Human Molecular Genetics, 2001, Vol. 10, No. 9 927-940
© 2001 Oxford University Press
A novel mutation in the coding region of the prosaposin gene leads to a complete deficiency of prosaposin and saposins, and is associated with a complex sphingolipidosis dominated by lactosylceramide accumulation
1Institute of Inherited Metabolic Disorders, Charles University, First Faculty of Medicine, Ke Karlovu 2, 128 08 Prague 2, Czech Republic, 2Institute of Pathology, Charles University, First Faculty of Medicine, Studnickova 2, 128 00, Prague 2, Czech Republic, 3Department of Chemical Pathology, Women’s and Children’s Hospital, North Adelaide, SA 5006, Australia, 4Institute for Brain Research, University of Tübingen, D-72070 Tübingen, Germany, 5Institute of Pathology, P.J. afarík University, Faculty of Medicine, Koice, Slovakia and 61st Medical Department—Clinical Department of Haematology and Nephrology, Charles University, First Faculty of Medicine, U nemocnice 2, 128 08 Prague 2, Czech Republic
Received 3 January 2001; Revised and Accepted 21 February 2001.
DDBJ/EMBL/GenBank accession no. AF307850.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A fatal infantile storage disorder with hepatosplenomegaly and severe neurological disease is described. Sphingolipids, including monohexosylceramides (mainly glucosylceramide), dihexosylceramides (mainly lactosylceramide), globotriaosyl ceramide, sulphatides, ceramides and globotetraosyl ceramide, were stored in the tissues. In general, cholesterol and sphingomyelin levels were unaltered. The storage process was generalized and affected a number of cell types, with histiocytes, which infiltrated a number of visceral organs and the brain, especially involved. The ultrastructure of the storage lysosomes was membranous with oligolamellar, mainly vesicular, profiles. Infrequently, there were Gaucher-like lysosomes in histiocytes. The neuropathology was severe and featured neuronal storage and loss with a massive depopulation of cortical neurons and pronounced fibrillary astrocytosis. There was a paucity of myelin and stainable axons in the white matter with signs of active demyelination. Immunohistochemical investigations indicated that saposins A, B, C and D were all deficient. The patient was homozygous for a 1 bp deletion (c.803delG) within the SAP-B domain of the prosaposin gene which leads to a frameshift and premature stop codon. In the heterozygous parents, mutant cDNA was detected by amplification refractory mutation analysis in the nuclear, but not the cytoplasmic, fraction of fibroblast RNA, indicating that the mutant mRNA was rapidly degraded. The storage process in the proband resembled that of a published case from an unrelated family. Saposins were also deficient in this case, leading to its reclassification as prosaposin deficiency, and her mother was found to be a carrier for the same c.803delG mutation. Both of the investigated families came from the same district of eastern Slovakia.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Prosaposin (PSAP) is a polyfunctional highly conserved glycoprotein. The human PSAP gene is located on chromosome 10 (10q22.1) (1) and probably spans >39 kb (2,3). Published sequences cover the promotor region plus exons 1 (3) and 2–14 (2). The PSAP mRNA encodes a 70 kDa polypeptide containing a signal peptide in addition to four homologous sphingolipid activator protein (SAP or saposin A, B, C and D) domains (2–5). In humans, an alternative splicing of exon 8 (within the SAP B domain) gives rise to three protein isoforms (524, 526 or 527 amino acids), with intracellular transport of the PSAP being isoform-dependent (6), but with little impact of the isoform on substrate turnover (7), despite effects on the binding affinity for sphingolipids of SAP B (8). Though ubiquitous, the level of PSAP expression in mammals is variable (4,9–14) and in rodents its expression was shown to be under developmental control (9–11).
The targeting and processing of PSAP varies in a tissue/cell-specific manner. Firstly, newly synthesized PSAP can enter the acidic endocytotic compartment via a mannose-6-phosphate-independent (15), but PSAP sequence-dependent (16) process in association with sphingolipids (17) and procathepsin D (18,19). Once in the lysosome, the dissociated mature protease, cathepsin D, participates in the maturation of PSAP (20). Alternatively, PSAP is secreted into a variety of extracellular fluids (10,21,22) and subsequently targeted to the lysosomal compartment via receptor-mediated endocytosis (23).
The precursor PSAP has many functions. Firstly, its binding affinity for gangliosides suggests that it has a role in glycosphingolipid transport (24). In extracellular fluids it may also act as a PSAP reservoir for surrounding tissues. Like the saposins it can facilitate enzymatic hydrolysis of certain sphingolipids (24) and it can also promote glycosphingolipid synthesis (25,26). A significant portion of PSAP is associated with gangliosides in the plasma membranes of neurons (27,28) and it has been shown to have neurotrophic, neuroprotective and reparative effects (29–34) as well as being myelinotrophic (35). At least in rodents, PSAP is thought to have specific effects on the development, maintenance and differentiation of male reproductive organs and may also play a role in lysosomal residual body degradation in Sertoli cells (36).
The saposins are important cofactors for the lysosomal degradation of sphingolipids and function by either activating the enzyme or solubilizing the substrate (37–41). The presence of mutations in both the SAP C (42,43) and SAP B (44–49) domains of PSAP has confirmed the critical role played by these saposins in sphingolipid hydrolysis.
To date, a complete deficiency of human PSAP has been described only in a single family (50,51), with the affected individuals being homozygous for a point mutation in the initiation codon of the PSAP gene (52). A mouse knock-out model for PSAP deficiency (PSAPD) (53,54) showed very similar biochemical features to the human disorder. We now present clinical, neuropathological, ultrastructural, histochemical, biochemical and molecular findings on a new case of complete PSAPD caused by a novel mutation within the SAP B domain of the PSAP gene. In addition, we also present evidence for reclassification of the case previously described as a lactosylceramide (LacCer)-storing variant of Niemann–Pick type C (NPC) (55) as PSAPD.
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RESULTS |
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Structural and histochemical findings
In patient PD1 (Materials and Methods) the storage process was predominantly expressed in the numerous macrophages infiltrating the spleen red pulp, liver sinusoids, adrenal cortex (especially the reticular zone) and pulmonary alveoles. Their cytology varied from a foamy to almost solid appearance with variantly expressed cytoplasmic Gaucher-like striations. The spleen was also rich in multinucleated storage histiocytes. Storage was histologically detectable in hepatocytes, renal tubular cells, glomerular podocytes, adrenal cortex (more towards the medulla), spleen sinus endothelium, brain neurons and in skin eccrine glands. Electron microscopy revealed variable storage in the vascular endothelium (most pronounced in the brain and just detectable in the heart capillaries), in fibrocytes, adipocytes, dermal Schwann cells and pancreas acinary exocrine cells, but more in the interlobular pancreatic ductules and in endocrine islet cells. The skin nerves contained well-myelinated fibres. The epidermis of the trunk was histologically practically normal with several loose keratin layers. The only unaffected cell type was cardiocytes.
The ultrastructure of the deposits was pleiomorphic and membranous (Fig. 1). Gaucher-like tubules were seen in macrophages intermingled with non-specific storage lysosomes (Fig. 1A). Frequently there were oligolamellar anular formations 
200 nm in diameter, which were often clustered (Fig. 1B). In some locations larger angulated deposits (especially in the glial cells) (Fig. 1C) or discrete spicules (in splenic macrophages) were seen, suggesting the presence of a crystalized lipid. The lysosomes varied greatly in size from <1 to 3 µm. The cerebral cortex (Fig. 2A) contained numerous reactive fibrillary astrocytes (strongly GFAP positive) (Fig. 2B) and lipid phagocytes (strongly stained with CD 68) (Fig. 2C) but there was a striking paucity of neurons (Fig 2A, insert). The neurons and lipid phagocytes displayed strong storage, whereas storage in the astrocytes was only detectable by electron microscopy. The white matter was astrogliosed and infiltrated with storing lipid phagocytes and, when compared with age-matched controls, axons (detected with Bodian) and myelin (absence of myelin birefringence and negative for Spielmayer staining) were both depleted, with the myelin, which was only rarely seen around persisting axons, showing a greatly reduced number of layers. The sample of basal ganglia displayed similar changes (massive fibrillary gliosis, paucity of myelin, excess of lipid phagocytes) but with a less pronounced loss of neurons (ballooned with storage).
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Lipid histochemistry showed an excess of neutral glycolipids. The staining for phospholipids was just detectable. Birefringence of the stored lipids ranged from minimal (macrophages, hepatocytes and neurons) to readily discernible (adrenal cortex and renal tubules). There was prominent accumulation of sulphatides in the renal tubules, mimicking the situation in sulphatidosis. Brain glial phagocytes were slightly stained with Oil red O but rich in cholesteryl ester solid crystals.
SAP immunohistochemistry gave completely negative results for all examined tissues, i.e. lung, kidney, spleen, liver and brain (Fig. 3, liver and brain), whereas controls from normal individuals and patients with unrelated lysosomal diseases were all immunoreactive. There was some non-specific staining of hypertrophic astroglia, a well known drawback of the combined antibody against SAPs A plus D (J. Tyynella, personal communication). Immunolabeling of Cathepsin D showed elevated staining of storage cells, consistent with its lysosomal localization (data not shown) and indicating that the deficiency of PSAP did not prevent lysosomal targeting of procathepsin D.
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Biochemical results
Cholesterol levels were almost normal in all extraneural tissues of PD1, thereby excluding the suspicion of NPC disease (Table 1) with the increase seen in brain derived from cholesterol esters, mentioned in the Discussion. Apart from the adrenal tissue, where it was increased three-fold, sphingomyelin was not greatly changed (Table 1), again differing from NPC patients. Instead, there was a massive general increase in shorter glycosphingolipids in all tissues analyzed, especially glucocerebroside (GlcCer) (Fig. 4A and B, kidney, brain and adrenals) and LacCer (Fig. 4B, adrenals; Fig. 5A, whole tissue series). In the kidney, large accumulation of galactocerebroside (GalCer) (Fig. 4A) and sulphatides (Fig. 4C) was found. Immunodetection confirmed that LacCer was the main component of the ceramide dihexoside fraction (Fig. 5A), with its concentration being highest in the adrenal gland and spleen. The ceramide trihexoside fraction, which contained globotriaosyl ceramide (Gb3Cer) (Fig. 5B), was also increased, with the highest amounts found in the kidney and adrenals (Figs 4B and 5B). The total concentration of simple glycolipids was significantly higher than sphingomyelin in PSAPD tissues, again contrasting with NPC samples (Table 1), and ceramides were increased several-fold in kidney, spleen and liver (Table 1). The ganglioside pattern in the gray and white matter of PD1 was notably changed with an increase in monosialogangliosides GM1, GM2 and GM3, accompanied by a virtual absence of polysialogangliosides (data not shown).
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Molecular findings
As only a very limited amount of the patient’s genomic DNA was available from blood spots, initial mutation screening was undertaken on his parents. The promotor region and all exons and intron/exon boundaries of the PSAP gene were sequenced from PCR products amplified from genomic DNA. Both parents carried a single base deletion (c.803delG) in exon 9 (Fig. 6A) which was confirmed by amplification refractory mutation system (ARMS) analysis (Fig. 6B). The PCR product containing the heterozygous mutation was cloned and clones containing each of the alleles were sequenced. No other differences from the published sequences were found in the sequenced parts of the gene, with the exception of the promoter region and exon 1 (see below). Subsequently, the patient was shown to be homozygous for the c.803delG mutation by sequencing of PCR products and by ARMS (Fig. 6B). The c.803delG mutation leads to a frameshift followed by a premature stop codon 27 bases later.
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Direct sequencing of overlapping RT–PCR products did not reveal any abnormalities in the region harboring the mutation for either of PD1’s parents, nor was the mutant allele detected by ARMS analysis of their cDNA (prepared from total RNA), even after 80 rounds of amplification (Fig. 6C). However, after only 35 amplification cycles both mutant and wild-type alleles were detected in cDNA prepared from the nuclear, but not the cytosolic, fraction of the parental cell lines (Fig. 6C).
The sequence of the promotor region and exon 1 differed in both parents from the published sequence in a number of positions. Sequencing of genomic DNA from 10 unrelated healthy control subjects gave identical sequencing results to that of the investigated parents. The changes in the promoter region do not affect the presumed regulatory domains. However, at the 3' end of exon 1 the published sequence contained three additional nucleotides which were not present in our data (data not shown). Based on our results, we have re-positioned the exon/intron boundary, the proposed new splice site corresponding well with the consensus splice site. Our revised sequence data for the promoter region and exon 1 has been submitted to Genbank (accession no. AF307850).
Revised diagnosis for the case previously thought to be a LacCer-storing variant of NPC (55)
The sphingolipid storage observed in PD2 (Table 1 and Fig. 5) displayed identical features to that found in PD1 (Table 1, Figs 4 and 5), and all of the saposins were immunohistochemically undetectable. DNA from the mother of PD2 showed that she was a carrier for the same PSAP mutation as found in PD1 (c.803delG). The families of PD1 and PD2, who denied any relationship, both live in eastern Slovakia near Kosice in villages 20 km apart.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our pathological findings for patient PD1 led to molecular analysis of his PSAP gene with the subsequent identification of a homozygous c.803delG mutation within the SAP B domain, the resulting frameshift introducing a premature stop codon. The identified deletion would be expected to disrupt expression of full-length SAP B as well as preventing expression of the more 3' PSAP domains encoding SAPs C and D. Immunohistochemical investigations indicated that, in addition to a deficiency of saposins B, C and D, SAP A was also deficient in tissues from PD1. The absence of all four saposins is supported by our molecular investigations in cells from the parents of PD1. Whereas both the mutant and wild-type alleles could be detected in cDNA prepared from the nuclear fraction, only the wild-type allele was detected in cDNA prepared from the cytosolic fraction, indicating specific loss of the mutant mRNA, presumably through the process of nonsense-mediated mRNA decay (56). The lack of cytosolic mRNA from the mutant allele predicts that, in addition to a deficiency of the individual saposins, the PSAP precursor protein, with its neurotrophic and other functions, will also be deficient in patient PD1.
At the biochemical level, the characteristic feature of our patients was a generalized lipid storage of unusual complexity; the spectrum of stored lipids being consistent with the absence of PSAP-derived saposins. Elevated levels of ceramide have been reported for both human and mouse PSAPD (51,53) and turnover studies demonstrated that its hydrolysis could be restored by addition of SAP D (57,58). In PD1 and PD2, a 4–10-fold increase in free ceramides was found in all examined extraneural tissues. However, it is probable that the reduced turnover of glycosphingolipids (see below) moderated its accumulation. Ceramide accumulation was most marked in the liver of PD1, where there was also a high proportion of the slower migrating fraction (20%) with an Rf corresponding to the hydroxy fatty acid (HFA)-ceramide standard. This fraction was not observed in the other PSAPD tissues examined here, nor in the liver of the PSAPD fetus investigated by Bradová et al. (51), and its composition is currently being characterized. Accumulation of GlcCer was prominent in all examined tissues from PD1 and correlated with the presence of storage histiocytes, the only cell type affected in Gaucher disease. Its storage can be explained by the absence of SAP C, a known activator of ß-glucocerebrosidase, with cases of isolated SAP C deficiency (59,60) showing a phenotypic range similar to Gaucher disease patients, where the primary defect is in the enzyme ß-glucocerebrosidase. GalCer was abnormally increased in the kidney, similar to human and animal models of Krabbe disease (61,62). In contrast to Krabbe patients, we also found some increase in GalCer in PSAPD brain white matter, despite the hypomyelination which is thought to prevent its accumulation in patients with a defective enzyme. Accumulation of GalCer can probably be attributed to the loss of SAPs A plus C, since the deficient turnover of this sphingolipid in PSAPD fibroblasts could be partially corrected by the addition of either of these saposins (63). The most conspicuous abnormality in the glycolipid pattern was the accumulation of LacCer, which was elevated 25–40-fold in all affected tissues. LacCer can be hydrolysed by both ß-galactosylceramidase and ß-galactosidase and, as discussed previously (64), its massive accumulation in PSAPD suggests that both of these activities are impaired, a view supported by the accumulation of LacCer in mice genetically deficient in both of these enzymes (65). The observation is also consistent with the role of SAP B in facilitating LacCer hydrolysis by ß-galactosidase (64) while activation of ß-galactosylceramidase is dependent on SAP A and/or C. The hydrolysis of Gb3Cer is known to be dependent on SAP B (66,67), its marked storage in the kidney and the notable storage in the vascular endothelium fitting well with the storage pattern in Fabry disease (FD) (68). There was also no detectable storage in cardiocytes, commonly affected in FD, even when storage in other tissues is absent (69,70). Another discrepancy was the prominent accumulation of Gb3Cer in the adrenal cortex, which is not affected in classical FD (M. Elleder, unpublished data). A possible explanation for the differences may be the cumulative impact of blocks in multiple degradative steps within the lysosomal compartment in PSAPD. Decreased turnover of Gb3Cer has been confirmed in cultured PSAPD skin fibroblasts (64). Sulphatides were increased 10-fold in the kidney of PD1. This, together with the histochemical findings, mimics the storage pattern in metachromatic leukodystrophy (71) and is consistent with the known role of SAP B in hydrolysis of sulphatide, as evidenced in isolated SAP B-deficiency (72,73). There was no sign of histochemical sulphatide storage in brain glial phagocytes, probably due to the paucity of cerebral myelin. The brain ganglioside pattern was changed substantially in PD1 with increases in GM1, GM2 and GM3 gangliosides. This contrasted with the nearly normal ganglioside pattern seen in the fetal case of PSAPD (51). This variance might reflect the difference in developmental age of the two cases, although an effect of formaldehyde fixation cannot be excluded in our case. In contrast to the brain, GM3 and GM2 gangliosides were increased in liver from the fetal PSAPD case (51) and monosialogangliosides were also elevated in brain from the PSAP knock-out mouse (53). Although turnover studies in PSAPD and SAP B-deficient fibroblasts did not support the previously held view that SAP B facilitates GM1 ganglioside degradation, they did indicate that SAP B facilitates the degradation of GM3 ganglioside (74), a view that was supported by in vitro studies (75). The accumulation of other gangliosides in PSAPD tissues may be secondary to the block in GM3 ganglioside turnover. The generally normal levels of sphingomyelin were consistent with earlier reports for human and mouse PSAPD (50,53,64).
One of the most remarkable aspects of the disease process in PD1 was the pronounced neuronal depletion, particularly in the cortical region, which was populated by abundant glial phagocytes and fibrillary astrocytes, suggesting a destructive/reparative sequence of events, the former strongly resembling the situation in neuronal ceroid lipofuscinosis (NCL) type 1 caused by protein palmitoyl thioesterase (PPT)-deficiency (76). Common to both disorders is the fact that both PPT and PSAP are under developmental control in neural tissues, which suggests a role in the development and maintenance of neurons (9,11,77). The destructive neuronal process may be facilitated by the absence of PSAP’s known neurotrophic functions, and the prominent astrocytic reaction is consistent with PSAP’s suppression of the glial reaction after wounding (34). The pronounced paucity of cerebral myelin may be due to a combination of hypomyelination and demyelination. The absence of the myelinotrophic effects of PSAP (35) would be consistent with hypomyelination; however, delayed myelination has also been observed in unrelated lysosomal disorders (78), so it could be a non-specific effect. The cholesteryl ester accumulation in glial phagocytes clearly supports the case for demyelination.
This report consolidates and, particularly in relation to its neuropathology, extends our knowledge of human PSAPD. In addition, it has at last provided a definitive diagnosis for patient PD2. This patient was previously diagnosed as a variant of NPC (55), because she had a similar neutral glycolipid storage pattern to an earlier case of lactosylceramidosis (79), which was subsequently shown to have NPC (80,81). However, in the case of PD2, the excessive accumulation of LacCer is now attributed to PSAPD.
Our findings have highlighted the need to consider the possibility of PSAPD in the differential diagnosis of neonatal patients with severe neurological manifestations and spleno- or hepatosplenomegaly resembling infantile Gaucher disease (Table 2). Ultrastructural and biochemical investigations which should aid in the diagnosis of PSAPD are indicated in Table 2. The levels of sphingomyelin, cholesterol, ceramide and the ratio of glycosphingolipids to sphingomyelin may be particularly helpful in distinguishing cases of PSAPD from NPC (Table 1) (82) or other disorders where LacCer is also increased. If a suspicion of PSAPD is raised, the above investigations can be followed by more specific indicators of PSAPD, such as immunochemical detection of PSAP or saposins (only informative if not detected; Table 2) and/or molecular analysis of the PSAP gene, the latter giving ultimate confirmation of a PSAP defect. It must also be noted that patients with mutations which result in only a partial deficiency of PSAP, or which impact on only certain domains encoded by the PSAP gene, would not be expected to show the full spectrum of features seen when PSAP is totally deficient.
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Cumulative evidence from three unrelated families indicates that a total deficiency of PSAP is associated with a rapid and fatal course with severe neurovisceral manifestations already evident at birth. The pathology may stem from a deficiency not only of saposins, with their additive effects on the hydrolysis of a number of sphingolipids, but also of the precursor protein with its independent functions. Finally, our data indicate that there is an increased incidence of this extremely rare disorder in a small district in Eastern Slovakia, suggesting the existence of a genetic isolate.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients
The first patient (PD1), a male (46XY), was the product of the second pregnancy of unrelated parents, their previous pregnancy having resulted in a spontaneous abortion during the first trimester. He was born by section, at term, after an uneventful pregnancy. He weighed 3420 g, was 51 cm in length and had an Apgar score of 9/10/10. At birth, moderate hepatomegaly and splenomegaly (4 cm below the costal margin) were apparent and generalized seizures developed within minutes. Routine biochemical, hematological and microbiological analyses gave normal results, but brain sonography revealed a diffuse periventricular and cortical atrophy as well as signs of atrophy in the brain stem and cerebellum. Microcephalus, atrophy of the optical nerve, a subcapsular cataract, a right-sided hydrocoela and a non-descendent left testis were additional findings. The skin was macroscopically unremarkable. During the neonatal period the patient, who was never fully conscious, never cried and often developed seizures in response to tactile stimuli. He died at the age of 3.5 months. Formalin-fixed (10% formaldehyde for one year) tissues (spleen, liver, lung, adrenal cortex, kidney, skin, pancreas, and basal ganglia and cortex) and a blood spot were available for analysis. His parents were not related in the previous three generations, but both came from the same region of eastern Slovakia near Koice. Cell cultures were established from skin biopsies taken from the parents and from the chorionic villus and amniotic fluid samples collected from the parents’ next pregnancy. The family’s medical history, including that for the previous three generations, was unremarkable with the exception of the mother’s sister, who suffered from epilepsy. All investigations were carried out with the family’s informed consent.
As part of our study we also reviewed a second patient (PD2). The clinical history of this female patient, who died in the neonatal period, has been published (55). She was thought to have a variant form of NPC with enhanced glycolipid (including LacCer) storage. However, only formalin-fixed spleen and maternal fibroblasts were available for biochemistry. For comparative purposes, liver from the original PSAPD fetus (PD3) (50) as well as samples from confirmed cases of NPC from two different complementation groups (NPC1 and NPC2), NCL and FD were also examined, together with a series of tissues from normal heathy controls (accidental deaths).
Tissue culture
Skin fibroblasts were cultured according to routine procedures in Dulbecco’s modified Eagle’s medium (Gibco BRL, Life Technologies GmbH) with 10% fetal calf serum (Gibco BRL, Life Technologies GmbH) in 25 cm2 culture flasks.
Structural and histochemical analyses
Frozen sections were prepared from fixed tissues for lipid histochemical investigations. The sections were stained with Sudan black B, Fettrot FB, ferric hematoxylin (for phospholipids), cresyl violet (for sulphatides and acidic lipids) and PAS with and without prior extraction with chloroform:methanol (C:M; 2:1; v/v) (glycolipids seen in non-C:M extracted samples), and unstained sections were analysed for birefringence (83). Paraffin sections were used for neuropathological studies and examined using Oil red O, hematoxylin and eosin (H&E), Bodian and modified Spielmayer stains.
Immunodetection of saposins was done on paraffin sections using antibodies recognizing SAP A plus D (rabbit anti-INCL-antiserum provided by Dr Jaana Tyynelä, Helsinki, Finland) (84), SAP C (rabbit antibody provided by Dr Helen Christomanou, Athens, Greece) and SAP B (goat antibody provided by Prof. K. Sandhoff, Bonn, Germany). The paraffin sections were stained after deparaffination, hydratation and proteolytic pretreatment (20–30 min, 37°C) with 0.1% w/v trypsin for Sap B detection, 4% w/v pepsin for detection of SAP A plus D (84) or without proteolytic pretreatment for optimal detection of SAP C. Detection of bound primary antisera was achieved using a Universal DAKO LSAB peroxidase kit with 3,3'-diaminobenzidine as substrate. Macrophages (including the glial ones) and astrocytes were detected using the monoclonal anti-human CD68 (clone PG-M1) and monoclonal anti-human glial fibrillary acidic protein (GFAP) (clone 6F2), both from DAKO, respectively. Cathepsin D was detected using rabbit anti-human antibody (DAKO).
For electron microscopy, samples were postfixed with osmium tetroxide, dehydrated with ethanol and then embedded in Araldite–Epon mixture.
Biochemical analyses
For lipid analyses, formalin-fixed tissue samples from patients and age-matched controls were exhaustively washed with water, then weighed, homogenized and extracted successively with mixtures of chloroform:methanol:water (C:M:W; 20:10:1; 10:20:1; 10:10:1; v/v/v) according to Natomi et al. (85). Purified pooled lipid extracts were analyzed on HPTLC Silica 60 plates (Merck). Sphingomyelin was determined by phosphorus analysis using the method of Bradova et al. (86). Ceramides were resolved by double development of the plate; first with chloroform:methanol (95:5; v/v) and then, after drying, with hexane:diethylether:glacial acetic acid (60:40:1; v/v/v), followed by detection with cupric sulfate/phosphoric acid (87). Glycosphingolipids were separated using C:M:W (65:25:4, v/v/v) on either untreated plates or, for the separation of GlcCer and GalCer, on plates impregnated with 1% (w/v) sodium tetraborate in methanol (87) followed by detection with orcinol. Parallel plates were sprayed with Azure A for specific detection of sulphatides (88).
Immunodetection of Gb3Cer and LacCer was performed as previously described (89,90) on Polygram Sil G sheets (Macherey-Nagel) using mouse monoclonal antibodies to Gb3Cer (provided by Dr Tadashi Tai, Tokyo Metropolitan Institute of Medical Science, Japan) and LacCer (provided by Dr Kristian Koubek, Institute of Hematology and Blood Transfusion, Prague, Czech Republic), respectively. Binding of primary antibodies was detected using Pierce anti-mouse IgG peroxidase conjugate (Pierce) or a Universal DAKO LSAB peroxidase kit for Gb3Cer, and Pierce anti-mouse IgM peroxidase conjugate (Pierce) for LacCer.
Chromatograms were evaluated densitometrically using a Camag TLC Scanner II (Cats3; Camag Scientific) in reflection mode and quantitation was based on comparison with known amounts of glycolipid standards applied to the same chromatogram. The following sphingolipid standards were prepared in our Prague laboratory and their identity confirmed by mass spectrometry: GalCer from human brain, LacCer from human spleen and erythrocytes, GlcCer from Gaucher spleen, Gb3Cer from Fabry myocardium, globotetraosyl ceramide (Gb4Cer) from human erythrocytes and sulphatides from human brain. Sphingomyelin from bovine brain, and non-hydroxy fatty acid (NFA)- and HFA-ceramides, were purchased from Sigma Chemical Company.
Molecular investigations
Genomic DNA was isolated by standard techniques from white blood cells and cultured skin fibroblasts or, for blood spots, according to Caggana et al. (91). Total RNA was extracted following the method of Chomczynski and Sacchi (92) and cDNA was prepared using Superscript II reverse transcriptase (Gibco BRL, Life Technologies GmbH) with oligo d(T)18 priming. Primers used for the amplification of genomic or cDNA are listed in Table 3. T7 or RP standard primers were added to the 5' ends of the primers as indicated. PSAP cDNA was amplified using two pairs of primers (PSAP-cDNA-S1-RP with PSAP-cDNA-A1-T7 and PSAP-cDNA-S2-T7 with PSAP-cDNA-A2-RP). For genomic DNA, the promoter region plus exon 1 was amplified using primers (PSAP-g1S-T7 and PSAP-g6A-RP) derived from the sequence of Sun et al. (3). The primers used for amplification of the other exons are listed in Table 3 and were based on the sequence of Rorman et al. (2). PCR products were gel purified and sequenced using ThermoSequenase (Amersham) or AmpliTaq FS polymerases (Perkin-Elmer) with fluorescently labeled T7 and RP primers. The sequencing reactions were analysed using an AlfExpress fluorescent sequencer (Amersham Pharmacia Biotech).
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To analyse RNA from the nuclear and cytosolic compartments, cultured skin fibroblasts (approximately 15 x 106 cells) were harvested and washed twice with phosphate buffered saline. The cells were pelleted and resuspended in 500 µl of ice-cold Triton X-100 lysis buffer [10 mM Tris–Cl pH 8, 150 mM NaCl, 1.5 mM MgCl2, 0.5 % Triton X-100 (v/v)]. The suspension was overlaid onto 300 µl of ice-cold Triton X-100 lysis buffer containing 0.3 M sucrose and centrifuged at 1000 g for 10 min at 4°C. The cytoplasmic upper phase was transferred to a fresh tube and mixed with a denaturing solution (Solution D) (92). The remainder of the sucrose phase was discarded and the pelleted nuclei were also solubilized in Solution D. Isolation of RNA from the nuclear and cytoplasmic lysates and preparation of cDNA were as described above.
ARMS analysis of the c.803delG mutation in genomic DNA and cDNA used primers 803delG-A-plus and primer 803delG-A-minus (Table 3) to specifically amplify the mutant and wild-type alleles. These primers were paired with primer 803delG-gDNA-S and PSAP-cDNA1S-RP (Table 3) for amplification from genomic and cDNA, respectively. The amplification mixture contained another set of primers amplifying a different part of the genome as an amplification control. The amplification reactions were performed in 50 mM Tris–HCl pH 9.1, with 2U of Klentaq 1 (GeneAge Technologies).
Electronic database information
Accession numbers and URLs for data in this article are as follows: Genbank, http://www.ncbi.nlm.nih.gov/Web/Genbank (accession nos M86181, AF057307 and AF307850).
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ACKNOWLEDGEMENTS |
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Dr Jaana Tyynelä, Dr Helen Christomanou, Prof. Konrad Sandhoff, Dr Tadashi Tai and Dr Kristian Koubek are thanked for their generous gifts of antibodies. This work was supported by grants from the Ministry of Education and Youth of the Czech Republic (VS 96127) and from the Grant Agency of the Charles University, Prague (GAUK 37/2000/C).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Institute of Inherited Metabolic Disorders, First Faculty of Medicine and University Hospital, Ke Karlovu 2, Bldg D, Division B, 128 08 Prague 2; Tel: +420 2 2491 8283; Fax: +420 2 2491 9392; Email: melleder@cesnet.cz
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REFERENCES |
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