Quantitation of plasmatic lysosphingomyelin and lysosphingomyelin-509 for differential screening of Niemann-Pick A/B and C diseases.

Acid sphingomyelinase deficiency (ASMd, Niemann-Pick disease A/B) and Niemann-Pick type C disease (NPC) share core clinical symptoms. Initial diagnostic discrimination of these two rare lysosomal storage diseases is thus difficult. As sphingomyelin accumulates in ASMd as well as NPC, lysosphingomyelin (sphingosylphosphorylcholine) and its m/z 509 analog were suggested as biomarkers for both diseases. Herein we present results of simultaneous LC-ESI-MS/MS measurements of lysosphingomyelin and lysosphingomyelin 509 in plasma and dried blood spots (DBS) collected from ASMd and NPC patients and suggest that the plasma but not DBS levels of the two analytes allow differential biochemical screening of ASMd and NPC.

X-Chromosome Inactivation Analysis in Different Cell Types and Induced Pluripotent Stem Cells Elucidates the Disease Mechanism in a Rare Case of Mucopolysaccharidosis Type II in a Female.

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder resulting from deficiency of iduronate-2-sulphatase activity. The disease manifests almost exclusively in males; only 16 symptomatic heterozygote girls have been reported so far. We describe the results of X-chromosome inactivation analysis in a 5-year-old girl with clinically severe disease and heterozygous mutation p.Arg468Gln in the IDS gene. X inactivation analysed at three X-chromosome loci showed extreme skewing (96/4 to 99/1) in two patient's cell types. This finding correlated with exclusive expression of the mutated allele. Induced pluripotent stem cells (iPSC) generated from the patient's peripheral blood demonstrated characteristic pluripotency markers, deficiency of enzyme activity, and mutation in the IDS gene. These cells were capable of differentiation into other cell types (cardiomyocytes, neurons). In MPS II iPSC clones, the X inactivation ratio remained highly skewed in culture conditions that led to partial X inactivation reset in Fabry disease iPSC clones. Our data, in accordance with the literature, suggest that extremely skewed X inactivation favouring the mutated allele is a crucial condition for manifestation of MPS II in females. This suggests that the X inactivation status and enzyme activity have a prognostic value and should be used to evaluate MPS II in females. For the first time, we show generation of iPSC from a symptomatic MPS II female patient that can serve as a cellular model for further research of the pathogenesis and treatment of this disease.

Rapid isolation of lysosomal membranes from cultured cells.

We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient. An enriched lysosomal membrane fraction was collected from the 20%/35% sucrose interface. The washed lysosomal membrane fraction was enriched 30 times relative to the homogenate and gave the yield of more than 8%. These results are comparable to lysosomal membranes isolated by magnetic chromatography from cultured cells (Diettrich et al., 1998). The procedure effectively eliminated mitochondrial contamination and minimized contamination from other cell compartments. The enriched fractions retained the ability to acidify membrane vesicles through the activity of lysosomal vacuolar ATPase. The method avoids non-physiological overloading of cells with superparamagnetic particles and appears to be quite robust among the tested cell lines. We expect it may be of more general use, adaptable to other cell lines and tissues.

Semisynthesis of C17:0 isoforms of sulphatide and glucosylceramide using immobilised sphingolipid ceramide N-deacylase for application in analytical mass spectrometry.

Sphingolipid ceramide N-deacylase (SCDase, EC 3.5.1.69) is a hydrolytic enzyme isolated from Pseudomonas sp. TK 4. In addition to its primary deacylation function, this enzyme is able to reacylate lyso-sphingolipids under specific conditions. We immobilised this enzyme on magnetic macroporous cellulose and used it to semisynthesise C17:0 glucosylceramide and C17:0 sulphatide, which are required internal standards for quantification of the corresponding glycosphingolipids (GSL) by tandem mass spectrometry. A high rate of conversion was achieved for both lipids (80% for C17:0 sulphatide and 90% for C17:0 glucosylceramide). In contrast to synthesis with a soluble form of the enzyme, use of immobilised SCDase significantly reduced the contamination of the sphingolipid products with other isoforms, so further purification was not necessary. Our method can be effectively used for the simple preparation of specifically labelled sphingolipids of high isoform purity for application in mass spectrometry.

Autopsy case of Gaucher disease type I in a patient on enzyme replacement therapy. Comments on the dynamics of persistent storage process.

We report a female patient with Gaucher disease (GD) type I on ERT (imiglucerase) for 5 years, which led to a significant general improvement. Aged 59 years she underwent an episode of altitude sickness followed by sepsis, disseminated intravascular coagulation, and multiorgan failure. She succumbed to a cerebral haemorrhage. Autopsy revealed liver cholestatic cirrhosis and multifocal liver carcinoma with immunophenotype compatible with cholangiocarcinoma. Analysis of the storage process revealed its absence or very low levels in the majority of liver and spleen macrophages. Gaucher cells (GCs) were seen only as occasional aggregates of various sizes in these organs. GCs were seen also in the leptomeninx of the cerebellum and as infrequent perivascular clusters in both the grey and white cerebral matters. Bone marrow was heavily infiltrated with GCs, especially in the adipocyte-rich part. GCs in this location displayed varied degrees of cytoplasmic vacuolation unrelated to the lysosomal compartment, caused by droplets of triglyceride, and interpreted as due to resorption of fragments of altered white adipocytes. All these observations point to the relative efficacy of ERT in covering the standard substrate load, which should not be exceeded as it would lead to the evolution of mature GCs. The results are discussed in relation to our recently published hypothesis on GD cell pathology.

Methods for a prompt and reliable laboratory diagnosis of Pompe disease: report from an international consensus meeting.

Pompe disease is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). It presents at any age, with variable rates of progression ranging from a rapidly progressive course, often fatal by one-year of age, to a more slowly, but nevertheless relentlessly progressive course, resulting in significant morbidity and premature mortality. In infants, early initiation of enzyme replacement therapy is needed to gain the maximum therapeutic benefit, underscoring the need for early diagnosis. Several new methods for measuring GAA activity have been developed. The Pompe Disease Diagnostic Working Group met to review data generated using the new methods, and to establish a consensus regarding the application of the methods for the laboratory diagnosis of Pompe disease. Skin fibroblasts and muscle biopsy have traditionally been the samples of choice for measuring GAA activity. However, new methods using blood samples are rapidly becoming adopted because of their speed and convenience. Measuring GAA activity in blood samples should be performed under acidic conditions (pH 3.8-4.0), using up to 2 mM of the synthetic substrate 4-methylumbelliferyl-alpha-D-glucoside or glycogen (50 mg/mL), in the presence of acarbose (3-9 microM) to inhibit the isoenzyme maltase-glucoamylase. The activity of a reference enzyme should also be measured to confirm the quality of the sample. A second test should be done to support the diagnosis of Pompe disease until a program for external quality assurance and proficiency testing of the enzymatic diagnosis in blood is established.

Tandem Mass Spectrometry of Sphingolipids: Applications for Diagnosis of Sphingolipidoses.

In recent years, mass spectrometry (MS) has become the dominant technology in lipidomic analysis. It is widely used in diagnosis and research of lipid metabolism disorders including those characterized by impairment of lysosomal functions and storage of nondegraded-degraded substrates. These rare diseases, which include sphingolipidoses, have severe and often fatal clinical consequences. Modern MS methods have contributed significantly to achieve a definitive diagnosis, which is essential in clinical practice to begin properly targeted patient care. Here we summarize MS and tandem MS methods used for qualitative and quantitative analysis of sphingolipids (SL) relative to the diagnostic process for sphingolipidoses and studies focusing on alterations in cell functions due to these disorders. This review covers the following topics: Tandem MS is sensitive and robust in determining the composition of sphingolipid classes in various biological materials. Its ability to establish SL metabolomic profiles using MS bench-top analyzers, significantly benefits the first stages of a diagnosis as well as metabolic studies of these disorders. It can thus contribute to a better understanding of the biological significance of SL.

Blood group B glycosphingolipids in alpha-galactosidase deficiency (Fabry disease): influence of secretor status.

Defect in degradation of blood group B-immunoactive glycosphingolipids in Fabry disease (deficiency of lysosomal alpha-galactosidase EC 3.2.1.22) has been studied using highly sensitive and specific TLC-immunostaining analysis of urinary sediments and tonsillar tissues of blood group B patients and healthy controls, secretors and nonsecretors. The B glycolipid antigens with hexasaccharide chains were consistently found increased (25- to 100-fold) in the urinary sediments of three Fabry patients, blood group B or AB secretors. Conversely, they were absent in the urinary sediment of one blood group B nonsecretor patient. In normal secretors, B glycosphingolipids were present only in traces. Moreover, significant increase in B glycolipid antigens (8-fold) was found in the tonsillar tissue of a Fabry patient blood group B secretor. We conclude that the secretor status is responsible for increased concentration of blood group B glycosphingolipids in both urinary cells and tonsils in alpha-galactosidase deficiency. The quantity of stored B-immunoactive glycosphingolipids, however, is much lower than that of the mainly accumulated glycosphingolipid Gb(3)Cer. The results clearly indicate that active or silent Se gene, which controls synthesis of B-antigen precursors, is responsible for notable difference in B-glycosphingolipids expression in Fabry patients - secretors and nonsecretors. Whether this novel aspect may be of prognostic significance, remains to be established.

Lactosylceramide in lysosomal storage disorders: a comparative immunohistochemical and biochemical study.

Immunohistochemical studies of the presence of lactosylceramide (LacCer) in lysosomal storage disorders (LSDs) were done using anti-LacCer monoclonal antibody of the CDw 17 type (clone MG-2). No sign of an association between LacCer and the lysosomal system in normal cells was observed, except for histiocytes active in phagocytosis. A comparative study of a group of LSDs showed a general tendency for LacCer to increase in storage cells in Niemann-Pick disease type C (NPC), and types A and B, GM1 gangliosidosis, acid lipase deficiency, glycogen storage disease type II and mucopolysaccharidoses. LacCer accumulated in storage cells despite normal activity of relevant lysosomal degrading enzymes. The accumulation of LacCer displayed variability within storage cell populations, and was mostly expressed in neurons in NPC. An absence of the increase in LacCer in storage cells above control levels was seen in neuronal ceroid lipofuscinoses (neurons and cardiocytes) and in Fabry disease. Gaucher and Krabbe cells showed significantly lower levels, or even the absence, of LacCer compared with control macrophages. Results of immunohistochemistry were corroborated by semiquantitative lipid thin-layer chromatography (TLC). It is suggested that different associations of LacCer with the lysosomal storage process may reflect differences in glycosphingolipid turnover induced by the storage-compromised lysosomal/endosomal system.

Testis - a novel storage site in human cholesteryl ester storage disease. Autopsy report of an adult case with a long-standing subclinical course complicated by accelerated atherosclerosis and liver carcinoma.

A case of long-standing subclinical cholesteryl ester storage disease (CESD) manifesting as hyperlipoproteinaemia type IIb without any hepatomegaly is described. The patient underwent surgical vascular interventions because of accelerated atherosclerosis, which dominated his middle age. CESD was an incidental finding when a liver biopsy specimen was taken because liver malignancy was suspected; the patient's condition proved to be due to a cholangiocarcinoma, which led to his death at the of age 52. The autopsy showed moderate-intensity storage in the set of cells characterized by constitutional high-level receptor-mediated LDL endocytosis (hepatocytes, adrenal cortical cells) and also revealed storage in the Leydig cells. The severity with which histiocytes were affected varied regionally, ranging from minimal detectable storage or none at all (gut, lymph nodes, spleen) to extreme lysosomal expansion by cholesteryl ester liquid crystals (bone marrow) or by ceroid (lung, testicular stroma), or by both (liver). The density of the histiocytic population did not correlate with the degree to which parenchymal cells were affected except in the testicular stroma, where it was prominent. The patient was a mixed heterozygote for the G934A and DeltaC(673-5) mutations.

Pulmonary storage with emphysema as a sign of Niemann-Pick type C2 disease (second complementation group). Report of a case.

A case is described of Niemann-Pick type C2 disease presenting an infantile pneumopathic phenotype known to occur in this recently established, second, minor complementation group of Niemann-Pick type C (NPC) disease. However, the pulmonary involvement was unique, being dominated, in addition to the usual storage macrophage infiltration of the alveolar and septal compartments, by irregular emphysema attributed to storage cell migration into the bronchiolar lumen. The latter modified considerably the X-ray findings and hindered the initial clinical diagnosis. Otherwise, the storage phenotype, including the range of stored lipids, storage distribution, and cell and organ pathology, was found to be identical to that in the whole Niemann-Pick type C disease group dominated by NPC1. The biochemical findings (cholesterol esterification level) corresponded to the classical biochemical phenotype. Emphysema should thus be considered as a variant of the pulmonary NPC2 storage process, governed most probably by an epigenetic mechanism responsible for storage macrophage migration into the bronchiolar compartment.

A case of type I Gaucher disease with cardiopulmonary amyloidosis and chitotriosidase deficiency.

Severe cardiopulmonary amyloidosis developed several months after a total splenectomy in a patient with type 1 Gaucher disease and led within a year to his death at 48 years of age. The autopsy findings were dominated by extensive pulmonary and cardiac amyloid infiltration. No Gaucher cells were found in the lungs. Aside from a glucocerebrosidase deficiency the patient was also deficient in chitotriosidase, an enzyme whose activity is usually greatly increased in the serum of Gaucher patients. Analysis of mutations in the glucocerebrosidase gene revealed heterozygosity for N370S and D409H mutations. The normal amount of glucocerebrosidase was found in the spleen by Western blotting. We suggest that amyloidosis should be considered in the differential diagnosis of severe cardiopulmonary disease in Gaucher patients.

Concentrations of polychlorinated biphenyls and chlorinated pesticides in human breast milk--a case study.

Concentrations of chlorinated pesticides (p,p'-DDE, lindane), hexachlorobenzene (HCB), sum of polychlorinated biphenyls (PCBs), and PCB congeners in breast milk during lactation and the distribution of the chlorinated compounds in fat tissue, blood serum and breast milk were pursued in five primi- or secundipara from the hinterland of a clinic of gynaecology and obstetrics in Brno in 1993. Capillary gas chromatography with ECD detection was used for the determination of the residues. The concentrations of HCB, lindane, p,p'-DDe, and sum of PCBs ranged from 42.5 to 238.4, from < 1.0 to 7.4 from 231.4 to 557.6, and from 661.3 to 2888.9 micrograms.kg-1 of milk fat, respectively. Seventeen PCB congeners were screened, of which 11 were identified in most of the milk samples (IUPAC numbers 28, 101, 118, 126, 128, 138, 153, 156, 170, 180, 194). Congeners 138, 153, 170, and 180 were the most abundant and their concentrations in microgram.kg-1 of milk fat ranged from 122.9 to 501.0 for 153, from 103.4 to 372.1 for 138, from 66.1 to 407.9 for 180, from 26.8 to 183.1 for 170, and from 10.0 to 41.7 for 118.

[Lysosomal sphingomyelinase deficiency: spectrum of phenotypes in Czech and Slovak patients]. / Deficit lyzozomální sfingomyelinázy: spektrum fenotypu u ceských a slovenských pacientu.

BACKGROUND: We present a series of 25 patients (from 21 families) with deficiency of lysosomal sphingomyelinase (acid sphingomyelinase, ASM), diagnosed during the last 30 years. METHODS AND RESULTS: Diagnosis was established by finding specific sphingomyelin storage pattern in bone marrow histiocytes and in some bioptical and postmortem tissues (presence of sphingomyelin liquid crystals) and finally by proving ASM deficiency in white blood cells and in cultured fibroblasts. Range of clinical manifestations of our patients notably exceeded the the known main (A,B) phenotypes described so far. In the group of type A patients (clinically overt neurovisceral symptomatology) there was significant tendency to prolonged course. Classical fulminant course with death between 5 to 45 months of age was seen only in a subgroup of 5 patients. In other type A patients (n = 8) the course was prolonged attaining 5 years of age, the end of the first (8, and 9 years), second (14, 17 years), third (22 years), fourth (32 years) and fifth decades (41 years). Three of these patients (aged 5, 22 and 41 years) are living. The series of patients with dominant visceral involvement (n = 12) consisted of three phenotypically different subgroups. One with chronic purely visceral affection and prolonged course (n = 4) corresponding to the classical type B, the second with chronic course and largely subclinical neurological affection (n = 4) and the third with accelerated fatal visceral affection (death in age range 31 months-9 years) without (n = 1) or with clinically minor signs of brain damage (n = 3). CONCLUSIONS: Study of the presented series of ASM deficient patients disclosed remarkable phenotypical variability. Two main factors seem to be responsible. Variability in the storage intensity in the two main tissue compartments (neuronal and visceral), and the absence of proportionality in their affection in some instances. The described phenotype variability enlarges significantly the known spectrum of phenotypes in ASM deficiency.

[Mucolipidosis II (I cell disease). First case report in the Czech Republic and prenatal diagnosis in a family]. / Mukolipidóza II (I cell disease). Popis prvého prípadu v Ceské republice a prenatální diagnóza v rodine.

The authors describe the first case of mucolipidosis II in the Czech Republic. The cause of this autosomal recessive hereditary disease is deficient synthesis of mannoso-6-phosphate ligand on precursors of lysosomal enzymes which normally make their transport into the lysosomal system possible. The diagnosis was proved by the presence of typical lysosomal cumulation in bioptic specimens and extremely elevated activity of lysosomal enzymes in the patient's serum caused by their non-regulated secretion and subsequent intracellular depletion. During the second pregnancy in the family prenatal diagnosis was made. A normal range of lysosomal enzyme activities in the supernatant of the amniotic fluid and in cultivated chorionic villi along with normal results of ultrastructural examination of the chorionic villus indicated the development of an intact foetus.

[Prenatal diagnosis of lysosomal enzymopathies in the Czech Republic]. / Prenatální diagnostika lysozomálních enzymopatií v Ceské Republice.

BACKGROUND: Prenatal diagnosisi represents the important fprm of prevention of the inherited metabolic diseases and its accessibility becomes the most effective assistance to involved families. The aim of the study was to introduce prenatal diagnosis of major inherited lysosomal disorders of the group of lipidoses, micopolysaccharidoses, glycoproteinoses, and mucolipidoses. METHODS AND RESULTS: Methodological approach is based on the activity estimation of the specific lysosomal hydrolases that are missing or inactive. Methods were extended by a set of supportive analyses, namely by ultrastructural identification of the lysosomal storage of the non-degraded substrate, DNA analysis showing mutation in the family or by biochemical analysis of the amniotic fluid. Uncultured cultured chorionic villi, cultured amniotic fluid cells yand samples of the amniotic fluid were examined. Altogether 17 pregnancies at risk for seven different lysosomal enzymopathies were followed: GM2 gangliosidosis (2 cases), Fabra disease (3 cases), Krabbe disease (1 case), Niemann-Pick disease type A (1 case), mucopolysaccharidosis I (5 cases), mucopolysaccharidosis II (4 cases), mucolipidosis II (I-cell disease) (1 case). Profound deficiency of enzyme activities (alpha-galactosidase A in fabry disease, galactocerebrosidase in Krabbe disease, alpha-iduronidase in mucopolysaccharidosis I) was identified in three pregnancies, which were terminated on the mother's decision. The diagnose was confirmed by the biochemical analysis of tissues of aborted foetuses. In two of them (Fabry disease, mucopolysaccharidosis I) ultrastructural sings of storage werw proved. In two cases the foetal heterozygote state was identified. In case at risk for Niemann-Pick disease type A, the diagnosis was confirmed also by DNA analysis. In the pregnancy at risk for Fabry disease, heterozygous state was confirmed indirectly according to the difference of alpha-galactosidase activities in cultured and uncultured cells. A set control values of enzyme activities in individual types of processed material (native and cultured chorionic villi, cultured amniocytes, and amniotic fluid supernatant) has been established. CONCLUSIONS: Inherited lysosomal enzymopathies represent important indication for prenatal diagnosis available now in our department. Condicio sine qua non is the biochemical or molecular genetic confirmation of diagnosis in the family involved.

[Postmortem diagnosis of Fabry disease in a female heterozygote leading to the detection of undiagnosed manifest disease in the family]. / Pitevní diagnóza Fabryho nemoci u heterozygotky, vedoucí k rozpoznání nediagnostikované manifestní nemoci v rodine.

The authors detected on necropsy in a 63-year-old woman with the clinical diagnosis of hypertension, atherosclerosis of the coronary and peripheral arteries, thromboembolism into the cerebral circulation and impaired cardiac conductivity lysosomal storage identified by histochemical and electronoptic analyses along with lipid chromatography as Fabry's disease. The stored lipids were neutral glycosphingolipids of the globo series globotriaosylceramide) and of the gala- series (galabiosylceramide) which accumulated as a result of deficient activity of the degrading enzyme alpha galactosidase A. Marked accumulation of these specific lipids was found in cardiomyocytes, in smooth muscles (of the media in arteries of the heart, kidneys, liver, spleen, lungs) in podocytes and mesangial cells of renal glomeruli, in epithelia of Henle's loop and in the distal tubules. In the vascular endothelium the storage was at the borderline of detectability. Accumulation did not lead to detectable organ disorders with the exception of the heart where it participated, no doubt, significantly in the cardiocyte hypertrophy. Examination of relatives revealed in the proband's son (age 41 years) a combination of renal, cardiac and skin changes typical for Fabry's disease which, however was not clinically diagnosed. The diagnosis was confirmed by proving of alpha-galactosidase A deficiency in the peripheral leucocytes and point mutation L293X in the VIth exon of the appropriate gene. In a granddaughter (age 15 years) biochemical and molecular genetic methods revealed the heterozygous state of Fabry's disease in preclinical stage.

[Enzyme therapy in children with severe forms of Gaucher's disease]. / Enzymová terapie u detí s tezkou formou Gaucherovy nemoci.

The enzyme therapy with Ceredase in patients with Gaucher's disease is at present probably the most expensive treatment in the whole world. One-year treatment of an adult patient with Gaucher's disease costs more than 7 million crowns. Indications for treatment in individual patients as well as financial provisions are so far problematic in the Czech Republic. From a total of 28 patients of varying age with Gaucher's disease diagnosed by the authors Ceredase was administered to two boys with a severe course of the disease. Within one year of treatment the health status of both children improved, growth became normal, the spleen diminished in size by 20-35%, haematological manifestations of hypersplenism are receding, there was a 32-46% decline of the activity of serum chitotriosidase and biochemical parameters of the disease improved.

[Lysosomal acid lipase deficiency. Overview of Czech patients]. / Deficit kyselé (lysozomální) lipázy. Prehled ceských pacientu.

Lysosomal lipase deficiency is a hereditary autosomal recessive enzymopathy leading to lysosomal storage of triacylglycerols (TAG) and cholesterol esters (CE). In particular cells with a permanently high receptor-mediated LDL endocytosis are affected (liver, kidneys). There are two basic phenotypes. The fatal infantile phenotype (Wolman's disease) with generalized storage of both types of apolar lipids. This form was diagnosed in this country only once. The opposite is the protracted, oligosymptomatic form encountered in all age groups. It is characterized by the storage of CE (which gave this entity the name of cholesteryl storage disease--CESD). Its main sign is affection of the liver (hepatomegaly, hepatopathy), which in some instances may lead to organ failure, directly or after cirrhotic transformation. Furthermore there is permanent hypercholesterolaemia (high LDL cholesterol) due to increased VLDL synthesis by hepatocytes, low HDL cholesterol and variably raised TAG. This constellation of blood lipids is a risk factor for the development of atherosclerosis. In the course of 25 years in the Czech Republic 13 cases of CESD were diagnosed in 11 families. Ten of these cases were characterized by clinically manifest hepatopathy with hepatomegaly, detected incidentally during medical examinations (at the age of 2-14 years). In three adult patients with permanent hypercholesterolaemia the storage process was subclinical and the diagnosis was established quite incidentally by examination of non-specific secondary and tertiary manifestations of the disease. The diagnosis was established in all cases of CESD at the tissue level (liver biopsy), at the biochemical (acid lipase deficiency) and molecular genetic level (mutation in enzyme locus). In all instances mutation of G934A was found leading to reduction and loss of the eighth exon. This mutation was present in five patients in a homozygous state. Six mutations were heterozygous. In one instance for technical reasons only one allele was analyzed. In three instances a point "missense" mutation was found: T323A (Trp74Arg), T4(75)A (Asp124Glu), A210T (Asp36Gl), in one instance a "nonsense" mutation: C233T (Arg44-stop) and twice a deletion mutation delta C673-5 and delta G1068-8 leading to impairment of the reading frame and to premature stop of the codon.

[Diagnosis of G(M2) gangliosidosis in routine practice]. / Diagnostika gangliosidosy GM2 v rutinní praxi.

Diagnosis of GM2 gangliosidosis and other most frequent thesaurismoses of the central nervous system was exposed on a case of a 2-year-old boy. Diagnostic process which exploited routine methods commonly used in every department of pathology enabled to choose chemical and enzymological investigation of brain tissue.