Evidence for clinical, genetic and biochemical variability in spinal muscular atrophy with progressive myoclonic epilepsy.

Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) is a recently delineated, autosomal recessive condition caused by rare mutations in the N-acylsphingosine amidohydrolase 1 (acid ceramidase) ASAH1 gene. It is characterized by motor neuron disease followed by progressive myoclonic seizures and eventual death due to respiratory insufficiency. Here we report an adolescent female who presented with atonic and absence seizures and myoclonic jerks and was later diagnosed as having myoclonic-absence seizures. An extensive genetic and metabolic work-up was unable to arrive at a molecular diagnosis. Whole exome sequencing (WES) identified two rare, deleterious mutations in the ASAH1 gene: c.850G>T;p.Gly284X and c.456A>C;p.Lys152Asn. These mutations were confirmed by Sanger sequencing in the patient and her parents. Functional studies in cultured fibroblasts showed that acid ceramidase was reduced in both overall amount and enzymatic activity. Ceramide level was doubled in the patient's fibroblasts as compared to control cells. The results of the WES and the functional studies prompted an electromyography (EMG) study that showed evidence of motor neuron disease despite only mild proximal muscle weakness. These findings expand the phenotypic spectrum of SMA-PME caused by novel mutations in ASAH1 and highlight the clinical utility of WES for rare, intractable forms of epilepsy.

[Cerebrotendinous xanthomatosis: a multicentric retrospective study of 15 adults, clinical and paraclinical typical and atypical aspects]. / Étude rétrospective multicentrique de 15 cas adultes de xanthomatose cérébrotendineuse : aspects cliniques et paracliniques typiques et atypiques.

INTRODUCTION: Cerebrotendinous xanthomatosis, a metabolic leukodystrophy with an autosomal recessive inheritance, is secondary to deficiency of sterol 27-hydroxylase, an enzyme involved in cholesterol catabolism. Classical symptoms include clinical or infraclinical xanthomas affecting the skin and tendons, early cataracts, neurological signs and diarrhea. Brain imaging reveals involvement of the dentate nuclei and periventricular white matter hyperintensities. The diagnosis is based on an increased cholestanol level in serum, confirmed by the presence of a mutation in the CYP27A1 gene. Treatment is based on chenodeoxycholic acid. METHOD: We report a retrospective multicentric study of 15 cases of cerebrotendinous xanthomatosis diagnosed in French adults. Clinical, molecular and MRI findings were recorded in all patients. RESULTS: The average age at diagnosis was 39years (range 27-65). Disease onset occurred in childhood in 73% of patients and in adulthood in 27%. All patients with a pediatric onset were diagnosed during adulthood (age range 28-65years). Clinical symptoms variably associated cerebellar syndrome, pyramidal syndrome, cognitive decline, epilepsy, neuropathy (sought in 10 of our patients, present in forms in 8), psychiatric disorders, cataract and xanthomas. One patient had an atypical presentation: monoparesis associated with xanthomas. Brain MRI was abnormal in all: findings consisted in T2-weighted hyperintensity of the dentate nuclei (47%), periventricular leuoencephalopathy (73%) which preferentially involved the posterior cerebral part (60%), leucoencephalopathy with a vascular pattern (7%), hyperintensity of the cortico-spinal tracts (53%), globi pallidi, corpus callosum and cerebral atrophy (33%). Serum cholestanol was elevated in 93% of patients. The most frequent mutation was 1183C>T (n=5/15). Under treatment with chenodeoxycholic acid, eight patients improved initially, followed by stabilization in five of them, and worsening in the others. Four patients died. CONCLUSION: Patients with the xanthoma-neurological disorder association should be tested for cerebrotendinous xanthomatosis. The disease often begins in childhood with a diagnostic delay but also in adulthood. Involvement of the dentate nuclei is specific but not sensitive and the supratentorial leucoencephalopathy is not specific but with an antero-posterior gradient. A vascular distribution and involvement of the corpus callosum are possible. Serum cholestanol assay is very reliable: an elevated level provides the diagnosis, which must nevertheless be confirmed by molecular biology.

[Mucopolysaccharidosis: A review]. / Mucopolysaccharidoses : quand y penser ?

Mucopolysaccharidosis are lysosomal storage diseases, secondary to the accumulation of mucopolysaccharides. Type 1 mucopolysaccharidosis is the most common form and affects between 0.69 and 1.66 newborns per 100,000. The severity of mucopolysaccharidosis is variable with lethal forms in utero and attenuated forms diagnosed in adults. The most common symptoms are short stature, facial dysmorphism, chronic articular pains that can mimic chronic inflammatory rheumatism, axial and peripheral bone involvement, hepatosplenomegaly and an early carpal tunnel. Depending on the type of mucopolysaccharidosis, corneal, cerebral or cardiac involvements are possible. Screening is based on the analysis of urinary glycosaminoglycans. The deficient enzyme assay and the gene analysis confirm the diagnosis. Mucopolysaccharidosis recognition is important for patient management and family screening. In addition, specific enzyme replacement therapy exists for certain types of mucopolysaccharidosis. Role of clinician is important to evoke and diagnose mucopolysaccharidosis.

Involvement of FAN in TNF-induced apoptosis.

TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.

Overcoming MDR-associated chemoresistance in HL-60 acute myeloid leukemia cells by targeting sphingosine kinase-1.

We examined the involvement of sphingosine kinase-1, a critical regulator of the sphingolipid balance, in susceptibility to antineoplastic agents of either sensitive or multidrug-resistant acute myeloid leukemia cells. Contrary to parental HL-60 cells, doxorubicin and etoposide failed to trigger apoptosis in chemoresistant HL-60/Doxo and HL-60NP16 cells overexpressing MRP1 and MDR1, respectively. Chemosensitive HL-60 cells displayed sphingosine kinase-1 inhibition coupled with ceramide generation. In contrast, chemoresistant HL-60/ Doxo and HL-60/VP16 had sustained sphingosine kinase-1 activity and did not produce ceramide during treatment. Enforced expression of sphingosine kinase-1 in chemosensitive HL-60 cells resulted in marked inhibition of apoptosis that was mediated by blockade of mitochondrial cytochrome c efflux hence suggesting a control of apoptosis at the pre-mitochondrial level. Incubation with cell-permeable ceramide of chemoresistant cells led to a sphingosine kinase-1 inhibition and apoptosis both prevented by sphingosine kinase-1 over-expression. Furthermore, F-12509a, a new sphingosine kinase inhibitor, led to ceramide accumulation, decrease in sphingosine 1-phosphate content and caused apoptosis equally in chemosensitive and chemoresistant cell lines that is inhibited by adding sphingosine 1-phosphate or overexpressing sphingosine kinase-1. F-12509a induced classical apoptosis hallmarks namely nuclear fragmentation, caspase-3 cleavage as well as downregulation of antiapoptotic XIAP, and release of cytochrome c and SMAC/Diablo.

Caspase-dependent and -independent cell death of Jurkat human leukemia cells induced by novel synthetic ceramide analogs.

Ceramide metabolism has emerged as a potential target for anticancer therapy. Here, the potential usefulness of two novel synthetic ceramide analogs as anti-leukemic drugs was investigated. Compounds AD2646 and AD2687 were able to dose-and time-dependently decrease the viability of Jurkat leukemic cells. This was accompanied by an accumulation of endogenous ceramide owing to perturbed ceramide metabolism. Cytotoxicity involved caspase activation but also necrotic-like features, as evidenced by phosphatidylserine externalization, membrane permeability, hypodiploidy, caspase processing and only partial protection from cell death by a pan-caspase inhibitor. Ceramide analogs also induced cell death in Jurkat mutants that are deficient in cell death signaling proteins, including FADD, caspase-8 and 10, and RIP. While overexpression of Bcl-xL did not suppress ceramide accumulation, it conferred robust protection from caspase activation and cell death. Altogether, these novel ceramide analogs are able to kill leukemic cells through distinct pathways implicating caspase activation and mitochondrial events, and represent a new group of bioactive molecules with potential applications in anticancer therapy.

[Adult leukoencephalopathy caused by alpha-mannosidosis deficiency]. / Leucoencéphalopathie de l'adulte associée à un déficit en alpha-mannosidase.

Adult leukoencephalopathy caused by alpha-mannosidosis deficiency (MIM248500) is a recessive inherited lysosomal storage disease associated with decreased activity of alpha-mannosidase. This enzyme degrades oligosaccharides and glycoproteins in neural and visceral tissues. There are two different disease phenotypes, type-I or severe infantile phenotype and type 2, which progresses more slowly and is compatible with survival into adulthood. We report the case of a 51-year-old man with gait disorders beginning at the age of 40 years associated with leukoencephalopathy due to alpha-mannosidosis deficiency.

[Papillon-Lefèvre syndrome: A new case]. / Le syndrome de Papillon-Lefèvre, à propos d'une nouvelle observation.

Papillon-Lefèvre syndrome (PLS) is a rare primary immunodeficiency, which combines severe periodontal disease with edentulism and palmoplantar keratosis (PPK). PLS is inherited as an autosomal recessive trait and is due to mutations in the cathepsin C gene. The biological properties of the neutrophils (PN) are altered, leading to a gingival dysbiosis and bacterial overgrowth, with intense inflammation of the periodontium. We report the observation of a 4-year-old girl who presented to the clinic with gingivitis, partial edentulism, and PPK, whose diagnosis, raised after a long delay, was suggested by null cathepsin C activity and confirmed by the presence of heterozygous mutations in exon 4: c.628C>T, pArg210* and in exon 7: c.1286G>A, p.Trp429*. A multidisciplinary approach transformed the functional and esthetic prognosis and psychological behavior of this child. This classical observation describes this poorly known phenotype.

Sphingolipid mediators in cardiovascular cell biology and pathology.

Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.

Sphingomyelin metabolites in vascular cell signaling and atherogenesis.

The atherosclerotic lesion most probably develops through a number of cellular events which implicate all vascular cell types and include synthesis of extracellular proteins, cell proliferation, differentiation and death. Sphingolipids and sphingolipid metabolizing enzymes may play important roles in atherogenesis, not only because of lipoprotein alterations but also by mediating a number of cellular events which are believed to be crucial in the development of the vascular lesions such as proliferation or cell death. Exogenous sphingolipids may mediate various biological effects such as apoptosis, mitogenesis or differentiation depending on the cell type. Moreover, several molecules present in the atherogenic lesion, such as oxidized LDL, growth factors or cytokines, which activate intracellular signaling pathways leading to vascular cell modifications, can stimulate sphingomyelin hydrolysis and generation of ceramide (and other metabolites as sphingosine-1-phosphate). Here we review the potential implication of the sphingomyelin/ceramide cycle in vascular cell signaling related to atherosclerosis, and more generally the role of sphingolipids in the events observed during the atherosclerotic process as cell differentiation, migration, adhesion, retraction, proliferation and death.

Modulation of subcellular distribution of doxorubicin in multidrug-resistant P388/ADR mouse leukemia cells by the chemosensitizer ((2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino]propyloxy)-benzenesulfonyl))indolizine.

The impact of the novel chemosensitizer ((2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino]propyloxy)benzenesulfonyl))indolizine (SR33557) on the intracellular distribution of doxorubicin (DOX) within the multidrug-resistant murine P388/ADR leukemia cell line was studied by fluorescence microscopy. We found that under conditions which modulated multidrug-resistant (30 microM SR33557 for 1 h), P388/ADR cells presented an original sequestration of DOX in large intracellular vesicles, where SR33557 is itself sequestered, as seen by colocalization studies. Colocalization experiments with lysosomal and mitochondrial probes suggest that these vesicles are neither mitochondrial in nature nor functional lysosomes. To investigate the biochemical basis for this effect, we studied the impact of SR33557 on the sphingolipid metabolism of P388/ADR cells. We observed that although P388/ADR cells normally catabolized exogenous [3H]sphingomyelin, when pretreated with SR33557 they showed almost complete inhibition of sphingomyelin breakdown. Finally, in order to demonstrate that the inability of P388/ADR cells to degrade sphingomyelin in the presence of SR33557 (which is a potent inhibitor of acid lysosomal sphingomyelinase) leads to phospholipid accumulation, we performed electron microscopy where we observed laminated inclusions. These morphological modifications are similar to those observed in Niemann-Pick disease lymphoblastoid cell lines which are inherently deficient in acid sphingomyelinase activity. The observation that, in the absence of SR33557, these Niemann-Pick disease cell lines presented similar DOX sequestration to that of SR33557-treated P388/ADR cells strongly suggests that DOX accumulates in SR33557-induced myeloid bodies. The redistribution of DOX within these vesicles, perhaps by preventing its expulsion by P-glycoprotein, may be a key in discovering the mechanism of action of SR33557.

The protein kinase C activators phorbol esters and phosphatidylserine inhibit neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin.

To address the role of protein kinase C (PKC) in the regulation of ceramide production, we evaluated the impact of the PKC activators 12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine on the apoptotic signaling pathway triggered by the chemotherapeutic drug daunorubicin. Treatment of U937 and HL-60 cells with 0.5-1 microM daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4-10 min with concomitant sphingomyelin hydrolysis and ceramide generation. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine inhibited daunorubicin-induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. In conclusion, PKC emerges as a potentially critical negative regulator of the anthracycline-activated sphingomyelin-ceramide apoptotic pathway.

Enhancement of ricin A chain immunotoxin activity by perhexiline on established and fresh leukemic cells.

In the perspective of increasing the clinical potential of ricin A chain immunotoxins (RTA-ITs), perhexiline (Pex) and four structural analogues (Pex 2, Pex 3, Pex 7, and Pex 11) were evaluated for their ability to enhance RTA-IT activity in vitro. Only perhexiline significantly enhanced the cytotoxic activity of anti-CD5 RTA-ITs, T101 and T101-F(ab')2, on CEM III cell line (30- to 2000-fold), and of anti-HLA-DR RTA-IT, HNC-241, on both RAJI cell line (greater than 100-fold) and two immortalized cell lines originating from patients suffering from B-cell chronic lymphocytic leukemia, EHEB and FS2 D5 (10-fold). On 16 consecutive fresh B-cell chronic lymphocytic leukemia cell samples, significant T101-F(ab')2 RTA-IT and HNC-241 RTA-IT enhancement was observed with perhexiline which was comparable to that of NH4Cl and monensin. Perhexiline almost completely blocked RTA-IT intracellular degradation and profoundly modified its routing. These observations were linked to perhexiline-induced lipidosis via inhibition of sphingomyelinase activity. In conclusion, since the concentrations used are relevant with the pharmacokinetics of this agent, perhexiline appears to be a promising agent for in vivo enhancement of ricin A chain immunotoxins.

In vitro and in vivo enhancement of ricin-A chain immunotoxin activity by novel indolizine calcium channel blockers: delayed intracellular degradation linked to lipidosis induction.

With regard to increasing the clinical potential of ricin A-chain immunotoxins (RTA-ITs), a novel class of calcium channel blockers, indolizines SR33557 [2-isopropyl-1-[4-(3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino)propyloxy)benzenesulfonyl))indolizine] and SR33287 [isopropyl-2-((1-butylamino-3-propyl)oxy-4-benzoyl)-3-indolizine], were evaluated for their ability to enhance RTA-IT activity in vitro and in vivo. Five microM SR33287 and 5 microM SR33557 were potent enhancers of both anti-Thy 1.2 AT15E RTA-IT (84- and 64-fold, respectively) on T2 cells and anti-CD5 T101 (622- and 538-fold) and T101 F(ab')2 RTA-IT (34- and 28-fold) on CEM III cells. This was superior to the effect achieved by both 10 microM verapamil and 10 mM NH4Cl, albeit slightly inferior to that of 50 nM monensin and 5 microM perhexiline. Murine T2 lymphoma cells bearing the Thy 1.2 antigen were injected i.v. in Thy 1.2 (-) BL. 1.1 mice (median survival time, 17.7 days). Intravenous treatment with 10 micrograms of AT15E RTA-IT prolonged the survival of mice (median survival time, 26.8 days). When 400 micrograms of SR33287 were coinjected i.v. with 10 micrograms of AT15E RTA-IT, mouse survival was further increased, with 5 of 6 mice surviving, disease free, over 42 days. SR33287 had a significant impact on the intracellular routing of 125I-AT15E RTA-IT, which induced a greater than 2-fold increase in intracellular intact AT15E RTA-IT at 90 min. This effect on RTA-IT half-life was distinctly different from that observed with either NH4Cl or monensin and may be linked to the inhibition of acid lysosomal sphingomyelinase by SR33287, leading to cellular lipidosis. In conclusion, indolizines appear to be promising agents not only for immunotoxin enhancement but also for increasing the activity of any number of targeted therapeutic agents where modifying either the intracellular routing or increasing the intracellular half-life of the ligand would be beneficial to its cytotoxic activity.

A sequential bioorthogonal dual strategy: ManNAl and SiaNAl as distinct tools to unravel sialic acid metabolic pathways.

Recent methodological developments in metabolic oligosaccharide engineering (MOE) pave the way for tremendous advances in glycobiology. Herein, we propose a Sequential Bioorthogonal Dual Strategy (SBDS) combining the use of two unprotected alkyne-tagged monosaccharide reporters (ManNAl and SiaNAl) with the bioligation of fluorescent probes by copper-catalysed azide-alkyne cycloaddition (CuAAC). With SBDS, we are able to shed light on trafficking and cellular uptake mechanisms of sialic acid. Using their corresponding analogues, we visualized that SiaNAl enters via endocytosis, whereas its biosynthetic intermediate ManNAl uptake is mediated by a yet unknown but specific plasma membrane transporter. Sialin, a lysosomal protein, is shown to be crucial for the export of exogenous sialic acid from lysosomes to the cytosol. Metabolic labeling with alkyne-tagged derivatives of N-acetylneuraminic acid (Neu5Ac) or N-acetylmannosamine (ManNAc) could thus be used to follow endocytosis in physiological vs. pathological conditions.

Uptake and intracellular degradation of fluorescent sphingomyelin by fibroblasts from normal individuals and a patient with Niemann-Pick disease.

Sphingomyelin, labelled with a fluorescent probe, pyrene, in the fatty acyl residue was associated with fetal calf serum; approx. 80% of the sphingomyelin was found in the low- and high-density lipoproteins. This was added to the growth medium of cultured human skin fibroblasts from normal individuals and a patient with Niemann-Pick disease type A, devoid of acid sphingomyelinase activity. The fluorescent sphingomyelin was taken up by both cell types, but only the former degraded it to produce fluorescent ceramide. Differences between normal and Niemann-Pick cells in sphingomyelin content or ceramide production were observed after several hours uptake. A more pronounced difference was noted when cells were incubated for 1 day with fluorescent sphingomyelin and then for two to three days in medium devoid of this compound. Under these conditions, the fluorescence intensity of the Niemann-Pick cells remained practically constant while that of their normal counterparts was almost completely eliminated from the cells. Comparison of fluorescence intensities of these two cell types could be made directly on aqueous suspensions of whole cells or, alternatively, on their lipid extracts. For evaluation of the degradation of fluorescent sphingomyelin to ceramide within the cells, several procedures were developed for the rapid isolation of the latter compound from the total lipid extract. The results suggest that when associated with the constituents of the fetal calf serum, sphingomyelin is taken up by the cells and transported into the lysosomal compartment where it is degraded to ceramide. Use of the fluorescent derivative of sphingomyelin provided a simple and rapid procedure for following the uptake by and degradation within the cultured cells. It also permitted the establishment of differences in the rates of degradation of the fluorescent sphingomyelin by cells with a normal metabolism and others lacking sphingomyelinase (i.e., Niemann-Pick disease type A cells).

Signalling sphingomyelinases: which, where, how and why?

A major lipid signalling pathway in mammalian cells implicates the activation of sphingomyelinase (SMase), which upon cell stimulation hydrolyses the ubiquitous sphingophospholipid sphingomyelin to ceramide. This review summarizes our current knowledge on the nature and regulation of signalling SMase(s). Because of the controversy on the identity of this(these) phospholipase(s), the roles of various SMases in cell signalling are discussed. Special attention is also given to the subcellular site of action of signalling SMases and to the cellular factors that positively or negatively control their activity. These regulating agents include lipids (arachidonic acid, diacylglycerol and ceramide), kinases, proteases, glutathione and other proteins.

Degradation of fluorescent and radiolabelled sphingomyelins in intact cells by a non-lysosomal pathway.

The aim of the present study was to investigate the role of the entitled neutral, sphingomyelinase in the non-lysosomal pathway of sphingomyelin degradation by intact cells (Spence et al. (1983) J. Biol. Chem. 258, 8595-8600; Levade et al. (1991) J. Biol. Chem. 266, 13519-13529). The uptake and degradation of sphingomyelin by intact living cells was studied using cell lines exhibiting a wide range of activity levels of acid, lysosomal and neutral sphingomyelinases as determined in vitro on cell homogenates by their respective standard assays. For this purpose, neuroblastoma, skin fibroblasts, lymphoid and leukemic cell lines, some of them derived from patients with Niemann-Pick disease (deficient in the acid, lysosomal sphingomyelinase) were incubated with radioactive, [oleoyl-3H]sphingomyelin or fluorescent, pyrene-sulfonylaminoundecanoyl-sphingomyelin. Either compound was taken up by a pathway which was not receptor-mediated and hydrolyzed by all intact cells, including those derived from Niemann-Pick disease patients. Moreover, their degradation by the intact cells was not inhibited by treatment with chloroquine, indicating hydrolysis by a non-lysosomal sphingomyelinase. The intracellular sphingomyelin degradation rates showed no correlation with the activity of the 'classical' neutral sphingomyelinase as determined in vitro. In particular, fibroblasts derived from Niemann-Pick patients lacking the lysosomal sphingomyelinase, and having no detectable in vitro activity of the 'classical' neutral sphingomyelinase, were able to degrade the exogenously supplied sphingomyelins. Indeed, in vitro these cells were shown to exhibit neutral, magnesium- and dithiothreitol-dependent sphingomyelinase activities, that might contribute to the non-lysosomal pathway for sphingomyelin degradation to ceramide in intact cells.

Cholesterol sulfate is not degraded but does not accumulate in Epstein-Barr virus-transformed lymphoid cells from patients with X-linked ichthyosis.

The metabolism of cholesterol sulfate (CS) was investigated in immortalized, Epstein-Barr virus-transformed lymphoid cell lines derived from normal individuals and patients affected with recessive X-linked ichthyosis (XLI). Normal lymphoid cells expressed arylsulfatase C and steroid sulfatase (including cholesterol sulfatase) activities, and these two sulfohydrolases showed the same enzyme properties as in other human cells, e.g., leukocytes or skin fibroblasts. XLI-derived lymphoid cell lines exhibited extremely deficient activity of both arylsulfatase C and steroid sulfatase. While normal and XLI intact, living lymphoid cells could take up exogenous radiolabelled CS through a non-receptor-mediated process. XLI cells were completely unable to degrade CS to cholesterol. However, despite their defect in CS degradation, steroid sulfatase-deficient cells did not accumulate CS because of outflux of this sterol. The potential implications of these findings to the pathogenesis of increased CS content in plasma and epidermis of XLI patients are discussed. This study also demonstrates that immortalized lymphoid cell lines may represent a useful experimental model system for the study of XLI.

Significance of two point mutations present in each HEXB allele of patients with adult GM2 gangliosidosis (Sandhoff disease) homozygosity for the Ile207-->Val substitution is not associated with a clinical or biochemical phenotype.

The molecular defects in the HEXB gene encoding the common beta-subunit of lysosomal beta-hexosaminidase A (beta-Hex A, alpha beta) and beta-Hex B (beta beta) were investigated in a Portuguese family affected with late onset Sandhoff disease (GM2-gangliosidosis variant 0). This family comprised two unaffected daughters and three affected sibs who developed at about age 17 cerebellar ataxia and mental deficiency. Their parents were consanguineous and clinically asymptomatic. There was no detectable beta-Hex B activity and a profound reduction in the activity of beta-Hex A in the leukocytes and transformed lymphoid cell lines from the affected sibs. The expected intermediate values were observed in the parents as well as in one daughter and her children. Western analysis revealed the presence of reduced, but detectable amounts of mature beta-chain protein in cell lysates from the probands and intermediate levels in the parents. Nucleotide sequencing of amplified, reverse-transcribed beta-chain mRNA demonstrated the presence of two single point mutations: an A619 to G transition in exon 5 (Ile207-->Val), and a G1514 to A transition in exon 13 (Arg505-->Gln). Both of these two mutations have been previously linked to the adult form of Sandhoff disease in compound heterozygote patients. All three affected sibs were found to be homoallelic for both mutations. Interestingly, while the mother was heterozygous for each mutation, the father was homozygote for the A619-->G substitution and heterozygote for the G1514-->A transition. Since the father is homozygote for the A619-->G mutation but expresses a biochemical phenotype consistent with a carrier of Sandhoff disease and is clinically asymptomatic, this substitution is likely a neutral mutation. We confirmed this hypothesis by finding this transition present in 4 of 30 alleles from normal individuals. We conclude that homozygosity for the G1514-->A mutation is exclusively responsible for the adult form of Sandhoff disease in this family, and that the A619-->G substitution is not a deleterious mutation but rather a common HEXB polymorphism.