A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases.

Sialic acid storage diseases (SASD, MIM 269920) are autosomal recessive neurodegenerative disorders that may present as a severe infantile form (ISSD) or a slowly progressive adult form, which is prevalent in Finland (Salla disease). The main symptoms are hypotonia, cerebellar ataxia and mental retardation; visceromegaly and coarse features are also present in infantile cases. Progressive cerebellar atrophy and dysmyelination have been documented by magnetic resonance imaging (ref. 4). Enlarged lysosomes are seen on electron microscopic studies and patients excrete large amounts of free sialic acid in urine. A H+/anionic sugar symporter mechanism for sialic acid and glucuronic acid is impaired in lysosomal membranes from Salla and ISSD patients. The locus for Salla disease was assigned to a region of approximately 200 kb on chromosome 6q14-q15 in a linkage study using Finnish families. Salla disease and ISSD were further shown to be allelic disorders. A physical map with P1 and PAC clones was constructed to cover the 200-kb area flanked by the loci D6S280 and D6S1622, providing the basis for precise physical positioning of the gene. Here we describe a new gene, SLC17A5 (also known as AST), encoding a protein (sialin) with a predicted transport function that belongs to a family of anion/cation symporters (ACS). We found a homozygous SLC17A5 mutation (R39C) in five Finnish patients with Salla disease and six different SLC17A5 mutations in six ISSD patients of different ethnic origins. Our observations suggest that mutations in SLC17A5 are the primary cause of lysosomal sialic acid storage diseases.

Clinical diversity in glycogenosis type II. Biosynthesis and in situ localization of acid alpha-glucosidase in mutant fibroblasts.

The molecular basis of clinical diversity in glycogenosis type II (Pompe's disease) was investigated by comparing the nature of acid alpha-glucosidase deficiency in cultured fibroblasts from 30 patients. Biosynthetic forms of acid alpha-glucosidase with different molecular mass were separated electrophoretically and identified by immunoblotting. Immuno-electron microscopy was employed to determine the intracellular localization of mutant enzyme. Our studies illustrate that maturation of acid alpha-glucosidase is associated with transport to the lysosomes. Deficiency of catalytically active mature enzyme in lysosomes is common to all clinical phenotypes but, in the majority of cases, is more profound in early onset than in late onset forms of the disease. Thus, the results suggest that the clinical course of glycogenosis type II is primarily determined by the amount of functional acid alpha-glucosidase. The role of secondary factors can, however, not be excluded because three adult patients were identified with very low activity and little enzyme in the lysosomes.

Isolation and structural characterization of sialic acid-containing storage material from mucolipidosis I (sialidosis) fibroblasts.

Sialic acid-containing storage material was isolated from cultured human mucolipidosis I (sialidosis) fibroblasts by gel permeation chromatography on Bio-Gel P-6 followed by medium-pressure anion-exchange chromatography on Mono Q. The structure determination of the isolated sialyloligosaccharides was carried out by 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis and analytical HPLC. The storage material showed completely sialylated mono-, di- and triantennary N-glycosidic N-acetyllactosamine oligosaccharides having the Man beta 1----4GlcNAc sequence at the reducing end in common. Heterogeneity occurred with respect to the linkages between terminal sialic acid and the penultimate galactose residues (alpha 2----3/alpha 2----6). It turned out that all the identified carbohydrate chains are consistent with the neuraminidase deficiency.

Determination of the structure of the carbohydrate chains of acid alpha-glucosidase from human placenta.

Acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz 1H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man5GlcNAcGlcNAc-ol and Man7GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man2-3GlcNAc[Fuc]0-1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.

Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts.

This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts.

Immunoelectron microscopical localization of lysosomal beta-galactosidase and its precursor forms in normal and mutant human fibroblasts.

Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In the lysosomes virtually all beta-gal exists as a high molecular weight multimer of mature enzyme. In the autosomal recessive disease GM1-gangliosidosis caused by a beta-gal deficiency and in galactosialidosis, associated with a combined deficiency of lysosomal neuraminidase and beta-gal, precursor forms of the latter enzyme are found in RER, Golgi and some labeling is present at the cell surface. The lysosomes remain unlabeled, indicative for the absence of enzyme molecules in this organelle. In galactosialidosis fibroblasts also no mature beta-gal is found in the lysosomes but in these cells the presence of the monomeric form can be increased by leupeptin (inhibition of proteolysis) whereas addition of a partly purified 32 kDa "protective protein" results in the restoration of high molecular weight beta-gal multimers in the lysosomes.

Methods for analysis of acid alpha-1,4-glucosidase activity in single hybrid cells.

Two methods are described which allow the quantitative assay of the lysosomal enzyme alpha-1,4-glucosidase in single fibroblasts. In the first procedure the substrate was maltose, and liberated glucose was measured with an enzymatic cycling procedure for reduced nicotinamide adenine dinucleotide phosphate. Single cultured fibroblasts were found to have enzyme activities in the range of 0.5-10 X 10(-13) moles glucose/hr. In the second procedure the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyransodie was used. It is hydrolyzed in a single step reaction to the fluorescent product 4-methylumbelliferone (MU). By reducing the incubation volume and by measuring the fluorescence in microdroplets with a microscope fluorometer, a sensitivity of 10(-14) moles MU could be obtained. Activities were found ranging from 0.5-10 X 10(-14) moles MU/hr/cell. Both procedures for single cell analysis proved to be reliable when compared with conventional assays on cell homogenates. Cocultivation and cell fusion studies were performed to demonstrate that these methods can be used to study the metabolic and genetic interaction between normal and enzyme-deficient fibroblasts derived from patients with glycogenosis II.

A fluorescence staining method for the demonstration and measurement of lysosomal enzyme activities in single cells.

A cytochemical fluorescence method is described that makes possible simple, rapid, and specific demonstration and measurement of the activities of a wide variety of lysosomal enzymes in single cells using 4-methylumbelliferyl derivatives as substrates. The validity of the method and a number of applications using normal and mutant human cells are presented.

Impact of first-trimester chromosome, DNA, and metabolic studies on pregnancies at high genetic risk: experience with 1,000 cases.

We describe the results and follow-up of chorionic villus studies in 1,034 pregnancies at risk for chromosome or metabolic disorders. Direct chromosome studies were successful in 99.7% and yielded results within a few days. Fifty pregnancies at risk for an unbalanced translocation, inherited from parents with many small reciprocal translocations, were a good test for the quality of the direct method. The 101 metabolic studies involving 28 disorders were correct in 99 pregnancies in the first trimester. In two cases a correct diagnosis was obtained by the confirmatory amniocentesis. DNA studies were carried out in pregnancies of male fetuses at risk for Duchenne muscular dystrophy and a few metabolic disorders. The abortion rate after chorionic villus sampling was 5.1% but more than half of the pregnant women were greater than or equal to 36 years old and have a spontaneous abortion rate of 10% between the 10th and 14th week according to Gustavii [Lancet 1:562, 1984]. Follow-up studies confirmed results of all chromosome studies after termination when there was fetal cell growth; the outcome of 504 consecutive continuing pregnancies showed no discrepancies of the phenotype after birth. It was concluded that first-trimester chorionic villi studies gave reliable results and were increasingly preferred by the patients, while the sampling can be considered a safe procedure based on the currently available data.

Factors influencing the reproductive decision after genetic counseling.

Here we report a follow-up study involving interviews with 164 couples 2-3 years after genetic counseling to assess the influence of various factors on their reproductive planning. The results show that the desire to have children and the absence of personal experience with the disorder (no close relative being affected) are important single factors for the decision to opt for having children after genetic counseling. The magnitude of the genetic risk is of relative importance in reproductive planning. Seventy percent of the couples with a high genetic risk (greater than 15%) opted for having children. When the disorder was perceived as severe and the risk was interpreted as high, 72% opted for having children. The availability of prenatal diagnosis became important only in combination with a high genetic risk (greater than 15%). Forty-seven percent of the couples with a high genetic risk refrained from having children when prenatal diagnosis was not available. In the absence of prenatal diagnosis, couples who had an affected child were more cautious about trying again than those who did not--50% versus 14% decided to abstain. This study has provided some insight into the complexity of reproductive decision-making after genetic counseling. The findings may help genetic counselors and clinical geneticists understand and support counselees in their decision-making process, which is "multi-factorial."

A comparative study of the accumulated sialic acid-containing oligosaccharides from cultured human galactosialidosis and sialidosis fibroblasts.

Sialic acid-containing storage material was isolated from cultured human galactosialidosis fibroblasts, by a combination of gel filtration and anion-exchange chromatography on Mono Q. The obtained sialyloligosaccharides were analyzed by 500-MHz 1H-NMR spectroscopy in combination with sugar analysis and analytical HPLC. The storage material consisted of a series of completely sialylated N-acetyl-lactosamine type of structures having Man beta 1-4GlcNAc at the reducing terminus in common, similar to those recently reported for human sialidosis fibroblasts. Comparison of the storage material from both sources revealed only differences in their relative amounts. In control fibroblasts these compounds could not be detected. The nature of the accumulated compounds is in accordance with the alpha-neuraminidase deficiency in both genetic diseases. The additional deficiency of beta-galactosidase in case of galactosialidosis is not reflected in the storage material.

Determination of Gaucher's disease phenotypes with monoclonal antibody.

Discrimination between the three clinical subtypes of Gaucher's disease based on the molecular forms of beta-glucocerebrosidase detected by monoclonal antibody is described. In normal fibroblast extracts, cross-reacting material (CRM) to human placental glucocerebrosidase is detected at Mr approximately equal to 63 000, 61 000 and 56 000. In Type 1 Gaucher's disease, the major fibroblast CRM has a Mr approximately equal to 56 000,, with less CRM seen at 61 000 and 56 000. Type 3 fibroblast extracts have a single CRM form at Mr approximately equal to 63 000. No CRM is found in Type 2 Gaucher's disease fibroblasts with monoclonal antiglucocerebrosidase antibody 8E4.

A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A).

4-Methylumbelliferyl-beta-D-galactopyranoside-6-sulphate was synthesized and used for the determination of galactose-6-sulphate sulphatase activity. Fibroblasts and leucocytes from 12 different Morquio A patients, showed 0.0-2.7% of mean normal galactose-6-sulphate sulphatase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-galactopyranoside-6-sulphate requires the sequential action of galactose-6-sulphate sulphatase and beta-galactosidase. Normal beta-galactosidase activity caused nearly complete hydrolysis of non-fluorescing 4-methylumbelliferyl-galactoside, formed during incubation. In cell extracts with a beta-galactosidase deficiency however, a second incubation in the presence of excess beta-galactosidase is needed to avoid underestimation of galactose-6-sulphate sulphatase activity.

Adult type GMl-gangliosidosis: a complementation study on somatic cell hybrids.

Two adult siblings with progressive pyramidal and extrapyramidal lesions, and generalized muscle atrophy had a profound deficiency of beta-galactosidase in all the cells and body fluids examined. Neuraminidase activity was normal in fibroblasts. The fused fibroblasts of infantile GMl-gangliosidosis and each of these adult patients had beta-galactosidase activity as expected for the average value in a mixture of equal numbers of parental cells. However, there was a remarkable increase in the activity of beta-galactosidase when the cells from each of these cases were fused with those from the beta-galactosidase-deficient adult with cherry-red spots, cerebellar ataxia, myoclonus and neuraminidase deficiency in fibroblasts. It was concluded that the two siblings represent a new genetic variant (adult type) of GMl-gangliosidosis.

[The stability of freeze-drying lysosomal enzymes].

OBJECTIVE: Enzyme assays for the diagnosis of lysosomal storage diseases have been underway in China only in a few laboratories, and the specimens for enzyme assay must not be inactivated by environmental factors. To solve the problem, the stability of freeze-drying lysosomal enzymes was tested. METHODS: Three pools for samples were set up: control leucocytes pool and plasma pool(40 individuals in each pool), control fibroblasts pool( 3 cell lines). 4 groups of different dispositions of samples were designed for the pools:(1)pellets or plasma directly stored at -80 degrees C as routine(non-freeze-drying group or N);(2)after freeze-drying, pellets or plasma immediately stored at -80 degrees C (0 week or 0W); (3) after freeze-drying, pellets or plasma stayed at room temperature for 1 week(simulated mail) then stored at -80 degrees C (1 week or 1W);(4) the same with 1W but 3 weeks at room temperature(3 weeks or 3W). Microassay for enzyme activity was employed: 20 enzymes in leucocytes pool, 14 enzymes in fibroblasts pool and 7 in plasma pool. RESULTS: Enzyme activities among 4 groups in each sample pool were compared. Single factor analysis of variance (F test) showed no significance of differences (P>0.05) among 4 groups in leucocytes and fibroblasts pool, but highly significant differences (P<0.01) among 4 groups in plasma pool. "t test" showed no significance of differences (P> 0.05) between N and 3W in leucocytes and also fibroblasts pool, but very significant differences (P<0.01) between N and 3W in plasma pool. CONCLUSION: The results suggest that freeze-drying samples from leucocytes and fibro- blasts can provide a stable resource for enzyme assay and be simple for transportation of the diagnostic samples. Though freeze-drying is not appropriate for the plasma samples, it is still applicable for wide use since only a few kinds of lysosomal enzyme being indicated to use plasma for enzyme assay.

[Clinical genetics in The Netherlands. I. Organization, activities and laboratory diagnosis]. / De klinische genetica in Nederland. I. Organisatie, activiteiten en laboratoriumdiagnostiek.

There are seven centres for clinical genetics in the Netherlands. In 1996, some 63,000 persons (patients and possible carriers of hereditary diseases) were tested. In centres for clinical genetics chromosomal studies, biochemical diagnostics of hereditary metabolic diseases and DNA diagnostics are integrated with genetic counseling and prenatal diagnosis. The borders between the three different forms of laboratory testing for congenital anomalies and hereditary diseases gradually diminish. The variations of the numbers of laboratory examinations, genetic advices and prenatal diagnoses over the last ten years show that there is no correlation between these activities and the method of funding. Owing to the low prevalence of the diseases involved, the total number of DNA diagnoses for monogenic diseases will not increase significantly. However, once genetic risk factors of diseases such as cancer, cardiovascular diseases, diabetes, asthma, rheumatism, some psychiatric disorders and Alzheimer dementia will have been mapped, DNA diagnostics will greatly expand and will have implications in a broad area of medicine.

[Clinical genetics in The Netherlands. II. Genetic counseling and prenatal diagnosis]. / De klinische genetica in Nederland. II. Erfelijkheidsadvisering en prenatale diagnostiek.

The main feature of clinical genetics is the involvement of close relatives in the diagnostics of a hereditary disorder, and the possible consequences of the findings for future generations. Complex genetic counseling is required in cases with different, possibly hereditary disorders or congenital anomalies in the family or by a syndrome with variable risks of recurrence, depending on the exact nature of the disorder; also the difficult, often emotionally charged choices with which counselees are faced demands the expertise of a clinical genetic centre. Results of follow-up studies after genetic counseling show that experience with a handicap or disease in the own environment and the presence of healthy issue are the main determinants for the decision about reproduction of persons with an enhanced genetic risk who request counseling. Because of the great variety in perception of risks and of the severity of a disorder, and because of the marked clinical heterogeneity, rigid legislation should be avoided in the field of prenatal diagnosis. In the future, the training of the clinical geneticist has to be adapted to the rapid progress in human genetics. Increasingly, the clinical geneticist will function in collaboration with other disciplines such as oncology, obstetrics and gynaecology, paediatrics and neurology; in connection with family testing and counseling, there will also be more collaboration with primary health care.

[Future developments in genetic research. I. Technological possibilities]. / Toekomstige ontwikkelingen in het erfelijkheidsonderzoek. I. Technologische mogelijkheden.

The attention in genetic research is shifting from the determination' of (rare) monogenic disorders to identification of genetic risk factors for important diseases at adult age. Mapping of all man's 80,000-100,000 genes will also provide more insight into the gene polymorphisms and mutations that are associated with various types of cancer, certain cardiovascular diseases, diabetes and neurodegenerative disorders, including Alzheimer dementia. Apart from new diagnostic possibilities, the DNA techniques create new prospects for the study of the pathogenesis of diseases and the devising of new strategies for treatment. Examples are familial hypercholesterolaemia, diabetes, breast cancer and colorectal carcinoma.

[Future developments in genetic research. II. Psychological and social aspects]. / Toekomstige ontwikkelingen in het erfelijkheidsonderzoek. II. Psychologische en maatschappelijke aspecten.

Regarding the determination of genetic risk factors for serious diseases, the main question for a patient's young relatives is 'to know or not to know'. The answer depends among other things on the assessment of the severity of the disorder and the magnitude of the risk in relation to the population risk and on the availability of therapeutic or preventive measures. Experience with the requests for and the coping with results of the presymptomatic DNA test for untreatable neurodegenerative chorea of Huntington show that expectations about the effect of predictive DNA studies are often different from reality. A problem of a different nature arises in multifactorial diseases such as mammary or colonic carcinoma, because irrespective of the DNA study it remains uncertain whether or not the disease will occur. Nevertheless more than half of the healthy relatives of a patient with breast cancer requests DNA testing and a large majority of the proven carriers of a BCRAI and BRCA2 mutation chose for bilateral mastectomy and oophorectomy. Many questions about psychosocial consequences of predictive DNA testing remain to be answered: what will be the effect of early medicalization, how will relations be affected and which effect will carriership of genetic risk factors have on behaviour? Dutch legislation provides adequate guarantees against the use by third parties of results of genetic testing. The question remains, however, how future internationalization will work out in this respect. The possibly far-reaching social and psychological consequences of genetic research make some people feel that restraints should be imposed on this research. However, there are more grounds for curiosity and enthusiasm to constantly find new solutions for the new problems.

[Current trends in medical genetics]. / Soucasné trendy lekarské genetiky.

Medical genetics made during the last decades immense advances and new findings from the sphere of fundamental research are increasingly penetrating into different clinical disciplines. The greatest attention was concentrated on investigations devoted to the molecular etiology of malignant diseases and the molecular mechanism of embryonic development. Mapping of the human genome is the prerequisite for identification of serious diseases at the gene level. Control of these problems opens possibilities of aimed diagnostics at the molecular level during the postnatal period and as well as the prenatal one. So far we possess knowledge required for genetic counselling and possible prevention. With the extension of our knowledge on gene mechanisms we find that originally apparently clear mechanisms of heredity are much more complicated. In the last three years also the possibility arose of gene therapy of some malignomas and other pathological conditions of the organism. Hitherto assembled experience is relatively optimistic although a major development of these techniques is foreseen only at the end of this millenium. Medical genetics advanced greatly in the prenatal diagnosis of inborn developmental and hereditary foetal diseases. Gradually different screening programmes were introduced to evaluate the status of the foetus in utero. At present also predictive tests are available for some serious diseases. All these methods and technologies are associated with many ethical, moral and psychosocial problems which must be gradually resolved.