as juvenile form. Delay of diagnosis was 6 ± 2.6 months in the infantile, 2.6 ± 3.79 years in the late-infantile, and 14 ± 3.48 years in the juvenile form. Coarse facial features, cherry red spots, and visceromegaly occurred only in patients with the infantile form. Patients with the late-infantile and juvenile forms presented with variable neurologic symptoms. Seventeen patients presented with dystonia and 14 with dysphagia. Laboratory analysis revealed an increased ASAT concentration (13/20), chitotriosidase activity (12/15), and pathologic urinary oligosaccharides (10/19). Genotype analyses revealed 23 causative or likely causative mutations in 19 patients, 7 of them being novel variants. In the majority, a clear genotype-phenotype correlation was found. CONCLUSIONS: Diagnosis of GM1 gangliosidosis often is delayed, especially in patients with milder forms of the disease. GM1 gangliosidosis should be considered in patients with progressive neurodegeneration and spastic-dystonic movement disorders, even in the absence of visceral symptoms or cherry red spots. ASAT serum concentrations and chitotriosidase activity may be of value in screening for GM1 gangliosidosis.

Potent GH20 N-Acetyl-ß-d-hexosaminidase Inhibitors: N-Substituted 3-acetamido-4-amino-5-hydroxymethyl-cyclopentanediols.

From 1,2;3,4-di-O-isopropylidene-d-galactopyranose, a preliminary series of highly functionalized amino(hydroxymethyl)cyclopentanes was easily available. These amine-containing basic carbasugars featuring the d-galacto configuration are potent inhibitors of the GH20 ß-d-hexosaminidases probed and may bear potential as regulators of N-acetyl-d-hexosaminidase activities in vivo.

A new type of pharmacological chaperone for GM1-gangliosidosis related human lysosomal ß-galactosidase: N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols.

N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of ß-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols: A new family of activity promotors for a GM1-gangliosidosis related human lysosomal ß-galactosidase mutant.

From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger on similar structures, these amine containing basic carbasugars are potent inhibitors of ß-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

A Morita-Baylis-Hillman based route to C-5a-chain-extended 4-epi-isofagomine type glycosidase inhibitors.

By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,ß-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of ß-galactosidases, is outlined. In line with reported data, the (C-5aR) epimer was found a highly potent experimental pharmacological chaperone for GM1-associated human lysosomal ß-galactosidase mutant R201C.

Screening for Fabry Disease by Urinary Globotriaosylceramide Isoforms Measurement in Patients with Left Ventricular Hypertrophy.

BACKGROUND: Left ventricular hypertrophy (LVH) is a frequent echocardiographic feature in Fabry disease (FD) and in severe cases may be confused with hypertrophic cardiomyopathy (HCM) of other origin. The prevalence of FD in patients primarily diagnosed with HCM varies considerably in screening and case finding studies, respectively. In a significant proportion of patients, presenting with only mild or moderate LVH and unspecific clinical signs FD may remain undiagnosed. Urinary Gb3 isoforms have been shown to detect FD in both, women and men. We examined whether this non-invasive method would help to identify new FD cases in a non-selected cohort of patients with various degree of LVH. METHODS AND RESULTS: Consecutive patients older than 18 years with a diastolic interventricular septal wall thickness of ≥12mm determined by echocardiography were included. Referral diagnosis was documented and spot urine was collected. Gb3 was measured by mass spectroscopy. Subjects with an elevated Gb3-24:18 ratio were clinically examined for signs of FD, α-galactosidase-A activity in leukocytes was determined and GLA-mutation-analysis was performed. We examined 2596 patients. In 99 subjects urinary Gb3 isoforms excretion were elevated. In these patients no new cases of FD were identified by extended FD assessment. In two of three patients formerly diagnosed with FD Gb3-24:18 ratio was elevated and would have led to further diagnostic evaluation. CONCLUSION: Measurement of urinary Gb3 isoforms in a non-selected cohort with LVH was unable to identify new cases of FD. False positive results may be prevented by more restricted inclusion criteria and may improve diagnostic accuracy of this method.

Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable ß-galactosidase inhibitors and activity promotors of GM1-gangliosidosis related human lysosomal ß-galactosidase mutant R201C.

From an easily available partially protected analog of 1-deoxy-L-gulo-nojirimycin, by chain-branching at C-4 and suitable modification, lipophilic analogs of the powerful ß-D-galactosidase inhibitor 4-epi-isofagomine have been prepared. New compounds exhibit considerably improved inhibitory activities when compared with the unsubstituted parent compound and may serve as leads toward new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

Synthesis of C-5a-chain extended derivatives of 4-epi-isofagomine: Powerful ß-galactosidase inhibitors and low concentration activators of GM1-gangliosidosis-related human lysosomal ß-galactosidase.

From an easily available partially protected formal derivative of 1-deoxymannojirimycin, by hydroxymethyl chain-branching and further elaboration, lipophilic analogs of the powerful ß-d-galactosidase inhibitor 4-epi-isofagomine have become available. New compounds exhibit improved inhibitory activities comparable to benchmark compound NOEV (N-octyl-epi-valienamine) and may serve as leads towards improved and more selective pharmacological chaperones for GM1-gangliosidosis.

Interfering parameters in the determination of urinary globotriaosylceramide (Gb3) in patients with chronic kidney disease.

INTRODUCTION: Globotriaosylceramide (Gb3, CD77) represents a pivotal part of the cell membrane. Measuring the urinary Gb3 content can be used to screen patients with chronic kidney disease (CKD) for Fabry disease, a disorder caused by hampered Gb3 degradation. However, little is known about factors influencing urinary Gb3 excretion other than Fabry disease. The aim of the present study was to identify routine diagnostic parameters as predictors of urinary Gb3 excretion in patients with CKD. METHODS: Our study included 609 subjects with CKD stage I-V. We analyzed the influence of age, gender, renal function, urinary cell content and chemical characteristics on urinary Gb3 concentrations (total Gb3, Gb3-24 isoform, and Gb3-24:18 isoform ratio), determined by direct electrospray ionization mass spectrometry. RESULTS: In 609 subjects the median total urinary Gb3 was 233 ng/mg and the Gb3-24:18 isoform ratio was 1.2. Twenty-one patients, none of whom had Fabry disease, had a Gb3-24:18 isoform ratio ≥2.3. Females excreted a higher total amount of Gb3, but the Gb3-24:18 isoform ratio was comparable to males. Renal function and age had no influence on total Gb3, Gb3 isoforms or the ratio. Only a distinct load of bacteria and leukocytes was associated with an increased Gb3 excretion. Urinary leukocytes, erythrocytes, bacteria, or protein content did not affect the Gb3-24:18 isoform ratio. CONCLUSION: The Gb3-24:18 isoform ratio is unaffected by several potential influencing variables and may thus be applied for screening for Fabry disease in unselected cohorts of patients presenting with CKD.

Distinct Niemann-Pick Disease Type C Clinical, Cytological, and Biochemical Phenotype in an Adult Patient With 1 Mutated, Overexpressed NPC1 Allele

Abstract Niemann-Pick disease type C (NP-C) is a rare autosomal-recessive neurovisceral lysosomal storage disease. We report on a juvenile onset, now 25-year-old female patient with typical neurologic symptoms, including vertical gaze palsy, of NP-C. The diagnosis was supported by a positive filipin test ("variant biochemical phenotype" of cholesterol accumulation) in cultured fibroblasts, high numbers of "Niemann-Pick cells" in the bone marrow, and 1 positive out of 3 NP-C biomarkers tested, but NP-C was not definitely confirmed genetically. She showed only 1 known NPC1 variant (3 bp deletion in exon 18; p.N916del); this allele, however, being distinctly overexpressed at the messenger RNA level as compared to the wild-type allele, as a not as yet clarified (copathogenic?) phenomenon. The patient's mother, also carrying the p.N916del allele but without overexpression, has a chronic inflammatory disease of the central nervous system classified as multiple sclerosis. However, her severe clinical phenotype includes some signs also consistent with NP-C. The laboratory diagnosis of NP-C can be challenging in detecting novel disease constellations.

Unrelated CD3/CD19-depleted peripheral stem cell transplantation for Hurler syndrome.

For patients with mucopolysaccharidosis type IH (MPS1-H; Hurler syndrome), early allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice. One boy and one girl aged 20.5 and 22 months, respectively, with MPS1-H received a conditioning regimen consisting of thiotepa, fludarabine, treosulfan, and ATG. Grafts were peripheral blood stem cells from unrelated donors (10/12 and 11/11 matched), that were manipulated by CD3/CD19 depletion and contained 20.3 and 28.2 × 10(6) CD34+ cells/kg body weight, respectively. Both patients achieved stable hematopoietic engraftment and stable donor chimerism. Neither acute or chronic graft-versus-host disease (GVHD) nor other severe transplant-related complications occurred. At a follow-up of 48 and 37 months, both patients are alive and well with normal levels of α-L-iduronidase and have made major neurodevelopmental progress. Treosulfan-based conditioning offers the advantage of reduced toxicity; the use of unrelated CD3/CD19-depleted peripheral stem cell grafts allows transfusion of high CD34+ cell numbers together with a "tailored" number of CD3+ cells as well as engraftment facilitating cells in order to achieve rapid hematopoietic engraftment while reducing the risk of graft rejection and GVHD. This regimen might be an additional option when unrelated donor HSCT is considered for a patient with MPS1-H.

Pyridoxine responsiveness in novel mutations of the PNPO gene.

OBJECTIVE: To determine whether patients with pyridoxine-responsive seizures but normal biomarkers for antiquitin deficiency and normal sequencing of the ALDH7A1 gene may have PNPO mutations. METHODS: We sequenced the PNPO gene in 31 patients who fulfilled the above-mentioned criteria. RESULTS: We were able to identify 11 patients carrying 3 novel mutations of the PNPO gene. In 6 families, a homozygous missense mutation p.Arg225His in exon 7 was identified, while 1 family was compound heterozygous for a novel missense mutation p.Arg141Cys in exon 5 and a deletion c.279_290del in exon 3. Pathogenicity of the respective mutations was proven by absence in 100 control alleles and expression studies in CHO-K1 cell lines. The response to pyridoxine was prompt in 4, delayed in 2, on EEG only in 2, and initially absent in another 2 patients. Two unrelated patients homozygous for the p.Arg225His mutation experienced status epilepticus when switched to pyridoxal 5'-phosphate (PLP). CONCLUSIONS: This study challenges the paradigm of exclusive PLP responsiveness in patients with pyridoxal 5'-phosphate oxidase deficiency and underlines the importance of consecutive testing of pyridoxine and PLP in neonates with antiepileptic drug-resistant seizures. Patients with pyridoxine response but normal biomarkers for antiquitin deficiency should undergo PNPO mutation analysis.

Positive outcome following early diagnosis and treatment of pyridoxal-5'-phosphate oxidase deficiency: a case report.

Pyridoxal-5'-phosphate oxidase (PNPO) deficiency is a rare autosomal recessive, vitamin-responsive metabolic disorder causing refractory neonatal seizures that respond to the administration of pyridoxal-5'-phosphate (PLP). There are currently few case studies that have documented the functional outcome in PNPO deficiency, which remains poor in the majority of cases. We present the case of a male infant born at 35 weeks gestation who promptly responded to oral administration of PLP, following resistance to common anticonvulsive therapy and to a pyridoxine trial. Neurological outcome at 21 months is favorable and illustrates the importance of standardized vitamin trials in the acute setting of "therapy-resistant" neonatal seizures. Early recognition of PNPO deficiency and appropriate intervention might be associated with a more favorable outcome than initially considered.

Acute cerebrovascular disease in the young: the Stroke in Young Fabry Patients study.

BACKGROUND AND PURPOSE: Strokes have especially devastating implications if they occur early in life; however, only limited information exists on the characteristics of acute cerebrovascular disease in young adults. Although risk factors and manifestation of atherosclerosis are commonly associated with stroke in the elderly, recent data suggests different causes for stroke in the young. We initiated the prospective, multinational European study Stroke in Young Fabry Patients (sifap) to characterize a cohort of young stroke patients. METHODS: Overall, 5023 patients aged 18 to 55 years with the diagnosis of ischemic stroke (3396), hemorrhagic stroke (271), transient ischemic attack (1071) were enrolled in 15 European countries and 47 centers between April 2007 and January 2010 undergoing a detailed, standardized, clinical, laboratory, and radiological protocol. RESULTS: Median age in the overall cohort was 46 years. Definite Fabry disease was diagnosed in 0.5% (95% confidence interval, 0.4%-0.8%; n=27) of all patients; and probable Fabry disease in additional 18 patients. Males dominated the study population (2962/59%) whereas females outnumbered men (65.3%) among the youngest patients (18-24 years). About 80.5% of the patients had a first stroke. Silent infarcts on magnetic resonance imaging were seen in 20% of patients with a first-ever stroke, and in 11.4% of patients with transient ischemic attack and no history of a previous cerebrovascular event. The most common causes of ischemic stroke were large artery atherosclerosis (18.6%) and dissection (9.9%). CONCLUSIONS: Definite Fabry disease occurs in 0.5% and probable Fabry disease in further 0.4% of young stroke patients. Silent infarcts, white matter intensities, and classical risk factors were highly prevalent, emphasizing the need for new early preventive strategies. Clinical Trial Registration Information- URL: http://www.clinicaltrials.gov.Unique identifier: NCT00414583.

Prevalence of tetrahydrobiopterine (BH4)-responsive alleles among Austrian patients with PAH deficiency: comprehensive results from molecular analysis in 147 patients.

Phenylketonuria (PKU, MIM 261600) is an autosomal recessive disorder caused by mutations of the phenylalanine hydroxylase gene (PAH, GenBank U49897.1, RefSeq NM_000277). To date more than 560 variants of the PAH gene have been identified. In Europe there is regional distribution of specific mutations. Due to recent progress in chaperone therapy, the prevalence of BH4-responsive alleles gained therapeutic importance. Here we report the mutational spectrum of PAH deficiency in 147 unrelated Austrian families. Overall mutation detection rate was 98.6 %. There was a total of 62 disease-causing mutations, including five novel mutations IVS4 + 6T>A, p.H290Y, IVS8-2A>G, p.A322V and p.I421S. The five most prevalent mutations found in patients were p.R408W, IVS12 + 1G>A, p.R261Q, p.R158Q and IVS2 + 5G>C. Neonatal phenylalanine levels before treatment were available in 114/147 patients. Prediction of BH4-responsiveness in patients with full genotypes was exclusively made according to published data. Among the 133 patients needing dietary treatment, 28.4 % are expected to be BH4 "non-responsive", 4.5 % are highly likely BH4-responsive, 35.8 % are probably BH4-responsive while no interpretation was possible for 31.3 %. The mutation data reflect the population history of Austria and provide information on the likely proportion of Austrian PKU patients that may benefit from BH4-therapy.

Fluorous iminoalditols act as effective pharmacological chaperones against gene products from GLB1 alleles causing GM1-gangliosidosis and Morquio B disease.

Unlike replacement therapy by infusion of exogenous recombinant lysosomal enzymes, pharmacological chaperones aim at a gain of function of endogenous gene products. Deficits resulting from missense mutations may become treatable by small, competitive inhibitors binding to the catalytical site and thus correcting the erroneous conformation of mutant enzymes. This may prevent their premature degradation and normalize intracellular trafficking as well as biological half-life. A major limitation currently arises from the huge number of individual missense mutations and the lack of knowledge on the structural requirements for specific interaction with mutant protein domains. Our previous work on mutations of the ß-galactosidase (ß-gal) gene, causing GM1 gangliosidosis (GM1) and Morquio B disease (MBD), respectively, characterized clinical phenotypes as well as biosynthesis, intracellular transport and subcellular localization of mutants. We recently identified an effective chaperone, DL-HexDGJ (Methyl 6-{[N(2)-(dansyl)-N(6)-(1,5-dideoxy-D-galactitol-1,5-diyl)- L-lysyl]amino} hexanoate), among a series of N-modified 1-deoxygalactonojirimycin derivatives carrying a dansyl group in its N-acyl moiety. Using novel and flexible synthetic routes, we now report on the effects of two oligofluoroalkyl-derivatives of 1-deoxygalactonojirimycin, Ph(TFM)(2)OHex-DGJ (N-(α,α-di-trifluoromethyl) benzyloxyhexyl-1,5-dideoxy-1,5-imino-D: -galactitol) and (TFM)(3)OHex-DGJ (N-(Nonafluoro-tert-butyloxy)hexyl-1,5-dideoxy-1,5-imino-D: -galactitol) on the ß-gal activity of GM1 and MBD fibroblasts. Both compounds are competitive inhibitors and increase the residual enzyme activities up to tenfold over base line activity in GM1 fibroblasts with chaperone-sensitive mutations. Western blots showed that this was due to a normalization of protein transport and intralysosomal maturation. The fact that the novel compounds were effective at very low concentrations (0.5-10 µM) in the cell culture medium as well as their novel chemical character suggest future testing in animal models. This may contribute to new aspects for efficient and personalized small molecule treatment of lysosomal storage diseases.

Broad spectrum of Fabry disease manifestation in an extended Spanish family with a new deletion in the GLA gene.

BACKGROUND: Fabry disease (FD) is an X-linked inherited disease based on the absence or reduction of lysosomal-galactosidase (Gla) activity. The enzymatic defect results in progressive impairment of cerebrovascular, renal and cardiac function. Normally, female heterozygote mutation carriers are less strongly affected than male hemizygotes aggravating disease diagnosis. METHOD: Close examination of the patients by renal biopsy, echo- and electrocardiography and MRI. Blood work and subsequent DNA analysis were carried out utilizing approved protocols for PCR and Sequencing. MLPA analysis was done to unveil deletions within the GLA gene locus. Quantitative detection of Glycolipids in patient plasma and urine were carried out using HPLC/MS-MS and ESI-MS. RESULTS: In the presented case, a female index patient led to the examination of three generations of a Spanish family. She presented with severe oto-cochlear symptoms and covert renal and cardiac involvement. While conventional sequencing failed to detect a causative mutation, MLPA analysis revealed a deletion within the GLA gene locus, which we were able to map to a region spanning exon 2 and adjacent intronic parts. The analysis of different biomarkers revealed elevated lyso-Gb3 levels in all affected family members. CONCLUSION: Our findings highlight the broad intrafamilial spectrum of symptoms of FD and emphasise the need to use MLPA screening in symptomatic females without conclusive sequencing result. Finally, plasma lyso-Gb3 proved to be a reliable biomarker for the diagnosis of FD.

A fluorescent probe for GM1 gangliosidosis related ß-galactosidase: N-(dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-1,5-imino-D-galactitol.

N-(Dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-1,5-imino-D-galactitol, a strong competitive inhibitor of ß-galactosidase, enhances residual ß-galactosidase activities in fibroblasts and serves as lead en route to diagnostic compounds for tracking the fate of mutant ß-gal as well as aberrant GM1 gangliosides by live cell imaging.