Effect of long term enzyme replacement therapy in late onset Pompe disease: A single-centre experience.

Late onset Pompe disease (LOPD) is a slowly progressive metabolic myopathy with variable clinical severity. The advent of enzyme replacement therapy (ERT) has modified the natural course of the disease, though the treatment effect on adult patients is modest compared to infants with the classic form. This study aims to describe the long-term clinical outcome of the Greek LOPD cohort, as assessed by 6 min walk test, muscle strength using MRC grading scale and spirometry. ERT efficacy was estimated using statistical methodology that is novel in the context of Pompe disease, which at the same time is well-suited to longitudinal studies with small samples and missing data (local non-linear regression analysis). Improvement over baseline was significant at 1 year for motor performance and muscle strength (p < 0.05), and at 2 years for FVC-U and FVC-S (p < 0.05). A subgroup analysis showed that the onset of the disease before adulthood (18 years), a male gender, and a latency of more than 2 years between the onset of symptoms and ERT administration are unfavorable prognostic factors. Conclusively, this study presents longitudinal data from the Greek LOPD cohort supporting previous observations, that therapeutic delay is related to worse prognosis and treatment effects may decline after several years of ERT.

Gaucher disease: Biochemical and molecular findings in 141 patients diagnosed in Greece.

Gaucher disease (GD) is characterized by a marked phenotypic and genetic diversity. It is caused by the functional deficiency of the lysosomal enzyme ß-glucocerebrosidase (GCase), which in most instances results from mutations in the GBA1 gene and over 500 different disease causing mutations have been described. We present the biochemical and molecular findings in 141 GD cases (14 were siblings) with the three types of the disorder diagnosed in Greece over the last 35 years. 111/141 (78%) GD patients were of Greek origin. The remaining patients were Albanian (24/141; 17%), Syrian (2/141; 1.4%), Egyptian (2/141; 1.4%), Italian (1/141; 0.7%) and Polish (1/141; 0.7%). Mutation analysis identified 28 different mutations and 37 different genotypes. Seven of the mutations were not previously reported (T231I, D283N, N462Y, LI75P, F81L, Y135S and T482K). The most frequent mutations were N370S, D409H;H255Q and L444P. Mutation D409H;H255Q was only identified in Greek and Albanian patients. Sixteen mutations, including the novel ones, were identified only in one allele. Although the N370S mutation was identified only in type 1 patients, not all of type 1 patients carried this mutation. Our results highlight the heterogeneity of Gaucher disease and support the Balkan origin of the double mutant allele D409H;H255Q.

Type I sialidosis, a normosomatic lysosomal disease, in the differential diagnosis of late-onset ataxia and myoclonus: An overview.

Lysosomal storage diseases (LSDs) are rare to extremely rare monogenic disorders. Their incidence, however, has probably been underestimated owing to their complex clinical manifestations. Sialidosis is a prototypical LSD inherited as an autosomal recessive trait and caused by mutations in the NEU1 gene that result in a deficiency of alpha-N-acetyl neuraminidase 1 (NEU1). Two basic forms of this disease, type I and type II, are known. The dysmorphic type II form features LSD symptoms including congenital hydrops, dysmorphogenetic traits, hepato-splenomegaly and severe intellectual disability. The diagnosis is more challenging in the normosomatic type I forms, whose clinical findings at onset include ocular defects, ataxia and generalized myoclonus. Here we report the clinical, biochemical and molecular analysis of five patients with sialidosis type I. Two patients presented novel NEU1 mutations. One of these patients was compound heterozygous for two novel NEU1 missense mutations: c.530A>T (p.Asp177Val) and c.1010A>G (p.His337Arg), whereas a second patient was compound heterozygous for a known mutation and a novel c.839G>A (p.Arg280Gln) mutation. We discuss the impact of these new mutations on the structural properties of NEU1. We also review available clinical reports of patients with sialidosis type I, with the aim of identifying the most frequent initial clinical manifestations and achieving more focused diagnoses.

Segmental and total uniparental isodisomy (UPiD) as a disease mechanism in autosomal recessive lysosomal disorders: evidence from SNP arrays.

Analyses in our diagnostic DNA laboratory include genes involved in autosomal recessive (AR) lysosomal storage disorders such as glycogenosis type II (Pompe disease) and mucopolysaccharidosis type I (MPSI, Hurler disease). We encountered 4 cases with apparent homozygosity for a disease-causing sequence variant that could be traced to one parent only. In addition, in a young child with cardiomyopathy, in the absence of other symptoms, a diagnosis of Pompe disease was considered. Remarkably, he presented with different enzymatic and genotypic features between leukocytes and skin fibroblasts. All cases were examined with microsatellite markers and SNP genotyping arrays. We identified one case of total uniparental disomy (UPD) of chromosome 17 leading to Pompe disease and three cases of segmental uniparental isodisomy (UPiD) causing Hurler-(4p) or Pompe disease (17q). One Pompe patient with unusual combinations of features was shown to have a mosaic segmental UPiD of chromosome 17q. The chromosome 17 UPD cases amount to 11% of our diagnostic cohort of homozygous Pompe patients (plus one case of pseudoheterozygosity) where segregation analysis was possible. We conclude that inclusion of parental DNA is mandatory for reliable DNA diagnostics. Mild or unusual phenotypes of AR diseases should alert physicians to the possibility of mosaic segmental UPiD. SNP genotyping arrays are used in diagnostic workup of patients with developmental delay. Our results show that even small Regions of Homozygosity that include telomeric areas are worth reporting, regardless of the imprinting status of the chromosome, as they might indicate segmental UPiD.

Correction: Stereodivergent synthesis of right- and left-handed iminoxylitol heterodimers and monomers. Study of their impact on ß-glucocerebrosidase activity.

Correction for 'Stereodivergent synthesis of right- and left-handed iminoxylitol heterodimers and monomers. Study of their impact on ß-glucocerebrosidase activity' by Fabien Stauffert et al., Org. Biomol. Chem., 2017, 15, 3681-3705.

Stereodivergent synthesis of right- and left-handed iminoxylitol heterodimers and monomers. Study of their impact on ß-glucocerebrosidase activity.

A library of dimers and heterodimers of both enantiomers of 2-O-alkylated iminoxylitol derivatives has been synthesised and evaluated on ß-glucocerebrosidase (GCase), the enzyme responsible for Gaucher disease (GD). Although the objective was to target simultaneously the active site and a secondary binding site of the glucosidase, the (-)-2-iminoxylitol moiety seemed detrimental for imiglucerase inhibition and no significant enhancement was obtained in G202R, N370S and L444P fibroblasts. However, all compounds having at least one (+)-2-O-alkyl iminoxylitol are GCase inhibitors in the nano molar range and are significant GCase activity enhancers in G202R fibroblats, as confirmed by a decrease of glucosylceramide levels and by co-localization studies.

The Spectrum of Niemann-Pick Type C Disease in Greece.

Niemann-Pick type C disease (NPC) is a neurovisceral lysosomal storage disease caused by mutations in either the NPC1 or the NPC2 gene. It is a cellular lipid trafficking disorder characterized by the accumulation of unesterified cholesterol and various sphingolipids in the lysosomes and late endosomes, and it exhibits a broad clinical spectrum. Today, over 420 disease-causing mutations have been identified in the NPC1 and the NPC2 genes. We present the clinical, biochemical, and molecular findings in 14 cases diagnosed in Greece during the last 28 years. Age at diagnosis ranged from 2.5 months to 48 years. Systemic manifestations were present in 7/14 patients. All developed neurological manifestations (age of onset 5 months to 42 years). Six patients are still alive (age: 5-50 years). Classical filipin staining pattern was observed in all but four patients (3 NPC1, 1 NPC2). The rate of LDL-induced cholesteryl ester formation was severely reduced in 4/7 and significantly reduced in 3/7 patients studied. Increased chitotriosidase activity was observed in 9/12 patients. Mutation analysis in 11 unrelated patients identified 12 different mutations in the NPC1 gene: eight previously described p.E1089K (c.3265G>A), p.F284Lfs*26 (c.852delT), p.A1132P(c.3394G>C), del promoter region and exons 1-10, p.R1186H (c.3557G>A), p.P1007A (c.3019C>G), p.Q92R(c.275A>G),p.S940L (c.2819C>T), and four novel ones: (p.N701K fs*13 (c.2102-2103insA), p.K1057R (c.3170A>G), IVS23+3insT(c.3591+3insT), p.C1119*(c.3357T>C); and the previously described IVS2+5G>A(c.190+5G>A) mutation in the NPC2 gene. All patients were of Greek origin. Assuming a birth rate of 100,000/year, a rough incidence estimate for NPC disease in Greece would be 0.5/100,000 births.

Investigation of original multivalent iminosugars as pharmacological chaperones for the treatment of Gaucher disease.

Multivalent iminosugars conjugated with a morpholine moiety and/or designed as prodrugs have been prepared and evaluated as new classes of pharmacological chaperones for the treatment of Gaucher disease. This study further confirms the interest of the prodrug concept and shows that the addition of a lysosome-targeting morpholine unit into iminosugar cluster structures has no significant impact on the chaperone activity on Gaucher cells.

The Spectrum of Krabbe Disease in Greece: Biochemical and Molecular Findings.

Krabbe disease is an autosomal recessive neurodegenerative lysosomal storage disease caused by the deficiency of ß-galactocerebrosidase. This deficiency results in the impaired degradation of ß-galactocerebroside, a major myelin lipid, and of galactosylsphingosine. Based on the age of onset of neurological symptoms, an infantile form (90% patients) and late-onset forms (10% patients) of the disease are recognized. Over 130 disease-causing mutations have been identified in the ß-galactocerebrosidase gene. We present the biochemical and molecular findings in 19 cases of Krabbe disease, 17 of them unrelated, diagnosed in Greece over the last 30 years. ß-Galactocerebrosidase activity assayed in leukocyte homogenates using either the tritium-labeled or the fluorescent substrate was diagnostic for all. Increased plasma chitotriosidase activity was found in 11/15 patients.Mutational analysis, carried out in 11 unrelated cases, identified seven different mutations, four previously described (p.I250T, c.1161+6532_polyA+9kbdel, p.K139del, p.D187V) and three novel mutations (p.D610A, c.583-1 G>C, p.W132X), and seven distinct genotypes. The most prevalent mutation was mutation p.I250T, first described in a patient of Greek origin. It accounted for 36.4% (8/22) of the mutant alleles. The second most frequent mutation was c.1161+6532_polyA+9kbdel that accounted for 22.7% (5/22) of the mutant alleles. The observed frequency was lower than that described in Northern European countries and closer to that described in Italian patients.

Selective chaperone effect of aminocyclitol derivatives on G202R and other mutant glucocerebrosidases causing Gaucher disease.

Gaucher disease is an autosomal recessive lysosomal disorder characterized by the accumulation of glucosylceramide as a result of a deficiency of the enzyme glucocerebrosidase. Several competitive glucocerebrosidase inhibitors are able to act as pharmacological chaperones for an efficient rescue of the mutated, misfolded forms of the enzyme. Along this line, we report in this work on the ability of several aminocyclitols to increase the residual glucocerebrosidase activity in patient fibroblasts with different genotypes. Some of the compounds were slightly active on fibroblasts bearing some mutations, including the highly prevalent N370S mutation. All compounds were highly active as enzyme activity enhancers on fibroblasts from Gaucher disease patients containing the G202R mutation. Moreover, using the novel tagged sphingolipid ω-azidosphingosine, a reduction in the tagged glucosylceramide accumulation was also observed for selected aminocyclitols. Attempts to explain the activity impairment observed in glucocerebrosidase bearing the G202R mutation by comparative molecular dynamic studies on wild type and the G202R mutated proteins (free and isofagomine-bound, in both cases) were unsuccessful. Under the simulation conditions used, no clear effect of the G202R mutation neither over the global structure of the protein nor on the loops that constitute the glucocerebrosidase active site was observed. Since the G202R residue is located on the protein surface, altered protein-membrane or protein-protein interactions could account for the observed differences. In conclusion, we have tested novel compounds that have shown some chaperone effect on particular glucocerebrosidase mutant enzymes, supporting the enhancement therapy as an alternative approach for Gaucher disease.

Glucocerebrosidase enhancers for selected Gaucher disease genotypes by modification of α-1-C-substituted imino-D-xylitols (DIXs) by click chemistry.

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9-DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α-1-C-propargyl-1,5-dideoxy-1,5-imino-D-xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as ß-glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX-28 (α-1-C-[(1-(3-trimethylsilyl)propyl)-1H-1,2,3-triazol-4-yl)methyl]-1,5-dideoxy-1,5-imino-D-xylitol), bearing the 3-trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM. Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non-competitive inhibitors, with the exception of the mixed-type inhibitor DIX-28.

Galactosialidosis: review and analysis of CTSA gene mutations.

BACKGROUND: Mutations in the CTSA gene, that encodes the protective protein/cathepsin A or PPCA, lead to the secondary deficiency of ß-galactosidase (GLB1) and neuraminidase 1 (NEU1), causing the lysosomal storage disorder galactosialidosis (GS). Few clinical cases of GS have been reported in the literature, the majority of them belonging to the juvenile/adult group of patients. METHODS: The correct nomenclature of mutations for this gene is discussed through the analysis of the three PPCA/CTSA isoforms available in the GenBank database. Phenotype-genotype correlation has been assessed by computational analysis and review of previously reported single amino acid substitutions. RESULTS: We report the clinical and mutational analyses of four cases with the rare infantile form of GS. We identified three novel nucleotide changes, two of them resulting in the missense mutations, c.347A>G (p.His116Arg), c.775T>C (p.Cys259Arg), and the third, c.1216C>T, resulting in the p.Gln406* stop codon, a type of mutation identified for the first time in GS. An Italian founder effect of the c.114delG mutation can be suggested according to the origin of the only three patients carrying this mutation reported here and in the literature. CONCLUSIONS: In early reports mutations nomenclature was selected according to all CTSA isoforms (three different isoforms), thus generating a lot of confusion. In order to assist physicians in the interpretation of detected mutations, we mark the correct nomenclature for CTSA mutations. The complexity of pathology caused by the multifunctions of CTSA, and the very low numbers of mutations (only 23 overall) in relation to the length of the CTSA gene are discussed.In addition, the in silico functional predictions of all reported missense mutations allowed us to closely predict the early infantile, late infantile and juvenile phenotypes, also disclosing different degrees of severity in the juvenile phenotype.

Danon disease: further clinical and molecular heterogeneity.

Two families of Greek patients with subclinical to severe cardiomyopathy are presented. The diagnosis of Danon disease was supported by a total lack of LAMP2 immunostaining in cultured skin fibroblasts and muscle biopsies. The LAMP2 mutation carried by one patient (c.928G>A) has already been reported but with different symptoms. The second patient had a novel point deletion. This has not been described previously, but it could be detected easily by restriction analysis. This mutation was also found in the patient's brother, and it was associated with severe cardiomyopathy leading to heart failure. Surprisingly, the proband also had partial reduction of alpha-galactosidase A activity, despite the absence of characteristic clinical features of Fabry disease. A substitution in the GLA gene (c.937G>T) was found, and its involvement in the cardiac disease is discussed.

Plasma lysosomal enzyme activities in congenital disorders of glycosylation, galactosemia and fructosemia.

BACKGROUND: Variable increases in the plasma activity of different lysosomal enzymes have been reported in patients with congenital disorders of glycosylation (CDG). In particular, elevated plasma aspartylglucosaminidase activity (AGA) has been found in the majority of CDG type I patients. We report on the plasma activity of AGA and other lysosomal enzymes in patients with different types of primary and secondary CDG defects. METHODS: AGA, alpha-mannosidase, beta-mannosidase and beta-hexosaminidase activities were assayed in the plasma of patients with CDGI (4CDGIa, 4CDGIx) and CDGIIx (5, all with a combined N- and O-glycosylation defect), classical galactosemia (GALT) (n=3) and hereditary fructose intolerance (HFI) (n=2). RESULTS: Increased AGA and beta-hexosaminidase activities were found in all and 7/8 of the GDGI patients respectively. All enzymic activities were normal in the CDGIIx patients. Elevated AGA and beta-hexosaminidase activity was also seen in GALT and HFI patients before treatment, when transferrin isoelectric focusing (TfIEF) patterns were also abnormal. CONCLUSIONS: Increased AGA plasma activity, although a consistent finding in CDGI patients, is not specific to this group of disorders since it is also observed in untreated cases of GALT and HFI. Furthermore, plasma AGA activity cannot serve as a marker for CDGII disorders. In conjunction with TfIEF it could be used in the follow up of GALT and HFI patients.

Serial magnetic resonance imaging and neurophysiological studies in multiple sulphatase deficiency.

We present serial clinical, magnetic resonance imaging (MRI) and neurophysiological findings of a patient with multiple sulphatase deficiency (MSD), who was first admitted at the age of 9 months, because of psychomotor retardation. MRI demonstrated extensive diffuse symmetrical high signal in the deep white matter of both cerebral hemispheres, as well as of the subcortical white matter and the brainstem, while there was additional enlargement of sulci and subdural spaces and mild atrophy. Assay of arylsulphatase A activity in white blood cell homogenates at the age of 29 months disclosed a marked deficiency of the enzyme, compatible with the diagnosis of early-infantile metachromatic leukodystrophy. During the course of a later admission, the presence of ichthyosis pointed out to the possible diagnosis of MSD; further assays of sulphatases in plasma, leukocytes as well as in cultured fibroblasts, combined with an abnormal excretion of mucopolysaccharides and sulphatides in urine confirmed the diagnosis. Molecular analysis identified a homozygous disease-causing mutation (R349W) of the SUMF1 gene. Serial neurophysiological and MRI studies demonstrated the progressive nature of the disorder (regarding both central and peripheral nervous system), correlating with the clinical deterioration (spastic quadriplegia, optic atrophy and epilepsy) with subsequent death at the age of 4 years.