Linda Berná

Prosaposin Deficiency and Saposin B Deficiency (Activator-Deficient Metachromatic Leukodystrophy): Report on Two Patients Detected by Analysis of Urinary Sphingolipids and Carrying Novel PSAP Gene Mutations

Prosaposin deficiency (pSap‐d) and saposin B deficiency (SapB‐d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65–70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A–D. We report on two new patients with PSAP gene defects; one, with pSap‐d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB‐d, who presented with a metachromatic leukodystrophy‐like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap‐d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap‐d patient was homozygous for a splice‐acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB‐d patient was a compound heterozygote with a splice‐acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap‐d mutation, on the other allele. Phenotypically, pSap‐d is a relatively uniform disease of the neonate, whereas SapB‐d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap‐d and the single saposin deficiencies is speculated.

Replacement of α-galactosidase A in Fabry disease: effect on fibroblast cultures compared with biopsied tissues of treated patients

The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant α-galactosidases A (agalsidases, FabrazymeTM, and ReplagalTM) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of (3H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with FabrazymeTM, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage.

Missense mutations as a cause of metachromatic leukodystrophy. Degradation of arylsulfatase A in the endoplasmic reticulum.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Biosynthesis studies of ASA with various structure‐sensitive monoclonal antibodies reveal that some epitopes of the enzyme form within the first minutes of biosynthesis whereas other epitopes form later, between 10 and 25 min. When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclonal antibodies revealed folding deficits in all 12 mutant ASA enzymes. Eleven of the 12 mutants show partial expression of the early epitopes, but only six of these show, in addition, incomplete expression of late epitopes. In none of the mutant enzymes were the late forming epitopes found in the absence of early epitopes. Thus, data from the wild‐type and mutant enzymes indicate that the enzyme folds in a sequential manner and that the folding of early forming epitopes is a prerequisite for maturation of the late epitopes. All mutant enzymes in which the amino acid substitution prevents the expression of the late forming epitopes are retained in the endoplasmic reticulum (ER). In contrast, all mutants in which a single late epitope is at least partially expressed can leave the ER. Thus, irrespective of the missense mutation, the expression of epitopes forming late in biosynthesis correlates with the ability of the enzyme to leave the ER. The degradation of ER‐retained enzymes can be reduced by inhibitors of the proteasome and ER α1,2‐mannosidase I, indicating that all enzymes are degraded via the proteasome. Inhibition of degradation did not lead to an enhanced delivery from the ER for any of the mutant enzymes.

Abnormal expression and processing of uromodulin in Fabry disease reflects tubular cell storage alteration and is reversible by enzyme replacement therapy

SummaryUromodulin (UMOD) malfunction has been found in a range of autosomal dominant tubulointerstitial nephropathies associated with hyperuricaemia, gouty arthritis, medullary cysts and renal failure—labelled as familial juvenile hyperuricaemic nephropathy, medullary cystic disease type 2 and glomerulocystic kidney disease. To gain knowledge of the spectrum of UMOD changes in various genetic diseases with renal involvement we examined urinary UMOD excretion and found significant quantitative and qualitative changes in 15 male patients at various clinical stages of Fabry disease. In untreated patients, the changes ranged from normal to a marked decrease, or even absence of urinary UMOD. This was accompanied frequently by the presence of aberrantly processed UMOD lacking the C-terminal part following the K432 residue. The abnormal patterns normalized in all patients on enzyme replacement therapy and in some patients on substrate reduction therapy. Immunohistochemical analysis of the affected kidney revealed abnormal UMOD localization in the thick ascending limb of Henle’s loop and the distal convoluted tubule, with UMOD expression inversely proportional to the degree of storage. Our observations warrant evaluation of tubular functions in Fabry disease and suggest UMOD as a potential biochemical marker of therapeutic response of the kidney to therapy. Extended comparative studies of UMOD expression in kidney specimens obtained during individual types of therapies are therefore of great interest.

Mucopolysaccharidosis Type I in 21 Czech and Slovak Patients: Mutation Analysis Suggests a Functional Importance of C-Terminus of the IDUA Protein

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme α‐L‐iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918_1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C‐terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C‐terminus of the IDUA protein.

Novel mutations associated with metachromatic leukodystrophy: phenotype and expression studies in nine Czech and Slovak patients.

Metachromatic leukodystrophy (MLD) is an inherited demyelinating disorder caused by the deficiency of arylsulphatase A (ASA). This defect leads to an accumulation of galactosylceramide I3‐sulphates (sulphatides) in lysosomes of different tissues. We report on mutations found in a group of nine patients from the Czech and Slovak Republics (former Czechoslovakia). Their diagnosis was confirmed by determination of the activity of arylsulphatase A in leukocytes and by abnormal urinary excretion of sulphatides. All alleles of the patients were identified and eight different mutations were found. They include four novel missense mutations in one infantile (D29N), one juvenile (C294Y), and three adult (C156R, G293S) patients. Four mutations were previously described sequence alterations (459 + 1G > A, G309S, I179S, and P426L). Polymorphisms characteristic for the ASA pseudodeficiency allele were not found in the patients. Substitutions of D29N, C294Y, and G293S in arylsulphatase A caused a severe reduction of enzyme activity in transient expression studies. In contrast, the C156R substitution reduces arylsulphatase A only to 50% of wild type ASA activity. Since no other mutations were found in this patient, the contribution of this mutation to the development of disease remains unclear.