Martin Hřebíček

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.

Sanfilippo syndrome type C: mutation spectrum in the heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT) gene†

Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl‐CoA (AcCoA): α‐glucosaminide N‐acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by α‐N‐acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death, causing neurodegeneration and severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice‐site mutations, 11 insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php?select_db=HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS IIIC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT. Hum Mutat 30, 918–925, 2009.

Natural history of the respiratory involvement in Anderson–Fabry disease

SummaryBackground:Anderson–Fabry disease (AFD) is an X-linked disorder caused by deficient activity of enzyme α-galactosidase A, resulting in the accumulation of glycosphingolipids within lysosomes. Pulmonary involvement in AFD has previously been documented, but until now has been studied only in a few series of patients without any longitudinal follow-up. The aim of this study was to compare spirometric changes in AFD patients with a matched control population and to follow the subsequent progression of the disease.Materials and methods:Fifty individuals (27 women, 23 men, mean age 40±14 years) with AFD from 14 families underwent a static spirometric examination under standard conditions. A set of indices was compared with that of the control population. Out of this cohort, 39 individuals not receiving enzyme replacement therapy were longitudinally evaluated (median follow-up time 24 months).Results:A clinically significant reduction in spirometric parameters, corresponding to mild to severe airway obstruction, was observed in 26% of women and 61% of men. During the serial follow-up, a significant (p<0.05) age-dependent reduction of predicted %FVC and %FEV1 values was observed in male patients, while the influence of age was not seen in female patients. The %FEF25–75 values decreased by similar degrees in men and women and in older and younger patients, indicating that progressive bronchial disease affects the small airways first.Conclusions:We have demonstrated a clinically relevant age- and sex-dependent progressive pulmonary involvement in AFD patients. The effects of enzyme replacement therapy on pulmonary involvement remain to be demonstrated.

Rotor-type hyperbilirubinaemia has no defect in the canalicular bilirubin export pump.

Background: The cause of Rotor syndrome (RS), a rare‐familial conjugated hyperbilirubinaemia with normal liver histology, is unclear. We hypothesized that RS can be an allelic variant of Dubin–Johnson syndrome, caused by mutation in ABCC2, and investigated ABCC2 (gene) and ABCC2 (protein) in two patients with RS.

Characterization of gana-1 , a Caenorhabditis elegans gene encoding a single ortholog of vertebrate α-galactosidase and α-N-acetylgalactosaminidase

BackgroundHuman α-galactosidase A (α-GAL) and α-N-acetylgalactosaminidase (α-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD) – Fabry (α-GAL deficiency) and Schindler (α-NAGA deficiency) diseases. Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins α-GAL and α-NAGA.ResultsBlastP searches for orthologs of human α-GAL and α-NAGA revealed a single C. elegans gene (R07B7.11) with homology to both human genes (α-ga lactosidase and α-N-a cetylgalactosaminidase) – gana-1. We cloned and sequenced the complete gana-1 cDNA and elucidated the gene organization.Phylogenetic analyses and homology modeling of GANA-1 based on the 3D structure of chicken α-NAGA, rice α-GAL and human α-GAL suggest a close evolutionary relationship of GANA-1 to both human α-GAL and α-NAGA.Both α-GAL and α-NAGA enzymatic activities were detected in C. elegans mixed culture homogenates. However, α-GAL activity on an artificial substrate was completely inhibited by the α-NAGA inhibitor, N-acetyl-D-galactosamine.A GANA-1:: GFP fusion protein expressed from a transgene, containing the complete gana-1 coding region and 3 kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. The GFP signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with Concanamycin A (CON A) or NH4Cl, agents that increase the pH of the cellular acidic compartment.Immunofluorescence detection of the fusion protein using polyclonal anti-GFP antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes.Inhibition of gana-1 by RNA interference resulted in a decrease of both α-GAL and α-NAGA activities measured in mixed stage culture homogenates but did not cause any obvious phenotype.ConclusionsGANA-1 is a single C. elegans ortholog of both human α-GAL and α-NAGA proteins. Phylogenetic, homology modeling, biochemical and GFP expression analyses support the hypothesis that GANA-1 has dual enzymatic activity and is localized in an acidic cellular compartment.

ABCB4 mutations underlie hormonal cholestasis but not pediatric idiopathic gallstones

AIM To investigate the contribution of ABCB4 mutations to pediatric idiopathic gallstone disease and the potential of hormonal contraceptives to prompt clinical manifestations of multidrug resistance protein 3 deficiency. METHODS Mutational analysis of ABCB4, screening for copy number variations by multiplex ligation-dependent probe amplification, genotyping for low expression allele c.1331T>C of ABCB11 and genotyping for variation c.55G>C in ABCG8 previously associated with cholesterol gallstones in adults was performed in 35 pediatric subjects with idiopathic gallstones who fulfilled the clinical criteria for low phospholipid-associated cholelithiasis syndrome (LPAC, OMIM #600803) and in 5 young females with suspected LPAC and their families (5 probands, 15 additional family members). The probands came to medical attention for contraceptive-associated intrahepatic cholestasis. RESULTS A possibly pathogenic variant of ABCB4 was found only in one of the 35 pediatric subjects with idiopathic cholesterol gallstones whereas 15 members of the studied 5 LPAC kindreds were confirmed and another one was highly suspected to carry predictably pathogenic mutations in ABCB4. Among these 16, however, none developed gallstones in childhood. In 5 index patients, all young females carrying at least one pathogenic mutation in one allele of ABCB4, manifestation of LPAC as intrahepatic cholestasis with elevated serum activity of gamma-glutamyltransferase was induced by hormonal contraceptives. Variants ABCB11 c.1331T>C and ABCG8 c.55G>C were not significantly overrepresented in the 35 examined patients with suspect LPAC. CONCLUSION Clinical criteria for LPAC syndrome caused by mutations in ABCB4 cannot be applied to pediatric patients with idiopathic gallstones. Sexual immaturity even prevents manifestation of LPAC.

Biochemistry of Fabry Disease

Fabry disease is a sphingolipid storage disorder resulting from a deficiency of the lysosomal hydrolase, α-galactosidase A. The deficiency leads to lysosomal accumulation of α-galactosidase A substrates, neutral glycosphingolipids with terminal α-galactosyl moieties, in multiple tissues. Globotriaosylceramide (Gb3Cer, CD77), the critical substrate of α-galactosidase A, is involved in cell signaling and associates itself with lipid rafts in the plasma membrane, where it also functions as a receptor for the Shiga-like toxins of E. Coli. Possible roles of Gb3Cer accumulation in the pathogenesis of Fabry disease, as well as biochemistry and function of minor α-galactosidase A substrates are discussed. There are two human lysosomal enzymes with the ability to hydrolyze substrates with terminal α-galactose, α-galactosidase A and α-N-acetylgalactosaminidase (NAGA). Both enzymes are active in vitro against saccharide, glycolipid and artificial substrates. α-N-acetylgalactosaminidase’s primary function appears to be hydrolysis of acetylated oligosaccharides and glycopeptides, while α-galactosidase A is the enzyme responsible for degradation of glycolipid substrates. α-Galactosidase A requires an activator protein, saposin B, for its activity in vivo. Properties of the above proteins and the biochemistry of their deficiencies in humans are also addressed.