et biophysica a~ta elsevier review lysosomal storage diseases · 2017-01-06 · elsevier biochimica...

ELSEVIER Biochimica et Biophysica Acta 1270 (1995) 103-136 et Biophysica A~ta

R e v i e w

Lysosomal storage diseases

Volkmar Giese lmann *

Department of Biochemistry II, Georg August Universitiit, Gi~ttingen, Germany

Received 31 August 1994; accepted 15 September 1994

Keywords: Lysosomal storage disease; Lysosomal enzyme

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

2. Lipidoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 2.1. Gu2 gangliosidoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Tay Sachs disease and G m gangliosidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Sandhoff disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 GM2 activator protein deficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

2.2. Gral gangliosidosis and morquio type B disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 2.3. Galactosialidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 2.4. Fabry disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 2.5. Niemann Pick disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 2.6. Metachromatic leukodystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 2.7. Gaucher disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 2.8. Sphingolipid activator protein deficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 2.9. Woiman disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 2.10. Krabbe disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

3. Disorders of glycoprotein degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 3.1. Fucosidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 3.2. Aspartylglucosaminufia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 3.3. Schindler disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

4. Mucopolysaccharidoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 4.1. Mucopolysacchafidosis type I/Hurler Scheie syndrome . . . . . . . . . . . . . . . . . . . . . . . . . 121 4.2. Mucopolysaccharidosis type H/Hunter syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 4.3. Mucopolysaccharidosis type IV/Morquio A syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . 122 4.4. Mucopolysaccharidosis type VI/Maroteaux Lamy syndrome . . . . . . . . . . . . . . . . . . . . . . 123 4.5. Mucopolysaceharidosis type VII /Sly syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 4.6. Glycogen storage diseases/Pompe disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 4.7. Pathogenesis of lysosomal storage diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 4.8. Therapy of lysosomal storage disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

* Corresponding author. Present address: Biochemisches Institut, Uni- versity of Kiel, OIshausenstraBe 40, 24098 Kiel, Germany. Fax: +49 431 880 2017.

0925-4439/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0925- 4 4 3 9 ( 9 4 ) 0 0 0 7 5 - 1

104 V. Gieselmann / Biochimica et Biophysica Acta 1270 (1995) 103-136

1. Introduction

Lysosomal storage disorders comprise a group of more than 30 different diseases. The common feature of these diseases is the deficiency of a lysosomal protein that is part of a catabolic pathway. The deficient proteins may be hydrolases or cofactors involved in the degradation of macromolecules or transporters, which deliver catabolic products to the cytosol. Most of lysosomal enzymes are exohydrolases acting in sequence, such that substrates are degraded by a stepwise removal of terminal residues. Thus, the deficiency of a single enzyme causes the block- age of the entire pathway, since the failure to remove a terminal residue makes the substrate inaccessible for further hydrolysis by other lysosomal enzymes. The lysosomal storage diseases can be classified depending on the pathway affected and the nature of the accumulated substrate. Ten different deficiencies can affect the degradation of mucopolysaccharides (mucopolysaccharidoses), five defects are known in the degradation pathways of glycoproteins, one for the intralysosomal storage of glycogen (glycogenesis) and eight deficiencies affect sphingolipid catabolism (lipidoses). However, there are frequent over- laps of storage material between those groups. Many lysosomal hydrolases are not specific for a particular compound, but rather for a terminal residue, which may be identical in, e.g., gangliosides and glycoproteins, so that both compounds accumulate when a commonly required enzyme is deficient. So far, no lysosomal storage disorders of nucleic acid or protein degradation have been described.

The synthesis and intracellular sorting of lysosomal enzymes have been reviewed in detail recently [1,2]. Solu- ble lysosomal enzymes follow a common intracellular route. A signal peptide mediates the cotranslational transport across the membrane into the lumen of the endoplas- mic reticulum (ER). In the lumen of the ER, the signal peptide is cleaved and the polypeptides cotranslationally receive N-linked oligosaccharide side chains. Subse- quently, lysosomal enzymes are transported to the Golgi apparatus where they are recognized by a phosphotransferase. This enzyme transfers N-acetylglucosamine (GlcNAc)-l-phosphate from UDP-GIcNAc to mannose residues of oligosaccharide side chains of lysosomal enzymes generating GlcNAc-l-phospho-6-mannose residues. In a second step, GlcNAc is removed by a N-acetylglucosamine- 1-phosphodiester-ot-N-acetylglucosaminidase leaving behind a mannose-6-phosphate residue. This residue binds to mannose-6-phosphate receptors, which mediate the further vesicular transport to the lysosomes. Two receptors of different molecular weight - 46 kDa and 300 kDa - are known, which have different binding properties and intracellular distribution [166]. Enzymes bind to receptors in a late Golgi compartment and dissoci- ate upon acidification in a prelysosomal compartment. The enzymes are delivered to the lysosomes while receptors

recycle. After arrival in the lysosome, most lysosomal enzymes undergo a limited proteolysis, which finally yields the mature enzymes. Although mannose-6-phosphate receptors play a major role in the intracellular transport of newly synthesized enzymes in fibroblasts, additional proteins may be required for targeting to lysosomes in other cell types. Thus, trafficking of lysosomal enzymes in hepatocytes, Kupffer cells and leucocytes is not severely impaired by a deficient phosphotransferase activity [325]. However, in many cells the critical step for the correct sorting of lysosomal enzymes is the recognition by the phosphotransferase. Since there are no apparent similarities among lysosomal enzymes it is still unknown what structure common to all lysosomal enzymes is recognized by this enzyme. The recognition domain is part of the protein backbone of the lysosomal enzymes and depends on their proper three dimensional folding [21]. In the lysosomal aspartylprotease cathepsin D two discontinuous sequences, each of which contributes to the phosphotransferase recognition domain, have been identified [3,4,305,306]. Other lysosomal enzymes like /3-o-galactocerebrosidase and /3- o-glucocerebrosidase are, though they are no transmembrane polypeptides, membrane associated and sorted independently of the mannose-6-phosphate receptor pathway [5], but details of their transport have not yet been elucidated. A third group are the transmembrane lysosomal proteins for which the classical lysosomal marker acid phosphatase is the best known example. This enzyme is synthesized as a transmembrane precursor and sequences in the cytoplasmic tail have been shown to be necessary and sufficient for lysosomal targeting [6]. In the lysosome the luminal domain is proteolyticaUy cleaved to yield the mature soluble acid phosphatase. Other lysosomal membrane proteins like LIMP and LAMP follow similar intracellular routes [7], but are not converted into soluble forms in the lysosome. Diseases due to mutations in these type of proteins have not been described and their function is not clearly understood.

Almost all lysosomal storage diseases show clinical variability. Most diseases vary with respect to the age at onset and progression of the symptoms. The later a disease begins the more protracted is the development of symptoms. Frequently, three different types of disease are distinguished, severe infantile, intermediate juvenile and mild adult forms, respectively. Although such a classification is useful, it should be realized that the spectrum of clinical severity is a continuum, and such imposed distinctions are artificial. In many lysosomal disorders symptoms in the early and late onset forms are similar, but in some diseases defects in the same gene may cause very different phenotypes. For example,/3-o-galactosidase deficiency can cause either GM1 gangliosidosis and Morquio type B disease. Whereas in GM1 gangliosidosis neurologic degeneration is the major symptom, patients with Morquio Type B disease have no neurologic involvement, but severe skeletal dys-

v. Gieselmann /Biochimica et Biophysica Acta 1270 (1995) 103-136 105

plasias. Similarly, in Gaucher disease depending on the involvement of the nervous system neuronopathic and non neuronopathic forms are distinguished.

During the last years much effort has been devoted to the cloning of genes of lysosomal enzymes and to the analysis of mutations [8]. A large amount of data has accumulated, so that this review will focus on the molecular biology of lysosomal storage diseases.

In the first part, I will review some of the molecular genetic data for each disease separately, whereas data on the pathogenesis and therapy of lysosomal storage diseases will be discussed in context in the second part. Since not all mutations can be reviewed in detail, I will focus on those which are frequent, important for the understanding of genotype-phenotype correlation or reveal some interesting biological aspect of the defective enzyme. The figures try to give condensed information about the gene structure and the mutations of most of the genes. References in the figures should allow convenient access to the original literature. Figures are only included for genes for which at least four different mutations have been described. Other- wise mutations are mentioned in the text. The frequencies of some alleles are indicated in the figures. It should be noted that these numbers may only have a limited validity, since in many cases they represent the results of only one study and may only be valid for certain ethnic groups. Some diseases will not be discussed, since no progress has been made in the cloning of the respective genes or elucidation of the genetic defect. These involve diseases caused by defects in lysosomal transporters, which have been covered by a recent review in this series (see Pisoni et al. [79]).

2. Lipidoses

2.1. GM2 gangliosidoses

This group of diseases is characterized by storage of G~a 2 ganglioside [138]. Three polypeptide chains encoded by three different genes are involved in the degradation of GM2 ganglioside. The synthesis of an a and fl-subunit for fl-hexosaminidase (/3-N-acetyl-D-hexosaminidase) allows for the formation of three isoenzymes trot, aft, and tiff, which are designated fl-hexosaminidase S, A, and B, respectively [138]. The fl-hexosaminidase isoenzyme A removes terminal N-acetylgalactosamine from GM2 ganglioside [314]. In order to be hydrolysed by fl-hexosaminidase A, the ganglioside has to be associated with a third polypeptide, the GM2 activator protein, which solubilizes the substrate for the action of the enzyme [312,314]. The storage of GM2 ganglioside can therefore be due to mutations in three different genes: (1) The fl-hexosaminidase a-subunit gene, which causes classical Tay Sachs disease or late onset GM2 gangliosidosis (variant B); (2) in the /3-subunit gene, which causes early or late onset

Sandhoff disease (variant 0); and (3) the GM2 activator protein (variant AB) [138].

These variant forms of disease are clinically similar, but differ in the composition of compounds which besides GM2 ganglioside are also found in the storage material. The presence of additional compounds is due to the fact that GM2 ganglioside is not the only substrate of fl-hexosaminidase A, and that the isoenzymes differ in their substrate specificities and dependence on the presence of the G m activator protein. The substrate specificities of the isoenzymes have either been demonstrated directly or have been concluded from the compounds found in the storage material. Generally, fl-hexosaminidases release terminal /3-glycosidically linked N-acetylgalactosamine or N- acetylglucosamine from a variety of substrates, fl- hexosaminidase A cleaves sphingolipids such as GM2 ganglioside [314], GDlaGalNAc [312], GA2 glycolipid [314] and globoside [314] as well as oligosaccharides [315,316]. In contrast fl-hexosaminidase B does not hydrolyse G m ganglioside [312] nor GDlaGalNAc and cleaves GA2 glycolipid only inefficiently [314]. Isoenzyme S has limited catalytic activity and is rapidly degraded [387]. Absence of isoenzyme A in variant B thus causes storage of GM2 ganglioside [313], GDlaGalNAc [312] and to lower extent of GA2 glycolipid [313], while oligosaccharides and globoside are hydrolysed by the unaffected isoenzyme B [317]. In variant O both isoenzymes A and B are deficient, so that besides Gra 2 ganglioside and GDlaGalNAc, globoside [314] and oligosaccharides [308] are stored and the amount of glycolipid GA2 is increased several fold compared to variant B [314]. Most of these compounds are stored in neurons, oligosaccharides are stored in viscera and brain and globoside is only found in the viscera [308,314]. In the variant AB only those compounds accumulate, which hydrolysis depends on the presence of the activator protein, such as GM2 ganglioside and GA2 glycolipid [314]. GDlaGalNAc and oligosaccharides are, in contrast to Tay Sachs and Sandhoff disease, not stored in the GM2 activator protein deficiencies. It has been shown in vitro, that hydrolysis of these compounds is activator protein-independent, which explains the lack of storage in the activator protein deficiencies [60]. Since all variant forms of disease are similar, the symptoms seem to be mainly due to the storage of G m ganglioside and not that of other compounds.

Tay Sachs disease and GM2 gangliosidosis Classical Tay Sachs disease is characterized by a rapidly

progressing neurological degeneration. The disease starts in between 3 to 5 months of age with progressive weakness and hyperirritability. A macular cherry red spot is seen in all patients. Progression of neurologic symptoms leads to the decerebrate, vegetative state in the second to fourth year of life [138]. In the juvenile form the onset of disease is later and the progression is slower. Adult GM2 gangliosidosis represents the mildest form of the disease,

106 V. Gieselmann /Biochimica et Biophysica Acta 1270 (1995) 103-136

which starts in 2nd to 3rd decade of life and presents with neurologic symptoms of spinocerebellar and lower mOtor neuron origin, like ataxias, dysarthrias and muscle weakness. Psychiatric symptoms are frequently present. A particular variant, which has been termed chronic, starts in between the age of 2 and 5, but patients survive well into adulthood [138].

Tay Sachs disease is most prevalent in the Ashkenazi Jewish population. Three defective /3-hexosaminidase a- subunit alleles account for 93% of all mutant alleles in this ethnic group [9]. One allele shows a 4 bp insertion in exon 11 [10], the second has a splice donor site mutation in intron 12 [11-13] and the third, which causes milder Gu2 gangliosidosis, is characterized by a Gly-269 > Ser substitution in exon 7 [14,15] (see Fig. 1). The insertion of 4 bp in exon 11, as well as the intron 12 splice site mutation result in a severe reduction of/3-hexosaminidase a-subunit mRNA and protein. The 4 bp insertion in exon 11 does not interfere with the processing of the primary nuclear mRNA transcript, but produces a frameshifl with premature termination 9 nucleotides downstream. Thus, this mRNA should be translated into a truncated polypeptide. However, the insertion causes instability of the mRNA demonstrating that the machinery of the cell is able to recognize a

nonfunctional mRNA at or prior to the level of translation, the underlying mechanism of which has yet to be elucidated. Not all transcript with premature termination codons are unstable, but similar phenomena have also been reported, e.g., for /3-hexosaminidase fl-subunit [300], fl-D- glucuronidase [213] and a-L-iduronidase transcripts with premature termination codons [254].

The Gly-269 > Set substituted a-subunit causes a reduction of the amount of functional heterodimeric enzyme, which is due to a decreased stability of the mutant a-subunit [282]. The enzymatic deficiency is, however, not complete, since the mutant a subunit still yields a low amount of functional heterodimeric /3-hexosaminidase A [16]. The genotype phenotype correlation in /3-hexosaminidase a-subunit deficiencies can be explained by the complete loss of functional enzyme in case of the 4 bp insertion in exon 11 and the intron 12 splice donor site mutation and the low amounts of residual activity associated with the Gly-269 > Set allele. Homozygosity for either of the first two alleles causes the severe classical Tay Sachs disease, whereas patients with adult GM2 gangliosidosis have been found to be genetic compounds for one of these alleles and the Gly-269 > Set allele [17]. One copy of the Gly-269 > Set allele is sufficient to confer the mild

1 kb I"----4

(27) Del 7,6 kb I

/ I

3/3

I ¢q

11/0 16/73 eClS

A A A A

Fig. 1. Structure of the fl-hexosaminidase a-subunit gene and mutations causing Tay Sachs disease and G m gangliosidosis. The figure shows the structure of the fl-hexosaminidase A a-subunit gene. Boxes indicate exons, lines introns hatched parts 5' or 3' untranslated sequences. Mutations are indicated and the numbers in parenthesis give the references [10,11,14,18-20,22-24,26,27,31-33,35-46,246] in which the mutations have been described. Numbers at the bottom depict the exons in which the mutations occur. Mutations which are associated with a juvenile or aduR phenotype are shown on the top, those which cause an early onset severe phenotype or which phenotype association could not be determined are shown on the bottom. B 1 mutations are depicted by italic letters and lines point to the location in the gene. Bold letters show pseudodeficiency alleles and the bold lines indicate the most frequent mutant alleles. Numbers give the frequencies (%) in the non Jewish/Jewish community, respectively. As far as alleles with mutations other than amino acid substitutions are concerned, the splice site mutations are indicated whether they occur in an exon (Ex) or intmn (Int), the number of the exon or intron in which they occur and whether they represent splice donor (SD) or splice acceptor (SA) site mutations. In case of small deletions (Del) the deleted bases are indicated and the following number either designates the codon in which this deletion has been found or the cDNA position of the deletion, depending on the designation in the original paper.

V. Gieselmann / Biochimica et Biophysica Acta 1270 (1995) 103-136 107

adult phenotype and the few patients identified being homozygous for this allele seem to have a more protracted, milder course of disease than those, who are genetic compounds [17].

The correlation of residual enzyme activity and severity of the clinical phenotype in /3.-hexosaminidase a-subunit deficiencies has also been demonstrated biochemically [234]. Cultured fibroblasts of patients with various clinical forms of disease were fed with the natural substrate GM2 ganglioside and the degradation rate was determined. Pa- tients with milder forms of disease had a higher turn over rate than those with severe forms. In the range of low residual enzyme activities GM2 ganglioside degradation increased steeply with slight increases of residual enzyme activity and it is estimated that about 10-15% of residual enzyme activity is sufficient to sustain a normal phenotype [234].

With one exception [246] the Gly-269 > Ser allele is so far the only allele that has been found in adult GM/ gangliosidosis patients. Most of the remaining defective alleles cause classical Tay Sachs disease and only three have so far been characterized, which lead to the juvenile intermediate type of disease. These alleles encode an a- subunit with an Arg-504 > His [18], an Arg-499 > His [18] and a Gly-250 > Asp substitution [19], respectively. The Arg-504 > His and the Gly-250 > Asp substituted a-subunits are misfolded and retained in the ER [18,283]. In fibroblasts of a patient with the Gly-250 > Asp substituted a subunit enzyme activities higher than those found in fibroblasts of infantile Tay Sachs patients have been demonstrated, when measured with an artificial substrate [283]. It is likely that low amounts of the mutant a-subunits are able to leave the ER and provide lysosomes with low residual enzyme activity, producing the juvenile phenotype of the patient.

Two other mutations affect codon 504, a deletion of Cytosine 1510 [20] and a Arg-504 > Cys substitution [22]. Introduction of the latter mutation into the normal /3- hexosaminidase cDNA and expression studies revealed that the effect of the mutation was similar to that of the Arg-504 > His substitution. However, although the same codon was substituted and data from expression studies were similar both alleles are associated with different forms of disease, juvenile GM2 gangliosidosis (Arg-504 > His) and classical Tay Sachs disease (Arg-504 > Cys), respectively [23].

The frequency of the exon 11 4 bp insertion, intron 12 splice donor site mutation and the Gly-269 > Ser allele has been determined in Askhenazi and non Jewish populations. The 4 bp insertion allele represented 73% of mutant alleles among Ashkenazi Jews, the intron 12 splice site mutation 15%, and the Gly-269 > Ser allele 3%. In the non Jewish population the exon 11 4 bp insertion allele accounted for only 16% of defective alleles, the intron 12 splice site mutation could not be detected and the frequency of the Gly-269 > Ser allele was identical to that found in Ashke-

nazi Jews [9]. Examination of defective alleles among non Jews revealed a donor splice site mutation in intron 9, which has a frequency of 11% and is thus the second most frequent allele among non Jews [25]. However, Tay Sachs disease among non Jews is far more heterogeneous than in the Ashkenazi population [26,246].

Two other ethnic groups, both descendants from French settlers, have a high incidence of Tay Sachs disease. One is the Cajun community in southwest Louisiana, in whom the 4 bp insertion in exon 11 was found in 11 of 12 mutant alleles examined [24]. The other group are the French Canadians in which a 7.6 kb deletion in the 5' region of the gene, which has occurred by illegitimate recombination between Alu repeats, accounts for more than 80% of defective alleles [27,28].

A particular form of /3-hexosaminidase A deficiency is the so called B1 variant, which is characterized by an altered substrate specificity of the enzyme. It displays normal activity towards non sulfated artificial substrates used in the diagnosis of Tay Sachs disease, but fails to degrade GM2 ganglioside, the natural substrate [29,30]. Five different mutations have been found in B1 patients. Three affect the same codon, causing the substitution of Arg-178 by His [31], by Cys [32] or Leu [33]. The Arg-178 > His allele, which has also been called DN allele, seems to be the most frequent B1 allele. This allele is found panethnicaUy, but with a particular high frequency in northern Portugal [34]. Although this mutation might have evolved independently in various parts of the world, it seems likely that its panethnic occurrence is at least in part due to Portuguese colonization. Two B1 mutations have been found in different codons, one is a Va1-192 > Leu [35] and the other a Asp-258 > His substitution [36]. The region of the a-subunit involved in the recognition of GM2 ganglioside is obviously larger, as was assumed on the basis of the initially identified Arg-178 > His mutation. As far as the genotype phenotype correlation is concerned, a genetic compound for a null allele and a B1 allele suffers from infantile Tay Sachs disease, whereas homozygosity for the Arg-178 > His allele was found in 10 juvenile patients [34].

Two other interesting mutations affecting substrate specificity are localized in the region in which the B1 mutations occur. Enzymes bearing an Arg-247 > Trp [37] or an Arg-249 > Trp substitution [38] have reduced activity towards an artificial substrate. However, for the Arg- 247 > Trp substitution normal activity towards the natural GM2 ganglioside has been demonstrated [37,307]. Since the diagnosis and heterozygote detection is mostly based on enzymatic assays with artificial substrates a healthy individual may appear /3-hexosaminidase A deficient in this assay. Likewise, in genetic counselling an individual heterozygous for this allele may be considered as a Tay Sachs disease carrier. Similar phenomena have also been described for other lysosomal enzymes [386]. Enzyme deficiencies, which do not cause disease, have been termed


pseudodeficiencies. Pseudodeficiencies are without clinical consequences for the carriers, but cause problems in the diagnosis and genetic counselling.

When Jewish and non Jewish suspected Tay-Sachs disease allele carriers, which were identified by enzyme activity determinations, were examined for the presence of these pseudodeficiency alleles, the Arg-247 > Trp and the Arg-249 > Trp allele was found in about 40% and 6% of non Jewish individuals, respectively [37,38,310]. The Arg- 249 > Trp allele was not found among Jewish suspected carriers [38], while conflicting data have been presented about the frequency - - 3% to 3 0 % - of the Arg-247 > Trp allele in this ethnic group [37,310,311].

Sandhoff disease Since the fl-subunit is part of two different /3-hexosa-

minidase isoenzymes, its deficiency causes the loss of isoenzymes A and B. Deficiencies of the /3-subunit have been termed Sandhoff disease [47]. The clinical phenotype and heterogeneity is similar to that found in a-subunit deficiencies. The cDNA [229] and the gene of the /3-subunit of fl-hexosaminidase have been cloned [55,156] The disease is rare, but there are some demographic isolates with a high frequency, e.g. in a region around the town of Cordoba in Argentina [48]. The most frequent defect found in the fl chain gene is a 16 kb deletion that removes the promoter and exons 1 to 5 (see Fig. 2). This deletion accounts for 27% of Sandhoff alleles and most likely arose from a recombinational event between two Alu repeats. Homozygosi ty of the allele causes the severe late infantile form of the disease [49].

As in Tay Sachs disease the milder forms have been found to be associated with mutations leading to the synthesis of enzymes displaying some residual activity [285]. In many of lysosomal storage diseases mutants, which result in residual enzyme activity, express amino

acid substitutions, that cause a substantial, but incomplete loss of enzyme activity. Sandhoff disease provides an example in which splice site mutations are still associated with some residual enzyme activity, because the normal splice site is still used, although at much lower frequency [50]. Nucleotide sequences at and near the splice site ensure the precision and effectiveness of intron removal. Mutations in these nucleotide sequences may affect splicing in a way that the effectiveness of splicing at the correct site is reduced, leaving most of the transcripts aberrantly spliced, while only a few transcripts will still be spliced correctly. These mutations cause a reduction, but not a complete deficiency of functional m R N A and therefore a reduced synthesis of a structurally unaltered enzyme. In the fl-hexosaminidase fl-subunit gene one of these mutations is not found within the splice acceptor consensus sequence, but in the coding region of exon 11, 8 nucleotides 3' of the splice acceptor site. It changes an amino acid (Pro-417 > Leu), but this substitution is benign, since it does not affect the function of the enzyme. This mutation, however, influences the use of the preceding splice acceptor site. Analysis of the fl-subunit m R N A revealed that in most, but not all transcripts exon 11 was skipped due to selection of a splice donor site further downstream [50]. The patient in whom this allele was found had the juvenile form of the disease and was homozygous for this allele.

A 57 year old patient with a very mild adult type Sandhoff disease was identified, who was a genetic compound for the frequent 16 kb deletion and the exon 11 splice site selection mutation [51]. Four of his fl-hexosaminidase deficient siblings were still healthy at an ad- vanced age (55 to 61 years of age). Compared to the homozygous juvenile patient mentioned above, there is an apparent discrepancy in the genotype phenotype correlation, the molecular basis of which is unknown. These cases

i kb

u !

(49)Del 16kb 27%

~ ^

Fig. 2. Structure of the /3-hexosaminidase fl-subunit gene and mutations causing Sandhoff disease. The designation of mutations is as described in Fig. 1, with references in parenthesis [48-50,52-54,284,285,300]. In this figure, the exon location of mutations is not given by numbers but the lines point to the location. Bold line depicts the most frequent allele. Number gives the frequency of the allele among patients. Bold letters depict a pseudodeficiency allele, which has also been termed Hexosaminidase Paris. Mutations causing late onset disease are shown on top, others at the bottom.

v. Gieselmann / Biochimica et Biophysica Acta 1270 (1995) 103-136 109

demonstrate that other factors can influence the outcome of the disease substantially. Both patients came from a different ethnic background, the juvenile patient being Japanese and the adult patient French Canadian. One can speculate that genetically determined differences in the splicing machinery might cause a more efficient selection of the normal splice site and thus lead to a higher residual enzyme activity in the French Canadian family. Late onset forms of disease, which are caused by similar splicing defects, have also been described in galactosialidosis [89] and Scheie type MPS I [256,266] (see respective para- graphs).

Two other mutations, which cause the generation of new splice sites, have been described: a G > A transition in intron 12 creates a new splice acceptor site [52,53] and leads to the insertion of 24 additional nucleotides, and a duplication including the splice acceptor site at the junction of intron 13 and exon 14 is the cause of an 18 nt insertion [53]. Since both insertions are in frame, an elon- gated /3 chain is synthesized. In both cases the mutant polypeptides are unstable and they do not reach the lysosome, since they are degraded in an early biosynthetic compartment [53]. The intron 12 splice site allele was found in a juvenile Sandhoff patient, who was a genetic compound with a null allele [52]. It is assumed that the normal splice site might still be used at low frequency in this patient and that the transcripts are translated into functional enzyme. An asymptomatic but /3-hexosaminidase deficient father and sibling of a juvenile Sandhoff patient was found to be a genetic compound for the intron 12 splice site allele and the duplication allele of the intron 13 / exon 14 junction [53]. The latter allele was initially termed /3-hexosaminidase Paris [309]. In spite of the duplication, low levels of normally spliced transcript were detectable in the cells of this individual [53]. The low amount of normal mRNA may supply the cell with sufficient enzyme to prevent the development of disease, which means that this allele by definition is a pseudodeficiency allele. Since the a-subunit is still produced in normal amounts, the low amounts of/3-subunit will predominantly form a/ /3 /3-hexosaminidase A heterodimers. Only very little fl//3 homodimer will be allowed to form. Since the carrier of this allele is healthy, it means that in vivo very low amounts of the fl-hexosaminidase B isoenzyme are sufficient to maintain a normal phenotype or the deficiency may be compensated for otherwise.

Four mutations causing premature termination codons in the /3-subunit mRNA have been examined in more detail [300]. Three of these lead to an about 100-fold decrease in mRNA steady state level and one to a 3-fold decrease. The latter 3-fold reduced mRNA and one of the severely diminished species have stop codons at adjacent amino acid positions 451 and 454. So far there is no explanation why premature termination codons occurring at about the same position within a mRNA can have such different effects on steady state mRNA levels.

The effects of the I1e-207 > Val and Tyr-456 > Ser substitution on the /3-hexosaminidase /3-subunit have been studied, because a patient with a peculiar clinical and biochemical phenotype was a genetic compound of these alleles. The symptoms in this adult patient started at the age of seven years with muscular weakness and a slowly progressing lower motor neuron disease [284]. Thus, the patient can be classified to suffer from chronic GM2 gangliosidosis. In the fibroblasts of this patient no /3-hexosaminidase B ( /3/ /3) polypeptides or catalytic activity could be detected. Surprisingly, about 30% to 50% of normal amounts of fl-hexosaminidase A ( a / / 3 ) protein and activity was found when measured with an artificial substrate [385]. It was demonstrated that the I1e-207 > Val substituted fl-subunit does not self associate to form /3//3 homodimers. Monomers are rapidly degraded explaining the lack of /3-hexosaminidase B. However, the mutant /3-subunit dimerizes with the a-subunit to form stable /3-hexosaminidase A heterodimers. The presence of the hexosaminidase A could explain the mild phenotype of the patient [385].

G M 2 activator protein deficiencies The third polypeptide involved in the degradation of

GM2 ganglioside is the GM2 activator protein. The deficiency of this protein is rare and causes a disease that is clinically similar to Tay Sachs disease. This small 24 kDa protein interacts with the a-subunit of the fl-hexosaminidase isoenzyme A [317] and solubilizes lipids such as GM2 ganglioside or GA2 glycolipid for the hydrolysis by /3- hexosaminidase A [314]. The cDNA and gene of this protein have recently been cloned [56]. Two mutations have been described so far. One is a Cys-138 > Arg [57,58] and the other an Arg-169 > Pro substitution [59].

2.2. GMZ gangliosidosis and morquio type B disease

Deficiency of the enzyme /3-D-galactosidase can cause very different clinical phenotypes, fl-o-galactosidase hydrolyses fl-glycosidically linked terminal galactose residues from a variety of different substrates. These substrates are lipids such as GM1 ganglioside [318], oligosaccharides with terminal galactose [319,326] or galactose containing intermediates of the degradation of the mucopolysaccha- ride keratan sulfate [319,327]. A complete deficiency of the enzyme results in the storage of all substrates [319,326,327]. Accordingly, GMI gangliosidosis presents clinically with neurologic symptoms, which can be attributed to the storage of GM1 ganglioside in neurons, while storage of keratan sulfate catabolic intermediates in the viscera results in hepatosplenomegaly and severe bone deformities typical for mucopolysaccharidoses. GM1 gangliosidosis may appear as a severe rapidly progressing infantile disease in which the neurologic symptoms domi- nate the clinical picture, but skeletal deformities, edema, hepatomegaly and coarse facial features are also present

110 v. Gieselmann /Biochimica et Biophysica Acta 1270 (1995) 103-136

[319]. The adult form of GM1 gangliosidosis shows mild, slowly progressing neurologic involvement like dysarthrias or gait disturbances and, except for vertebral dysplasias, little involvement of the skeleton [319]. Since GM~ ganglioside metabolism seems to be normal in Morquio type B disease [377], patients have no neurologic involvement, but demonstrate progressive skeletal dysplasias. Intermedi- ate phenotypes between these clinical extremes also exist and it has therefore been suggested that the term /3- galactosidoses describes diseases due to the deficiency of /3-D-galactosidase more comprehensively [321].

The fl-D-galactosidase cDNA [61] and the gene [62] has been cloned and a variety of mutations have been characterized (see Fig. 3). Defective alleles are heterogeneous and no single mutation with a particular high frequency has been found. So far 20 amino acid substitutions [63-65], two duplications of 23 nt [66] and 165 nt [64] and three splice donor site mutations [276,279,320] have been described in GM1 gangliosidosis patients. As in other lysosomal storage diseases, homozygosity for a null allele (e.g., the 165 nt duplication) was found in patients with the severe infantile form of the disease [64]. Juvenile patients were found to be genetic compounds or homozygous for an allele with an amino acid substitution (Arg-201 > Cys). Expression of a cDNA carrying this mutation in trans- formed h~man fibroblasts showed some residual enzyme activity, when measured with an artificial substrate [64]. The Arg-201 > Cys substituted enzyme does not associate with the protective protein, which is thought to stabilize /3-D-galactosidase in vivo (see paragraph on galactosialidosis), and is therefore rapidly degraded [322].

Although the disease is genetically heterogeneous [63,64] one allele prevails among patients with adult GM1 gangliosidosis. When 16 Japanese patients were examined, 13 were homozygous for an allele carrying an Ile-51 > Thr substitution [64,67]. One patient was a genetic compound

for this allele and an Arg-457 > Gin substitution, and the remaining two were homozygous for the latter. The Ile-51 > Thr allele showed residual enzyme activity towards artificial substrates in transfection studies with mutated cDNAs and in the patients fibroblasts [64]. Only a small amount of the Ile-51 > Thr substituted enzyme is transported properly to the lysosomes and expresses some enzyme activity. For the majority of the mutant enzyme transport seems to be disturbed at or prior to the Golgi apparatus [322]. Although all patients homozygous for the Ile-51 > Thr allele suffered from late onset GM1 gangliosidosis, there was still a remarkable heterogeneity in the clinical phenotype, demonstrating once more that there are other factors influencing the phenotypic expression of lysosomal disease [67].

In Morquio type B disease three different mutant alleles found in two patients have been detected, characterized by a Trp-273 > Leu, Arg-482 > His and Trp-509 > Lys substitution [65]. Both patients were genetic compounds for the Trp-273 > Leu allele and one of the two other alleles, respectively. In the same study, these alleles were not found among patients with GM1 gangliosidosis, suggesting that these alleles were specific for Morquio Type B diseases. An Italian Gra 1 gangliosidosis patient was identified, who was homozygous for the Arg-482 > His allele [68]. Thus, only the Trp-273 > Leu allele can therefore be considered specific for Morquio type B disease. This mutation may affect the substrate specificity of the enzyme in a way that affinity or hydrolysis for keratan sulfate is decreased, but normal for GM1 ganglioside [299,377]. This may solely lead to the storage of keratan sulfate, but would not affect GM1 ganglioside metabolism. The Trp-273 > Leu substituted enzyme is normally processed, transported to the lysosome and stabilized by the protective protein [322]. It has a reduced specific enzyme activity towards an artificial substrate [322], but its substrate specificity has

o ~ . ~

I I[ |1 i ii

n ~ N ,

I ~ I I I J I I

v

I i

!J\

eq eq

< L9

Fig. 3. Structure of the fl-D-galactosidase gene and mutations causing GM1 gangliosidosis or Morquio type B disease. Labelling of mutations is as described before, with references in parenthesis [63-67,276-279,320]. The bold letters depict a mutation causing Morquio type B disease. / / indicates that the intron size is not known.

V. Gieselmann /Biochimica et Biophysica Acta 1270 (1995) 103-136 111

not yet been analysed in detail. Theoretically, heterozygosity for a GM1 gangliosidosis and a Morquio type B allele should result in Morquio type B syndrome, since the normal catalytic activity of the Morquio allele towards GM1 ganglioside should compensate the defective GM1 gangliosidosis allele.

2.3. Galactosialidosis

This disease is characterized by the combined deficiency of two enzymes fl-D-galactosidase and N-acetyl-a- neuraminidase [319,328]. This is caused by the deficiency of a 'protective protein' [379], which associates with these two enzymes and forms a high molecular weight (> 600 kDa) complex [87]. Complex formation leads to the stabi- lization of fl-D-galactosidase [82,332] and the activation of neuraminidase [83,331,332]. Neuraminidase hydrolyses a-glycosidically linked neuraminic acid from oligosaccharides and glycolipids [334]. Oligosaccharides with terminal neuraminic acid are therefore one of the major stored compounds in galactosialidosis patients [335,336], but oligosaccharides terminating with galactose have also been detected [336]. In tissues of the nervous system a variety of lipids containing neuraminic acid and galactose has been found in the storage material (e.g. GM1, GM2, GM 3 gangliosides) [337].

Patients with the infantile form of disease suffer from edema, ascites, skeletal deformities visceromegaly and variable involvement of the nervous system [333]. The phenotype ranges from severe forms fatal at birth to late infantile and juvenile/adult forms. Symptoms in the late onset forms are distinct. Patients suffer from slowly progressing neurologic disease, dysmorphism and skeletal dysplasias and may survive into adulthood [333]. Most cases of the galactosialidosis have been found in Japan [88]. The majority of these Japanese patients suffer from a late onset form of the disease [88]

Cloning of the cDNA of the protective protein showed a high degree of homology to serine carboxypeptidases from yeast and plants [84]. Expression studies of the cDNA revealed that the protective protein has serine esterase, deamidase and carboxypeptidase activity [281]. Surpris- ingly, a deamidase released from human platelets upon thrombin stimulation appears to be identical to the protective protein [85,329]. This enzyme deamidates and inacti- vates a variety of biologically active peptides, whose C terminus is blocked by an amide bond (e.g. substance P, oxytocin, endothelin) [329]. It has two enzymatic activities with different pH optima. A deamidase activity is optimal at neutral pH and a carboxypeptidase activity at acidic pH. Further studies revealed that the protective protein is most likely identical to lysosomal cathepsin A [86,85,329]. The catalytic activity of this protease and its protective function for fl-D-galactosidase and neuraminidase activity are distinct, since active site mutants are still able to restore the activity of these enzymes in galactosialidosis fibroblasts

[86]. The stoichiometry of the three proteins in the complex is unclear. /3-D-galactosidase deficiencies, in which no cross reacting material is found, demonstrate that this enzyme is not an essential factor of the complex, since neuraminidase is active in these cells. Cross reacting material negative mutants have not yet been identified among neuraminidase deficient cell lines, since neuraminidase deficiencies have not been analysed at the molecular level yet. The role of neuraminidase in complex formation is therefore unknown. The existence of different pools of fl-D-galactosidase and protective protein has been noted in several studies [86,87]. The majority of these proteins are present in the non complexed form, whereas only 20% are found within the complex. The fraction of protective protein, which coimmunoprecipitates with fl-o-galactosidase, correlates with the amount of protective protein present in high molecular weight complexes as determined by gel filtration. The existence of different pools of protective protein and fl-D-galactosidase may be a consequence of their function in different reactions. Within the complex the enzyme activities may act together to degrade glycoproteins. Uncomplexed protective protein might act as a protease in the degradation of non glycosylated proteins and, depending on the local pH, as a deamidase in the inactivation of biologically active peptides. Uncomplexed /3-D-galactosidase may be involved in the degradation of sphingolipids, such as GM1 ganglioside and the mu- copolysaccharide keratan sulfate. However, the existence of different pools of/3-D-galactosidase is speculative, since the current data cannot explain, why fl-D-galactosidase should be stable outside the protective protein complex.

The complete structure of the gene of protective protein has not yet been published although a report of a partial gene structure has been reported [88]. Among late onset Japanese patients a splice donor site mutation of intron 7 causing the skipping of exon 7 has been identified and it accounted for 28 of 38 mutant alleles examined [89]. However, this mutation seems to allow the production of a small amount of normal mRNA, explaining the mild phenotype of the patients. Patients homozygous for this allele had a milder course of disease than genetic compounds with a null allele, a genotype phenotype pattern resembling that of other lysosomal storage diseases. Several mutations have been identified (see Fig. 4) and some have been analysed in more detail. A Phe-412 > Val was found in two late infantile patients [90]. This substitution renders the protective protein inactive with respect to its cathepsin activity and prevents dimerization of the protein. The lack of dimerization may be the cause for the retention and premature degradation of the mutant precursor in the ER. The retention, however, is not complete since some mutant protein becomes phosphorylated on mannose-6-phosphate residues and is properly sorted to the lysosomes [90]. Residual amounts of mature protective protein are present in lysosomes and this may explain the mild clinical phenotype of the patient. When a cDNA having a mutation

112 V. Gieselmann / Biochiraica et Biophysica Acta 1270 (1995) 103-136

N[

74% ~,

/ /

\\ ]c

Fig. 4. Structure of the protective protein eDNA and mutations causing galactosialidosis. N and C depict the amino and carboxyterminus, respectively. Mutations associated with mild phenotypes are shown on top, others at the bottom. Bold line depicts the most frequent allele found among Japanese patients. Numbers gives the frequency among Japanese patients. References are in parenthesis [89,90,286,287,330,395]

found in a mild late infantile case (Tyr-249 >Asn) was transfected into deficient fibroblasts, a partial restoration of /3-D-galactosidase and neuraminidase activity could be demonstrated [287], indicating that low amounts of residual protective protein may predispose for a milder course of disease.

2.4. Fabry disease

Fabry disease is an X linked disorder in which the enzyme C~-D-galactosidase A is deficient. This causes the storage of glycosphingolipids with a terminal o~-glycosidically linked galactose, such as globotriaosylceramide, gal- abiosylceramide [323] and blood group B glycolipids [324]. Deposition of these lipids occurs predominantly in vascu- lar endothelial, smooth and myocardial muscle cells and causes angiokeratomas of the skin and visceral disorders with renal and cardiovascular symptoms [378]. Pathology appears to be due to obstruction of small blood vessels by lipid storage and accumulation of lipids in myocytes. There is no involvement of the central nervous system, but patients typically suffer from episodic crisis of burning pain in the distal extremities, severe acroparesthisia and hypohidrosis caused by the involvement of the autonomic nerves. The onset of the disease is in childhood or adoles- cence [378]. Heterozygous female carriers may be healthy or display milder forms of disease.

The eDNA [70] and the gene [71] have been cloned. The gene has 12 exons and is 14 kb long. It is characterized by an exceptionally high content of Alu sequences. Twelve repetitive Alu sequences have been identified, which cover more than 30% of the genomic a-D-galactosidase sequence. Since several deletions were detected in mutant alleles, it was assumed that the gene was prone to deletions due to illegitimate recombinational events be-

I kb

I I

\ \ / /

{ DeL (72,77)

7

I Del (72,77) [ L D*I (72,77) I

Del (72,77)

I Del (72,77) I

Dupl (72) { I

Fig. 5. Structure of the a-D-galactosidase A gene and mutations causing Fabry disease. Bars at the bottom indicate deletions or duplications. Mutations causing cardiac variant of Fabry disease are shown on the top. References are in parenthesis [69,72,75-78,80,81,247,301-303]. Numbers below the brackets indicate the exon in which the mutation is found.


tween Alu sequences. However, of 6 deletions analysed only one could be explained through Alu recombination, whereas the others occurred at short direct repeats [72,77] (see Fig. 5).

In a particular form of Fabry disease the storage of lipids is confined to the myocardium. These patients may develop a hypertrophic cardiomyopathy or may only suffer from angina pectoris with otherwise normal hemodynamic functions [73-75]. Five different mutations of a-D-galactosidase A have so far been described in these late onset patients, a Asn-215 > Ser [303], a Gin-279 > Glu [69], a Met-296 > Val [75], a Arg-301 > Gin substitution [76] and an 3 bp in frame deletion causing the loss of Arg-404 or 405 [303], respectively. All these mutations are localized in the carboxyterminal halve of the molecule. A considerable amount of residual enzyme activity (up to 25% when measured with an artificial substrate) has been found in these patients with the cardiac variant of Fabry disease [75]. Overexpressed a-D-galactosidase A bearing the Gin- 279 > Glu or the Arg-301 > Gin substitutions still has 5% and 15% of enzyme activity towards an artificial substrate, respectively [69]. Thus, residual enzyme activity seems to predispose for the cardiac variant. It may suffice to prevent storage in endothelial and renal cells, but is insufficient to protect the heart.

The Gin-279 > Glu substituted enzyme has been characterized in more detail. While its kinetic properties are similar to the normal enzyme, the mutant is thermolabile in vitro at 37°C and neutral pH, but shows an increase in stability at lower temperatures or acidic pH. The thermola- bility is not just an in vitro phenomenon, since three times more enzyme activity can be expressed from the mutant cDNA transfected into COS cells, when these cells were grown at 30°C. Since these two conditions - 37°C and neutral pH - also apply to the prelysosomal compartments in vivo, the in vitro observations may be explain an instability of the mutant enzyme in an early biosynthetic compartment [280].

2.5. Niemann Pick disease

Niemann Pick disease type A and B are caused by a deficiency of the enzyme acid sphingomyelinase [340]. This enzyme hydrolyses sphingomyelin into ceramide and phosphorylcholine. In addition a nonlysosomal neutral sphingomyelinase has been described, but this enzyme is not affected in Niemann Pick disease [341]. Deficiency of the lysosomal enzyme causes storage of sphingomyelin [340]. Two types of disease can be distinguished an acute type A and a chronic type B. (Recently, a new designation has been suggested, in which type A is termed IA and type B is termed IS) [340]. The acute form is a severe disorder of infancy characterized by progressive psychomoter retardation, massive visceromegaly and death by 3 years of age. The chronic form is characterized by a visceromegaly, but little involvement of the nervous system and survival into

I ! kb I

- - 1

II 23% 7.6%

I 30%

Fig. 6. Structure of the sphingomyelinase gene and mutations causing Niemann Pick Type A and B disease. Labelling of mutations is as indicated in Fig. 1 and 2. Bold lines indicate the most frequent defective alleles among Ashkenazi Jews. References are in parenthesis [101- 104,248-250]. Numbers give frequencies. Niemann Pick Type B mutations are indicated on top.

adulthood. Accordingly, the two types of disease are termed neuronopathic (type A) and nonneuronopathic (type B). The disease is panethnic, but has a higher frequency among Ashkenazi Jews. The cDNA [99] and the gene [100] of sphingomyelinase have been cloned. Three mutant alleles have been found to be most frequent among Ashkenazi Jews (see Figs. 6 and 7). Two alleles show amino acid substitutions - Leu-302 > Pro and Arg-496 > Leu - and account for 23% and 30% of mutant alleles, respectively [101]. The third has a deletion of a C in codon 330 causing premature termination and represents about 8% of defective alleles in Ashkenazi Jewish patients. Introduction of the amino acid substitutions into the wild type cDNA and their expression revealed a complete loss of enzymatic activity. These alleles have, so far, not been found outside the Jewish population. Patients, who were homozygous or genetic compounds for these alleles, always suffered from the acute type A form of Niemann Pick disease.

Three mutations have been found in milder Niemann Pick type B disease. A deletion of a single codon eliminat- ing Arg-608 has initially been characterized in Ashkenazi Jews [102]. Two Ashkenazi type B patients were studied and both were genetic compounds for this allele. The same allele was shown to be the prevalent mutation in Niemann Pick type B patients of Northern African origin [288]. The allele could not be detected among type A patients. In fibroblasts of patients being homozygous or genetic compounds for this allele a considerable residual enzyme activity could be detected towards the natural substrate in vitro and in situ in cultured fibroblasts [288]. A Japanese patient was found to be homoallelic for a Ser-436 > Arg substitution [250] and a European type B patient was a


25%

~ ^ ~ ~

~r

I 25%

Fig. 7. Structure of the arylsulfatase A gene and mutations causing metachromatic leukodystrophy. Bold lines depict the most frequent alleles among Caucasians. The mutations of the pseudodeficiency allele are indicated in bold letters. Both mutations are found in the same allele. Disease causing mutations found on the background of the pseudodeficiency allele are shown in italics. Mutations associated with late onset disease are shown on top, others at the bottom. References are in parenthesis [106,109,110,113,115-119,244,251,289,290,297,298,344].

genetic compound for a Niemann Pick type A allele and an allele carrying a Gly-242 > Arg substitution [103]. In vitro mutagenesis of the sphingomyelinase cDNA and expression studies of the Gly-242 > Arg substituted enzyme revealed a substantial residual enzyme activity (40% of control determined with a synthetic substrate) encoded for by this allele [103]. Thus, it seems that the nonneuronopathic form is found in patients being homozygous or genetic compounds for alleles encoding for mutant enzyme with some residual activity.

In addition to Niemann Pick type A and B there is a third clinically similar form termed type C. Cells from these patients show an impaired intracellular transport of cholesterol from lysosomes, but the primary defect is not in the sphingomyelinase gene and has so far not been elucidated (for review see [342]).

2.6. Metachromatic leukodystrophy

The deficiency of arylsulfatase A causes metachromatic leukodystrophy. The substrate of this enzyme is cerebroside-3-sulfate (sulfatide). This glycolipid is mainly found in the myelin sheaths of the nervous system. Deficiency of arylsulfatase A causes storage of sulfatide in various organs, but the disease mainly affects the nervous system [343]. Patients suffer from progressive demyelination, which leads to a variety of neurologic symptoms and finally causes death. Typically the disease starts with gait disturbances at the age of 18 to 24 months, in the further

course of disease patients develop a spastic tetraparesis, seizures and dementia. In the milder late onset forms psychiatric symptoms frequently prevail initially before neurologic symptoms become apparent [343].

Arylsulfatase A is encoded in a small gene [105], and the entire coding sequence of 1.5 kb is distributed over 8 exons and encompasses not more than 3 kb of genomic sequence (see Fig. 7). Two mutant alleles are particularly frequent and account each for about 25% of mutant alleles among Caucasian patients [106,107]. One of these alleles has a splice donor site mutation at the exon/intron 2 border and causes a complete loss of arylsulfatase A mRNA. In the other allele a Pro-426 > Leu substitution is found, which allows for the normal synthesis of catalytically active enzyme, which is highly unstable upon arrival in the lysosome [108]. The high frequencies of these alleles have allowed their detection in a number of patients and have revealed a simple genotype phenotype correlation. Homozygosity for the splice donor site mutation causes the severe late infantile form of the disease, heterozygosity for the splice donor site mutation and the Pro-426 > Leu allele mitigates the course of the disease to the juvenile form and homozygosity for the Pro-426 > Leu allele is mostly found in the mildly affected adult patients. This genotype-phenotype correlation can be explained by the low residual enzyme activity associated with the Pro-426 > Leu allele, in which one copy of this allele mitigates the course of the disease and the gene dosage effect of two alleles causes the still milder adult form of metachromatic leukodystrophy. A correlation between the clinical phenotype and the residual enzyme activity has also been demonstrated in a study in which the residual enzyme activity was determined by feeding sulfatide to cultured fibroblasts of patients with different severity of disease. Higher residual enzyme activities were found in cells of mildly affected adult patients [234].

Except for the Thr-274 > Met allele, which has been found in high frequency among Australians of Lebanese origin [251] all other alleles characterized so far have only been found in one or few patients, which shows that the disease is genetically heterogeneous [109].

Although residual enzyme activity is one of the important determinants of the clinical phenotype one patient has been identified, who suffered from the severe late infantile form of the disease but had considerable residual enzyme activity. When tested with the natural substrate of arylsulfatase A, cultured fibroblasts of this patient metabolized cerebroside sulfate at higher rates than fibroblasts of patients with the mild adult forms of the disease. The apparent discrepancy of the residual enzyme activity and the phenotype of this patient may be explained by cell type specific differences in the expression of the defect. Since metachromatic leukodystrophy is a disease of oligodendrocytes, the expression of the defect may be different in those cells than in cultured fibroblasts. The mutation found in this patient was a Gly-309 > Ser substitution, which

V. Gieselrnann /Biochimica et Biophysica Acta 1270 (1995) 103-136 115

caused a reduced intralysosomal stability of the mutant enzyme [110].

Deficiency of arylsulfatase A has also been demonstrated in healthy individuals at high frequency (up to 2.6% of the population) [107,345]. The deficiency is substantial, but since it is not complete it does not cause a disease and is another example of lysosomal enzyme pseudodeficiency [111]. As indicated above, pseudodeficiencies have also been described for various lysosomal enzymes [386] such as /3-hexosaminidase a and fl-subunits, and seem to be quite frequent in the /3-D-galactocerebrosidase gene [112], which is defective in Krabbe disease. Arylsul- fatase A pseudodeficiency is caused by homozygosity for a frequent allele that due to a mutation in a polyadenylation signal supports only the synthesis of about 10% of arylsulfatase A compared to the normal allele [113]. The frequency of this allele is estimated to be 7-15% [107,345]. Whereas homozygosity for the pseudodeficiency allele is not associated with a disease, it has been a matter of debate whether heterozygosity for a pseudodeficiency allele and a metachromatic leukodystrophy allele, may be the cause of neurologic disease in these persons. The allele frequencies of metachromatic leukodystrophy alleles and the pseudodeficiency allele allow to calculate that about 1 in 1000 individuals should be a genetic compound for these two alleles. Sixteen such genetic compounds have been examined clinically and radiologically. Neurologic symptoms or radiologic alterations have been found in three of the probands. However, neither the neurologic symptoms nor the radiologic findings resembled those seen in metachromatic leukodystrophy patients. This study demonstrates that the majority of these genetic compounds will not develop a progressive neurologic disease [114]. However, since the oldest person examined was 56, it can

not be excluded that some might develop a senile form of metachromatic leukodystrophy in old age.

In comparison, /3-hexosaminidase a subunit pseudodeficiencies and, on the other hand, /3-hexosaminidase /3- subunit and arylsulfatase A pseudodeficiencies are funda- mentally different. Whereas /3-hexosaminidase A pseudodeficiency is caused by a reduced activity towards the artificial substrates and can thus be attributed to a change of catalytic properties [37,38], /3-hexosaminidase /3-subunit and arylsulfatase A pseudodeficiencies are due to diminished mRNA levels causing reduced synthesis of enzymes [53,113].

2.7. Gaucher disease

Gaucher disease is caused by the deficiency of /3-0- glucocerebrosidase [347]. The enzyme hydrolyses glucose from the sphingolipid glucosylceramide [347]. Glucosylce- ramide is an intermediate compound in the synthesis and degradation of complex glycosphingolipids. Deficiency of the enzyme causes storage of glucosylceramide mainly in cells of the monocyte/macrophage system [348]. Gener- ally, the disease affects the bone marrow, bone, spleen, and liver, causing anaemia, thrombocytopenia, bone lesions and hepatosplenomegaly. In the less frequent, more severe forms of the disease, the central nervous system is also affected and, as in Niemann Pick disease, these variants have been termed neuronopathic. The severe neuronopathic form (type II disease) is rare and occurs panethnically. It presents with hepatosplenomegaly and neuronal complications [348]. The clinical presentation is quite uniform and death usually occurs before 2 years of age. The mild nonneuronopathic form (type I disease) is frequent among Ashkenazi Jews and is clinically heterogeneous.

1 kb I I

I

70%/23%

I

<

10%/0% / 0%/5% 3%/51%

Fig. 8. Structure of the fl-D-glucocerebrosidase gene and mutations causing Gaucher disease. Designation of mutations is as indicated in Fig. 1. Mutations causing mild type of disease are shown on top. Bold lines depict the most frequent alleles and numbers give the frequency of these alleles in Jewish/non Jewish communities, respectively. References are in parenthesis [123,126,129,131,135-137,139,237,304,385,393-396].

116 V. Gieselmann / Biochiraica et Biophysica Acta 1270 (1995) 103-136

Symptoms may develop in childhood or not until old age and many patients are only diagnosed by chance since their symptoms are so mild that they do not seek medical advice [125]. In this mild Gaucher type I disease, the most frequent sign is hepatosplenomegaly with pancytopenia and in the course of the disease disabilities due to bone deformities develop [348]. A third intermediate form of Gaucher disease (type III) is a so called subacute neuronopathic form, which, due to its high frequency in a Swedish isolate, has also been termed Norbottnian type Gaucher disease [140]. Patients show neuronal as well as visceral involvement, but the neurological symptoms develop later and more slowly than in the acute neuronopathic form.

The cDNA, the gene and pseudogene of /3-D-glucocerebrosidase have been cloned [120,121]. The pseudogene is tightly linked to the normal gene, but is smaller due to several mainly intronic deletions. Thirty six different mutations have been characterized [349] (see Fig. 8). Most of these mutations are rare and some are limited to single families. However, five of the mutant alleles account for more than 98% of defective/3-D-glucocerebrosidase alleles among Ashkenazi and for about 70% of mutant alleles among non Jews [123,292].

Homozygosity or heterozygosity for the Asn-370 > Ser allele precludes the development of neurologic symptoms and is always associated with the type 1 nonneuronopathic form of the disease [349]. Asn-370 > Ser homozygotes have late onset of disease -median 30.8 years, (combination of data from [122] and [124])-, whereas genetic compounds for this allele and the 84GG allele, which has a G insertion causing a frameshift and is thought to be a null allele, develop symptoms earlier with a median of 7.4 years. However, among patients with identical genotypes the range of age of onset can be enormous: 3-73 years for Asn-370 > Ser homozygotes and 2-28 years Asn-370 > Ser /84GG heterozygotes. Thus disease of a Asn-370 > Ser homozygote can be more severe than that of a genetic compound. It has also been shown that about two thirds of the Asn-370 > Ser homozygotes have such a mild form of disease that they do not seek medical advice and remain undiagnosed [125].

Among non Jews the most frequent allele has a Leu-444 > Pro substitution [123]. Homozygosity for this mutation has been found in patients suffering from all three types of disease. From a total of 44 homozygous patients - summary of patients mentioned in references [126], [127], [140-144] and [291] - 11 had type I, 9 type II and 24 type III disease. These numbers show that the majority of homozygotes do have neurologic symptoms, but why about 25% suffer from the nonneuronopathic type I disease is not clear. Given identical fl-D-glucocerebrosidase alleles the genetic background may influence the type of disease which will develop. Most of the Leu-444 > Pro homozygous type I patients were of Japanese origin, whereas most with type III disease were of Swedish specifically Norbott- nian origin. It has been objected that the Japanese type I

patients were diagnosed at young age and that it cannot be excluded that they will develop neurologic symptoms in the near future, which would classify them as type III patients [349]. However, the fact that patients from different ethnic origin suffer from different type of disease or at least from a different course of the same variant of the disease points to other genetic or epigenetic factors that may influence the outcome of disease. This may resemble the situation in other lysosomal storage diseases such as Sandhoff disease, where the same allele in a Japanese and a French Canadian patient was associated with similarly varying phenotypes [50,51].

In many alleles the Leu-444 > Pro substitution is present as a single point mutation. However, the same amino acid substitution can be found in the /3-D-glucocerebrosidase pseudogene. Recombination or gene conversion events between the gene and the pseudogene can introduce this mutation into the fl-19-glucocerebrosidase gene. Since the Leu-444 > Pro substitution is not the only difference between the gene and the pseudogene, a number of other mutations will be introduced at the same time. The number depends on the site of recombination. The existence of two such recombination alleles - termed RecNci and Rec TL - carrying the Leu-444 > Pro substitution has been demonstrated [127-129]. These alleles show recombination at different sites and introduce 1 (Ala-456 > Pro) or 2 (Ala- 456 > Pro and Asp-409 > His) additional mutations into the gene. These alleles have been termed complex alleles.

It is interesting to note, that five patients [127,130] who have been identified as genetic compounds for a Leu-444 > Pro and a complex allele suffered from severe type II disease, but that this kind of heterozygosity has so far not been described among type I and type III patients. Further- more, not a single patient with type II disease has been identified, who has unambiguously been shown to be a Leu-444 > Pro homozygote [349]. Although the number of patients in which Leu-444 > Pro carrying alleles have been differentiated is low, preventing definitive conclusion to be drawn, the data suggest that a genetic compound of a Leu-444 > Pro point mutation allele and complex allele may suffer from a more severe type of disease.

The 84GG allele [131], characterized by an insertion of a G in exon 2 is thought to be a null allele, since the mutation causes a frameshift, which prevents the synthesis of any functional /3-D-glucocerebrosidase. Interestingly, no 84GG homozygotes have yet been described. The Gaucher mice that have recently been generated by homologous recombination of murine embryonic stem cells, are homozygous for a null allele [132]. They displayed such a severe phenotype such that they died one day after birth. Severe neonatal forms of Gaucher disease have been described [265] and assuming a similar genotype-phenotype correlation in mice and man, it can be speculated that severe neonatal forms of Gauchers may be homozygous for null alleles (e.g. 84GG alleles).

The effects of the Asn-370 > Ser and the Leu-444 > Pro


mutations on the fl-D-glucocerebrosidase enzyme have been investigated by expression of cDNA's encoding polypeptides with these substitutions. The Leu-444 > Pro substituted enzyme is highly unstable and has a 10-fold higher Vm~ x towards an artificial substrate [133]. Cells transfected with the Asn-370 > Ser cDNA showed /3-D-glucocerebrosidase polypeptides, the specific activity of the enzyme towards a synthetic and a natural substrate was 2-5-fold decreased [134]. For optimal enzymatic activity fl-D-glucocerebrosidase has to be activated by an activator protein (see chapter on saposin activator proteins). The mode of action is not a solubilization of the substrate as in the case of GM2 activator protein, but rather a direct stimulation of enzyme activity. It could be demonstrated that in vitro the Asn-370 > Ser substituted enzyme is less effectively stimulated by the activator than the normal enzyme [133].

2.8. Sphingolipid activator protein deficiencies

Several enzymes involved in the degradation of sphingolipids depend on the assistance of small acidic proteins to hydrolyse their substrates. These Sphingolipid Activator Proteins are now called saposins (SAPosin). Five different saposins are encoded by two different genes. One gene codes for the GM2 activator protein and has already been discussed in the GM2 gangliosidoses section. The other four saposins, which have been designated A, B, C and D are encoded by a single gene [145,146]. They are synthesized as a common precursor polypeptide (prosaposin), which is proteolytically cleaved in the lysosome to yield the four independent, but structurally homologous proteins [147]. The mechanism by which saposin B acts resembles that of the GM2 activator protein, in that it solubilizes sulfatide for the desulfation by arylsulfatase A [148]. How- ever, saposin B seems not to be specific for the degradation of sulfatide by arylsulfatase A, since it has also been shown to activate the hydrolysis of other lipids by other enzymes [357]. Saposin C has been shown to activate /3-D-glucocerebrosidase, however, as indicated above this occurs not by solubilization of the substrate, but via direct

interaction and stimulation of the enzyme [149]. The activation of arylsulfatase A and fl-D-glucocerebrosidase by saposin B and C, respectively, has initially been shown in vitro, but its in vivo significance has been demonstrated by the existence of genetic diseases due to mutations affecting the saposin B and C domain of the precursor protein causing selective deficiencies of one of these saposins (see Fig. 9). Mutations in saposin B cause a variant form of metachromatic leukodystrophy, whereas mutations in the saposin C domain have been found in a patient with a variant form of Gaucher disease [160,164]. Metachromatic leukodystrophy caused by saposin B deficiency is clinically similar to an arylsulfatase A deficiency. However, the fact that saposin B also activates the hydrolysis of other lipids by different enzymes is reflected by the composition of the storage material, which in addition to sulfatide contains globotriaosylceramide and GM 3 ganglioside in saposin B deficiencies [358]. Saposins A activates the hydrolysis of glucosylceramide by /3-D-gluco- sylcerebrosidase and of galactosylceramide by fl-D-galactocerebrosidase [149,357]. Saposin D has been considered as an sphingomyelinase activator [356], but it has recently also been shown to inhibit this enzyme [273]. Since deficiencies of these proteins, which should cause variant forms of Gaucher and Niemann Pick disease, respectively, have not been described their in vivo function remains unclear. Because saposin C deficiencies cause Gaucher disease in the presence of saposin A, one can conclude that the latter cannot complement the defective saposin C and does not interact with fl-D-glucocerebrosidase in vivo. Further clues about the in vivo function have come from a patient in whom, due to a mutation in the initiation codon, the entire precursor protein and, thus, all four saposins are deficient [150,151]. Shortly after birth this patient presented with hyperkinesia, respiratory insufficiency and hepatosplenomegaly and died at the age of 16 weeks. Fibroblasts showed a substantial deficiency of /3-D-galactocerebrosidase, fl-D-glucocerebrosidase and ceramidase. Storage of the substrates of these enzymes could be demonstrated in the patients liver [150]. Sphingomyelinase

lkb i I

/

Fig. 9. Structure of and mutations in the prosaposin gene. Mutations causing a variant form of metachromatic leukodystrophy are shown in italics, the one causing Gaucher disease in bold letters. Question mark indicates that exon one has not been analyzed on the genomic level yet. References are in parenthesis [151,160,162-164]

118 V. Gieselmann /Biochiraica et Biophysica Acta 1270 (1995) 103-136

activity was normal and there was no storage of sphingomyelin, which suggest that, in vivo, saposins are not essential for sphingomyelinase activity.

Evidence is accumulating, that saposins and particularly the prosaposins, may have functions other than just the activation of hydrolases. Prosaposin has been identified in various secretory fluids like cerebrospinal fluid, seminal fluid, bile and milk. In the latter the amount changed during the lactation period [152]. Prosaposin is secreted by platelets upon stimulation with thrombin [153]. In vitro studies have shown that saposin B is able to transfer a variety of sphingolipids from donor to acceptor liposomes in vitro [154]. Prosaposin binds gangliosides with high affinity and is also able to transfer gangliosides from donor to acceptor membranes [155]. Thus, these proteins may act in a more general way as lipid transfer proteins.

Saposins have been shown to accumulate in tissues of patients with lysosomal storage disease. As much as 100- fold levels of saposin A were found in the liver of fucosidosis patients [263]. Saposins accumulated to various ex- tents in patients with GM1 gangliosidosis, Tay Sachs, Sandhoff, Gaucher and Niemann Pick disease. It is not clear whether the accumulation is due to a reduced break- down or to an enhanced synthesis of the prosaposin molecule [263].

The cDNA and the gene for the saposin precursor have been cloned [145,157-159]. Five mutations causing three different diseases have been described (see Fig. 9). As indicated earlier a mutation in the initiation codon Met-1 > Leu leads to a deficiency of all saposins causing a severe disease with early death of the patient [150,151]. Three mutations have been found in the saposin B domain. All these mutations cause a variant form of metachromatic leukodystrophy. These include the loss of a potential glycosylation site due to a Thr-217 > lie substitution [160,161] and the substitution of a cysteine (Cys-241 > Ser) [162], which is conserved among all four saposins. A point mutation in intron 7 creates an alternative splice site causing a 33 bp insertion [163]. Finally, a point mutation in the saposin C domain has been found in a patient with Gaucher disease [164]. Like in the mutant saposin B allele substitution of a conserved cysteine (Cys-385 > Phe) might destroy a functionally important disulfide bridge.

2.9. Wolman disease

Deficiency of lysosomal acid lipase results in intralysosomal storage of cholesteryl esters and triacylglycerols and causes two clinically distinguishable diseases: Wolmans disease and cholesteryl ester storage disease (CESD) [346]. Whereas Wolmans disease becomes apparent in early infancy by hepatosplenomegaly, various gastrointestinal problems, anaemia, calcification of the adrenal glands and is usually lethal in the first year of life, CESD is milder and age at onset is later. A variety of different symptoms have been described in CESD patients [346]. However, all

patients showed hepatomegaly, which in some cases lead to hepatic fibrosis and liver failure [346]. Due to the low abundance, the purification of the enzyme and the cloning of the cDNA has been difficult, but has recently been achieved [170]. The cDNA of acid lipase is 2.6 kb long and the open reading frame predicts a protein of 372 amino acids. It shows a high homology when compared to human gastric lipase. When both enzymes are compared 58% of amino acid residues are identical. The gene of lysosomal acid lipase is divided into 10 exons and encompasses about 36 kb of genomic sequence [359,360]. So far three mutations causing Wolman disease or CESD have been described. A splice donor site G > A substitution at the 3' end of exon 8 has been found in a patient with CESD resulting in a 72 bp exon deletion coding for amino acid 254-277 [361]. A patient with Wolman disease was a genetic compound of an allele, which due the insertion of a T is associated with premature termination of the mutant protein, and an allele with a Leu-179 > Pro substitution [360]. The reasons why some patients develop Wolman disease and others CESD are not yet clear.

2.10. Krabbe disease

Krabbe disease or globoid cell leukodystrophy is due to the deficiency of fl-D-galactocerebrosidase [371]. The enzyme releases galactose from galactocerebroside, one of the major membrane lipids of the myelin sheaths. Like in metachromatic leukodystrophy patients suffer exclusively from neurologic symptoms [371]. They present in the first six months of life with hyperirritability towards external stimuli, hyperaesthesia, spasticity and regression of neurologic development. Patients usually die before their second year of life in a decerebrate state [371]. In addition late onset forms have been described. The cloning of the gene has been difficult due to the low abundance and hydro- phobicity of the enzyme, but has recently been achieved [296,405]. The purified enzyme has a molecular weight of 51 kDa. The cloned cDNA has 3795 nucleotides, 2007 of which represented an open reading frame, predicting a protein of 669 amino acids [296]. Although the substrates of /3-D-glucocerebrosidase and /3-D-galactocerebrosidase are structurally similar, no sequence homology between the two enzymes has been found. A nonsense mutation was found at codon 369 (GAA > TAA) in the coding sequence of cDNA amplified from cultured skin fibroblast mRNA from a patient with typical Krabbe disease [405].

3. Disorders of glycoprotein degradation

3.1. Fucosidosis

Fucosidosis is caused by a deficiency of the enzyme C~-L-fucosidase, which cleaves fucose residues in various substrates. Fucose is found in N- and O-linked oligo-

V. Gieselmann / Biochimica et Biophysica A cta 1270 (1995) 103-136 119

i1 kb I

--D I D l D

/ v v ~

I (174)Del

\

I 20%

Fig. 10. Structure of the o~-L-fucosidase gene and mutations causing fucosidosis. Designation is as described in Fig. 1 and 2. Bold line depicts the most frequent allele. References are in parenthesis [174-176,236,252,253]

saccharide side chains of sphingolipids, blood group anti- gen containing glycolipids and glycopeptides and the mu- copolysaccharide keratan sulfate [171,388]. Intermediates of N-linked oligosaccharide degradation represent the majority of storage compounds in fucosidosis [171]. Most of the intermediates are fucosylated glycopeptides and gly- coasparagines, while few are oligosaccharides [171]. In- creased urinary excretion of keratan sulfate has also been reported [388]. Among the stored glycolipids blood group H containing glycolipids have been identified [171]. Two types of fucosidosis can be distinguished. Type I starts in the first year of life and shows a rapid neurological deterioration, coarse facies, dysostosis multiplex and causes death within the first decade of life. Type II fucosidosis is more frequent than type I, and develops later in life. Symptoms are similar to type I, but progress more slowly and in addition the patients typically develop angiokeratoma diffusum. These patients may survive into the fourth decade of life [171]. Intermediate types of disease have also been described [171]. The cDNA [172], the gene and a

pseudogene of a-L-fucosidase have been cloned [173]. So far, 12 defective Ot-L-fucosidase alleles have been described (see Fig. 10). The most frequent allele has a premature termination codon at amino acid position 422 (Gin > ter) and accounts for 20% of all mutant C~-L- fucosidase alleles [175]. In accordance with the existence of both types of disease in the same family, patients homozygous for the Gin > ter-422 allele have been found among type I as well as type II patients [171]. Interest- ingly, so far only one allele with an amino acid substitution Gin-281 > Arg has been found [176], another has a splicing defect [253] and all others show premature termination due to the small deletions or introduction of termination codons [174,252].

3.2. Aspar ty lg lucosaminur ia

The final step in the hydrolysis of N-linked oligosaccharide side chains of proteins is the cleavage of the bond between N-acetylglucosaminylamine and asparagine.

1 kb I I

"~ "d

o o o o

I 98%

D D

o o o o o o

/

I I DeI3'UTR (183)

Fig. 11. Structure of the L-aspartyl-D-glucosaminidase gene and mutations causing aspartylglucosaminuria. The bold line indicates the mutation in Fins and its frequency in Finnish patients. References are in parenthesis [178,182,185-187].

120 v. Gieselmann / Biochiraica et Biophysica Acta 1270 (1995) 103-136

This reaction is catalysed by glycoasparaginase (N4-( fl- N-acetylglucosaminyl)-L-asparaginase). Deficiency causes the accumulation of aspartyl-N-glucosamine in various tissues [380]. Patients typically suffer from mental retardation, which becomes apparent in between the age of 1 and 4 years. Patients usually die of pulmonary complications at the age of 30 to 50 years [380]. The clinical phenotype is uniform and no clinical subtypes can be distinguished, which in this respect makes the disease unique among lysosomal storage disorders [380]. The disease occurs panethnically, but shows a particularly high frequency in the Finnish population [178,380]. The cDNA [177,178] and the gene of the enzyme have been cloned [179] and a number of mutations have been described [180] (see Fig. 11). The mutant allele found in the Finnish isolate has two amino acid substitutions one codon apart (Arg-161 > Gin and Cys-163 > Ser) and accounts for 98% defective alleles among Fins [178]. Expression studies revealed that only the Cys-163 > Ser substitution is responsible for the enzyme deficiency, whereas the Arg-161 > G l n substitution is a polymorphism [181]. Cys-163 is involved in the formation of a disulfide bond in the normal enzyme and its substitution is likely to cause a misfolding of the polypeptide [362]. In 9 non Finnish patients of different ethnic origin, 9 different mutant alleles have been found demonstrating the heterogeneity of the disease [182]. Eight of the patients were homozygous, which suggests that most came from consanguineous marriages. In one patient a deletion occurred in the 3' untranslated region of the cDNA [183]. No mutation was found in the coding sequence. Although the mutant RNA was smaller, its amount was comparable to normal RNA levels. However, glycoasparaginase polypeptides could not be detected, suggesting that the mutant RNA was translationally inactive. Expression of the cDNA carrying the same deletion in COS cells yielded normal enzyme activities. Thus, the mechanism by which this mutant causes the deficiency remains obscure [183].

The pattern of proteolytic maturation of glycoasparaginase is unique among lysosomal enzymes. Lysosomal enzymes are usually proteolytically cleaved within the lysosome, but processing of glycoasparaginase starts already in the ER [184]. Shortly after synthesis the inactive 42 kDa precursor is cleaved into the active 27 kDa/17 kDa dimeric enzyme. After the arrival in the lysosome the 27 kDa subunit is further trimmed by a removal of 10 amino acids at the carboxyterminus [184]. Since the enzyme is also active at neutral pH, the activation of the enzyme in the ER could also be functionally important.

3.3. Schindler disease

a-N-acetylgalactosaminidase has long been characterized as a lysosomal activity [352,406], but patients suffering from a lysosomal storage disease due to the deficiency of this enzyme have only recently been de-

scribed [91,93,407]. The enzyme hydrolyses a-glycosidically linked N-acetylgalactosamine residues from synthetic substrates, oligosaccharides, glycolipids and glycopeptides [339]. Such residues can be found in glycolipids containing blood group A specifying trisaccharides [350], keratan sulfate from cartilage [351], N-linked [352,353] and O-linked oligosaccharides [352,353].

Initially urine from the older affected German sibling was analyzed and a 5-fold elevated accumulation of blood group A trisaccharide was detected [354]. Since the brother of this patient was blood group 0 and did thus not excrete blood group A trisaccharide, but suffered from an even more severe type of disease, the storage of blood group A trisaccharide cannot be a major determining factor in the pathogenesis of the disease [355]. Further analysis of patients urine identified an approx. 100 times elevated urinary excretion of compounds in which oligosaccharides were O-glycosidically linked to amino acids or peptides [94,355]. Thus, c~-N-acetylgalactosaminidase deficiency is a disorder of O-glycan catabolism. Interestingly, the major oligosaccharides had no ot-N-acetylgalactosaminyl residues at their nonreducing terminals, but neuraminic acid [94,355]. Thus, in vivo, the enzyme may act as an endogly- cosidase and neuraminidase may only desialylate oligosaccharides after endoglycolytic cleavage has occurred, however no satisfying explanation has been found.

In 1989 the disease was described in two sons of a consanguineous [91] couple and has been called Schindler disease [378]. The patients showed a variety of neurologic symptoms. The disease started by a loss of acquired skills and a psychomotor regression in the second year of life. Patients developed cortical blindness, seizures and spasticity. Examination of the central and peripheral nervous tissue revealed spheroid bodies in terminal axons, which classified the disease as a neuroaxonal dystrophy [91]. Already in 1987, a Japanese 46-year-old woman was described, who suffered from disseminated angiokeratoma, but showed no neurologic involvement [92]. 3 years later the patient was shown [93,407] to be ot-N-acetylgalactosaminidase deficient and thus suffered from late onset form of the disease. These examples demonstrate the remarkable clinical variability of the disease. The cDNA [96,97,408] and the gene [95] of the enzyme have been cloned. The nucleotide sequence predicts a protein of 411 amino acids. The amino acid sequence and the gene structure shows a strong homology to Ot-D-galactosidase A suggesting a common evolutionary origin of both enzymes [96,97,408]. Analysis of mRNA of two affected children revealed a Glu-325 > Lys substitution. No enzyme activity could be expressed from a cDNA encoding an enzyme with these mutations [98]. Biosynthetic studies in fibroblasts revealed defective phosphorylation of the precursor polypeptide, absent maturation and rapid intracellular degradation [409]. The mutation in the adult onset patient is a single C > T transition resulting in the amino acid substitution Arg-329 > Trp [410].


4. Mucopolysaccharidoses

4.1. Mucopolysaccharidosis type I /Hurler Scheie syndrome

Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of a-L-iduronidase. This enzyme cleaves terminal iduronic acid residues from the glycosaminoglycans heparan and dermatan sulfate [381]. Deficiency causes storage and urinary excretion of these undegraded or partially degraded mucopolysaccharides [381]. The spectrum of clinical phenotypes is broad. In the severe form, patients typically show symptoms in the first year of life presenting with hepatosplenomegaly, skeletal dysplasias, corneal clouding, short stature and mental retardation. These severe forms have been termed Hurler syndrome. At the other end of the clinical spectrum are the patients with mild form of disease termed Scheie syndrome. They have little or no neurologic involvement, have mild hepatosplenomegaly, joint stiffness and deformities, heart valve problems, but may have a normal lifespan. Intermediate phenotypes have accordingly been termed Hurler/Scheie syndrome [381].

Several studies have been undertaken to correlate the biochemical parameters with the clinical phenotype of the disease. Although no absolute correlation has been found, it seems that most patients with the severe disease have no detectable a-L-iduronidase protein, whereas protein as well as a-L-iduronidase activity can be detected in fibroblasts of most, although not all of Scheie patients [188]. The cDNA and the gene of the a-L-iduronidase have been cloned [189,190]. Southern blot analysis of more than 40 patients detected no gross abnormalities of the gene, show- ing that deletions are not frequent in MPS I. Several point

mutations have been described (see Fig. 12). Among Euro- pean patients an allele with a premature termination codon at position 402 (Trp-402 > ter) accounts for 47% of mutant a-L-iduronidase alleles and an allele with a termination codon at position 70 (Gin-70 > ter) for 15% [398]. Patients homozygous for these premature termination alleles suffered from the most severe type of disease and, in most cases, died before the age of 4 years [191]. In fibroblasts of these patients no a-L-iduronidase protein was detected. Homozygotes for a Pro-533 > Arg allele and a Trp-402 > ter/Pro-533 > Arg genetic compound showed a somewhat milder intermediate course of disease; the latter died at the age of 12 years [192]. In fibroblasts of a Trp-402 > ter/Pro-533 > Arg heterozygote, low levels of a-L- iduronidase protein and activity were detected, and Pro- 533 > Arg homozygotes seem to be less severely affected than genetic compounds for a null allele. Thus, it seems that the Pro-533 > Arg allele is associated with a less severe form of the Hurler type MPS I.

A patient suffering from the mild Scheie form of MPS I was found to be a genetic compound for the frequent Trp-402 > ter allele and an allele that had a splice acceptor site mutation in intron 5 [256,266]. Due to this mutation most transcripts use a cryptic splice site 28 nucleotides 5' of the normal splice site. However, the latter is still used with low efficiency, so that low amounts of RNA transcripts remain correctly spliced. These transcripts are translated into low amounts of a-L-iduronidase, which explains the mild phenotype of the patient. Thus, this mutation resembles the splice acceptor site mutation in exon 11 of the /3-subunit of /3-hexosaminidase, which causes a mild type of Sandhoff disease [51].

The emerging pattern of genotype-phenotype correlation in MPS I thus resembles that of other lysosomal

A ^

// B-0

I 15%

S I

47%

Fig. 12. Structure of the a-L-iduronidase gene and mutations causing mucopolysaccharidosis type I/Hurler and Scheie syndrome. The most frequent alleles are indicated by bold lines. Numbers give the frequencies. Mutations associated with mild disease are shown on top. References are in parenthesis [191,192,254-256,266,268,398].


storage diseases, such that homozygosity for null alleles causes severe disease and alleles associated with some residual activity allow for a milder course of the disease. Although not yet analysed at the molecular level, one mutation is accompanied by a phenomenon that is so far unique among lysosomal storage disorders. In fibroblasts of a patient suffering from severe type of disease six times more a-L-iduronidase protein could be detected than in normal fibroblasts [193]. The enzymatically inactive mutant a-L-iduronidase is sorted normally to the lysosome, but its proteolytic processing to the mature enzyme is delayed. It is not yet clear, whether the increase in a-L- iduronidase protein is due to a prolonged half life of the mutant polypeptide or to an enhanced synthesis. The latter would suggest a feedback mechanism by which the enzyme or its substrate controls the transcription or translation of a-L-iduronidase mRNA.

4.2. Mucopolysaccharidosis type H/Hunter syndrome

Mucopolysaccharidosis type II (MPS II) is caused by the deficiency of a-L-iduronate-2-sulfate sulfatase. The enzyme desulfates iduronate-2-sulfate residues, which can be found in heparan and dermatan sulfate. Deficiency of the enzyme causes accumulation of degradation intermediates [381]. MPS II is the only mucopolysaccharidosis that is inherited as an X linked trait. Clinically, it resembles MPS I, with skeletal abnormalities, hepatosplenomegaly, but less severe mental retardation and no corneal clouding. The spectrum of clinical severity is broad, t~-L-iduronate- 2-sulfate sulfatase cDNA has been cloned and the structure of the gene has been determined [194,261,274]. The cDNA sequence predicts a protein of 550 amino acids and the deduced amino acid sequence shows a strong homology to

other lysosomal and non lysosomal sulfatases. The gene seems to be prone to deletions, which have been detected in about every fourth patient [195,196]. Complete deletions of the gene can be found in 8% of patients [197], and those always suffer from the most severe type of MPS II as do patients with other gross rearrangements. Thirty one point mutations have been described which cause severe, intermediate or mild type of disease (see Fig. 13). Most of the mutations are private and their effects on the enzyme have not yet been studied [293]. No correlation between mutant proteins having some residual activity and milder forms of disease have been reported. Conclusions about genotype- phenotype correlation cannot be drawn yet, but the fact that on one hand large deletions are always associated with severe form of disease and on the other hand such deletions have never been found among mildly affected patients, would fit into the pattern of genotype phenotype correlation seen in other lysosomal storage diseases. MPS II provides another example for mutations occurring in the same codon, but which lead to different clinical phenotypes. An Arg-468 > Trp substitution was in a mildly affected patient [338], while the same amino acid was changed to glutamine in a patient suffering from severe type of disease [258].

4.3. Mucopolysaccharidosis type IV / Morquio A syndrome

Mucopolysaccharidosis type IV (MPS IV)is caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. This enzyme cleaves sulfate residues from sulfated galactose found in keratan sulfate or sulfated N-acetylgalactosamine found in chondroitin-6-sulfate [381]. MPS IV is characterized by corneal clouding, severe skeletal deformities such as dwarfism, odontoid hyperplasia,

l kb I I

r~ ~

~ A A A

.1

~ X A - -

DelEx3-8(267) I I

Fig. 13. Structure of the Ot-L-iduronate-2-sulfate sulfatase and mutations causing mucopolysaccharidosis type II /Hunter syndrome. Mutations causing milder phenotypes are shown on top. Deletions have been found in about 20% of patients. Since they have not been exactly defined, they are not shown in this figure. References are in parenthesis [197,198,257,258,267,293,294,338].


NI ]

\ t . q a ~

// 1 2 3 f

¢q

e ~

4 1 5 16F 71

A oO tr~ ¢'4

[- ,

8 I f

Fig. 14. Schematic structure of the coding sequence of the arylsulfatase B cDNA and mutations causing mucopolysacchar idosis type VI/Maroteaux-Lamy syndrome. N and C depict the amino and carboxyterminus, respectively. Since the structure of the gene has not been completely resolved, only the exon boundaries and numbers are indicated. Mutations causing mild phenotype are shown on top. References are in parenthesis [204-207,245,260].

kyphosis, hyperlordosis, dysostosis multiplex, but the absence of neurologic involvement [381]. A broad spectrum of clinical variability has been described. The cDNA [199] and the gene [363] have been cloned. The cDNA has 2339 bp and contains an open reading frame which predicts a protein of 522 amino acids [199]. As for ot-L-iduronate-2- sulfate sulfatase the deduced amino acid sequence shows strong homology to other sulfatases of different function. The gene contains 14 exons and is about 50 kb long [363]. Two point mutations have been characterized. A deletion of two nucleotides causes a frameshift at position 1342 of the cDNA and was found in a patient with the severe form of the disease. In a patient with the mild form of disease, an Asn-204 > Lys substitution has been found. Since this amino acid is part of a potential N-glycosylation site the

mutation leads to the loss of an oligosaccharide side chain [2001.

4.4. Mucopolysaccharidosis type V1/Maroteaux Lamy syndrome

Mucopolysaccharidosis type VI (MPS VI) is caused by a deficiency of arylsulfatase B. This enzyme catalyses the hydrolysis of the sulfate ester in N-acetylgalactosamine-4- sulfate. Due to this deficiency, patients are unable to metabolize chondroitin-4-sulfate and dermatan sulfate [381]. There is a wide range of clinical severity. Patients have no neurologic involvement, but multiple skeletal malformations, hepatosplenomegaly, corneal clouding and frequently aortic valvular dysfunction [381]. Biochemical studies have been performed to correlate the residual enzyme activity with the clinical phenotype of the disease [201]. In these studies a trisaccharide substrate resembling the oligosaccharides found in dermatansulfate has been used. No arylsulfatase B protein or enzyme activity could be detected in the majority of cell lines from severely affected patients. All mildly affected patients showed some enzyme activity and the highest activity of about 5% of normal being present in a patient, who is still healthy at the age of 45 [201].

Cloning of the cDNA revealed high homology to other sulfatases [202]. The gene is large and has only partially been characterized [203]. A variety of mutations have been identified, but none of the mutant alleles is frequent show- ing genetic heterogeneity for this disease (see Fig. 14). Two mutations have been studied in detail. A Gly-137 > Val substitution was found in homozygosity in a patient with the intermediate type of disease [204]. The mutant enzyme was catalytically normal, but was highly unstable. In another patient with the intermediate form of the disease the termination codon was substituted by Gin. The mutant protein had a 50 amino acid C terminal extension. Surpris- ingly, this mutant enzyme showed a 20 times higher

1 kb I

I

/ / U l U

; >

$-

g

Fig. 15. Structure of the fl-o-glucuronidase gene and mutations causing mucopolysaccharidosis type VII/Sly syndrome. Designation of mutations are as described in Fig. 1 and 2. Mutations associated with hydrops fetalis are shown in bold letters. References are in parenthesis [208,211-213,295].

124 V. Gieselmann /Biochimica et Biophysica Acta 1270 (1995) 103-136

maximal velocity of substrate turnover, while g m was only two times higher. Thus the mutant enzyme is catalytically more efficient than the wild type enzyme. The deficiency of the enzyme activity in the patient could be explained by a rapid degradation of the mutant enzyme in the ER [205].

4.5. Mucopolysaccharidosis type VII~Sly syndrome

Mucopolysaccharidosis VII (MPS VII) is caused by the deficiency of the enzyme fl-D-glucuronidase. This enzyme hydrolyses terminal glucuronic acid residues from glycosaminoglycans dermatan and heparan sulfate, chondroitin-4 and -6 sulfate [381]. The clinical picture shows a wide spectrum of severity and resembles that of MPS I and MPS II. Patients suffer from skeletal dysplasias, hepatosplenomegaly, and moderate mental retardation. Intelli- gence can be normal in late onset cases [381]. In addition a severe neonatal from of MPS VII exists, which already presents during pregnancy as a hydrops fetalis [208]. The cDNA [210] and the /3-D-glucuronidase gene have been cloned [209]. So far, seven mutations have been described (see Fig. 15). Homozygosity for an Arg-382 > Cys substitution was found in an intermediately affected patient [211] and homozygosity for Ala-619 > Val in a mildly affected individual [212]. Both substituted amino acids are highly conserved among glucuronidases of different species (mouse, rat, E. coli). Three alleles are associated with hydrops fetalis, the most severe form of disease [208,295]. The mutations of these alleles cause an Arg-216 > Trp, an Ala-354 > Val and an Arg-611 > Trp substitution, respectively [295]. The effects of two of these substitutions have

been studied in more detail. The Arg-611 > Trp substitution causes a complete loss of enzyme activity. The inactive enzyme is unstable and fails to form tetramers with wild type monomers from other species [208]. When the Ala-354 > Val substituted enzyme is expressed in COS ceils or in glucuronidase deficient murine fibroblasts a surprisingly high residual enzyme activity of 30-40% when measured with an artificial substrate was found. These findings contrasted with the severe form of disease and the low enzyme activity measured in the patients fibroblasts. The reason for this discrepancy remains unclear, although it was suggested that even a slight overexpression of the mutant enzyme may drive folding reactions in the ER towards a properly folded polypeptide and that this reaction may not occur in the patients fibroblasts where the amount of enzyme expressed is much lower [208].

A similar phenomenon was also observed for a Trp-627 > Cys substituted /3-D-glucuronidase, for which it could be proven experimentally that the level of residual enzyme activity in transfection experiments correlates with the degree of overexpression [213]. These findings are not unique for /3-D-glucuronidase, but have also been described for /3-hexosaminidase A [389]. Although the disease is rare, it has attracted considerable attention since it was the first lysosomal storage disease for which a spontaneous mouse model existed and the cDNA of the defective gene was available. Thus, several therapeutic trials involv- ing gene transfer have been performed in this model of lysosomal storage disease, the results of which will be discussed in the section on therapy of lysosomal storage diseases.

i I k b i

1¢3

k---q

DelEx 18(399,400)

/ ~ ^

Fig. 16. Structure of the ot-o-glucosidase gene and mutations causing Pompe disease. Mutations causing adult type of glycogenosis type II are shown on

top. //indicates that the size of the intron is unknown. References are in parenthesis [216,217,219,220,259,399].


4.6. Glycogen storage diseases / Pompe disease

Pompe disease is the only lysosomal disease among the glycogen storage disorders and results from the deficiency of OZ-D-glucosidase, which hydrolyses a- l ,4 and a-l ,6 glycosidic linkages. Deficiency of the enzyme results in intralysosomal storage of glycogen in nearly all tissues, but symptoms are mainly due to functional impairment of skeletal and heart muscle and the nervous system [382]. The severe infantile form starts in the first months life with muscle weakness, macroglossia, hepatomegaly and cardiomyopathy [382]. Death ensues before the second year of life. Adult forms of Pompe disease are mild and symptoms may be confined to skeletal muscle weakness. Between these extremes, there is a spectrum of intermediate forms of disease [382]. The cDNA and the gene of a-o-glucosidase have been cloned [214,215]. The amino acid sequence deduced from the cDNA revealed strong homology to non lysosomal enzymes isomaltase, maltase and sucrase [214]. The phenotypic variation in Pompe disease is most likely explained by different levels of residual enzyme activity. Such correlation was already apparent before mutations in the a-D-glucosidase gene were analysed [241]. Eight mutant alleles have been characterized. A deletion of exon 18 and the adjacent intronic sequences seems to be the most frequent mutation among Dutch patients and has been found in the heteroallelic form in severely and mildly affected patients (see Fig. 16). A Met-318 > Thr substitution, which creates an additional N-glycosylation site [216] and a Glu-521 > Lys substitution, which is located in the neighbourhood of the catalytic site at position Asp-518 have been found in patients suffering from the infantile form of disease [217,218]. Both amino acid residues are conserved among the homologous enzymes mentioned before.

So far two patients with the adult form of Pompe disease have been examined and both were genetic compounds for two defective O~-D-glucosidase alleles. In one allele, an Arg-854 > ter substitution was identified and biochemical studies revealed that no protein is expressed from this allele [220]. Three amino acid substitutions were found in the other allele, an Asp-645 > Glu, a Val-816 > lie, and a Thr-927 > Ile substitution [219,220]. Two, Val- 816 > lie and Thr-927 > lie, are polymorphisms. The latter abolishes a glycosylation site, but does not affect the function of the enzyme [220]. Asp-645 > Glu has been identified as the deficiency causing mutation [220]. Trans- fection studies revealed that no enzymatic activity measured with the natural substrate can be expressed from a cDNA encoding this mutant enzyme [220]. The second late onset patient had a Gly-643 > Arg substitution on one allele and a Arg-725 > Trp on the other [259]. Studies in the biosynthesis of a-D-glucosidase substituted with these amino acids showed that in cultured cells both mutant polypeptides are retained and degraded in the ER [220,259]. Although genetically different, both adult patients have

one common feature. In both patients a discrepancy was noted between the very low enzyme activity found in cultured fibroblasts, cultured muscle cells and in transfection studies and the mild phenotype of the patient [219,220,259]. This may be explained by differences in the expression of the defect under different conditions. When cultured fibroblasts of this patient were examined electron- microscopically, a D-glucodiase was detected in the dilated ER. This was also found in a biopsy of muscle fibers, but in addition in muscle tissue it appeared that fully matured intralysosomal enzyme was more abundant than in the cultured cells [264]. The apparent discrepancy of residual activity and phenotype may be due to differences in the processing of the mutant enzyme under different conditions, such that it is largely degraded in the ER in cultured cells whereas some enzyme reach the lysosome in vivo [264].

4. 7. Pathogenesis of lysosomal storage diseases

The analysis of disease causing mutations is important for the understanding of a disease but tells little about the pathogenesis. In some lysosomal storage diseases, part of the symptoms can simply be explained by the macromolec- ular effects of storage material. In Maroteaux-Lamy syndrome, storage of dermatan and chondroitin sulfate in the heart valves causes valvular insufficiency and stenosis due to thickening of the endothelial walls, which leads to cardial insufficiencies in the patients. On the other hand, symptoms may be a consequence of effects that the storage material exerts on the metabolism of a single cell. These may simply be due to mechanical effects of massively enlarged lysosomes, which may rather unspecifically per- turb various intracellular functions. However, there are several examples which demonstrate that more specific effects of the storage material itself may explain the pathogenesis more adequately. In several sphingolipidosis, e.g., metachromatic leukodystrophy [221], Krabbe disease [222], GM2 gangliosidosis [223] and Gaucher disease [224] an abnormal accumulation of lysosphingolipids has been demonstrated. Lysosphingolipids represent deacylated forms of sphingolipids in which the acyl residue bound to the N of the sphingosine is missing. Lysosphingolipids have been shown to be lytic and to be potent inhibitors of protein kinase C [225]. In brains of metachromatic leukodystrophy patients, 50 to 100 times more lysosul- fatide has been detected and it is conceivable that via inhibition of protein kinase C, this compound interferes with a variety of cellular functions [221]. In the brain of twitcher mice, which are a model for human Krabbe disease, the accumulation of lysogalactocerebroside (psychosine) has been demonstrated. When dorsal root gan- glion cells from these mice were cultured in vitro they grew poorly and died. Compared to normal cells 70 times more psychosine was found in affected cells [226].

Already many years ago, it has been noted that pyrami-


dal neurons in GM2 gangliosidoses develop large neuronal processes, which have been termed meganeurites [227]. Although all cells store GM2 ganglioside only the pyramidal neurons develop meganeurites. In addition the axonal hillock of pyramidal cells gives rise to spiny ectopic dendrites, which are able to form ectopic synapses [227]. It is obvious, that such ectopic connections interfere with information processing in a neuronal network and may explain, in part, the neurological symptoms of the patients. The storage of GM2 ganglioside is not restricted to GMZ gangliosidosis but, for reasons that are not understood, occurs to a lesser extent also in c~-mannosidosis [364,365]. In a feline model of this disease, only a few pyramidal neurons present ectopic neurites. It was recently demonstrated that only those cells that store GM2 ganglioside develop additional neurites and that the storage of GM2 ganglioside precedes the development of abnormal dendrites [228]. Gangliosides have been implicated in a variety of cellular functions. They can act as receptors, or ligands [366] and may play key roles in differentiation, since their pattern of expression shows characteristic changes during embryonic development [367]. If one speculates that GM2 ganglioside may play a physiological role in neurite formation of pyramidal cells, its pathologic accumulation in GM2 gangliosidosis or c~-mannosidosis may continuously trigger parts of developmental processes resulting in the de novo formation of additional, ectopic dendrites.

Caprine /3-mannosidosis is an example where the amount of accumulated storage material does not correlate with the severity of the lesions. The main symptom of these animals is a myelin deficiency, which is severe in cerebral hemispheres, but only moderate in the spinal cord [275]. When the amount of storage material was determined, there was no correlation of oligosaccharide accumulation and demyelination and it even seemed that more storage material was present in the less severely affected spinal cord [400]. When oligodendrocytes of affected animals were cultured an increased number of immature glial progenitor cells were found, suggesting a defect in oligo- dendrocyte differentiation [230].

In Gaucher disease the actual amount of glucocerebroside in liver tissue is in between 0.1 and 18 mg per gram of liver tissue [269,270]. For this reason, the hepatomegaly cannot simply be explained by the accumulation of glucocerebroside. Pathologic examinations revealed, that an increase in cell number explains the organomegaly in this disease [368]. It has been shown, that macrophages can be stimulated in vitro to secrete cytokines in a dose-dependent response upon addition of glucocerebroside [271]. These cytokines may be secreted by macrophages in Gaucher disease and may stimulate other cells to divide.

This summary of pathophysiologic correlates of various lysosomal storage diseases may seem like a random collec- tion of phenomenology with questionable relevance for pathogenetic mechanisms. They indicate, however, that there may a lot more to learn from lysosomal storage

diseases. The elucidation of the pathogenesis of lysosomal storage diseases is greatly facilitated by the availability of animal models. For various diseases naturally occurring models exist for example in dogs [370], cats [369], or rats [384]. Spontaneous murine models have been described for mucopolysaccharidosis type VII [404], for Krabbe disease [383], and for Niemann Pick type C [342].

Recently, the generation of a mouse model by homologous recombination of the /3-D-glucocerebrosidase locus in embryonic stem cells was reported [132]. Homozygous mutant mice were already severely affected at birth and died within the first day of life. They showed hyperkera- totic skin, were cyanotic and did not move and feed as their normal littermates. Examination of the animals revealed a storage of glucocerebroside in macrophages of several organs. However, the storage was not extensive enough to explain the rapid death of the mice. Thus, it has been speculated that the accumulation of toxic compounds may be responsible for the severe phenotype of affected animals. The skin in these mice was found to be hyperker- atotic. This symptom in mice resembled that found in humans with neonatal form of Gaucher disease, which due to their ichthyotic appearance of the skin have been termed collodion babies [265].

4.8. Therapy of lysosomal storage disease

The first lysosomal storage disorder for which treatment was available has so far not been discussed in this review, since the protein which is defective has not yet been purified. This disease is cystinosis. It is caused by the deficiency of a lysosomal cystine transporter, which trans- ports cystine that has been liberated in the process of lysosomal protein degradation back into the cytosol (see Pisoni et al. [79]). Due to the deficiency of this transporter cystine accumulates in the lysosomes. Since cystine is poorly soluble, crystals form easily in the tissues. The main symptom is a nephropathy causing renal failure. Cysteamine is a weak base, which enters the lysosome, reacts with cystine to form a disulfide linked cystine cysteamine compound. This compound can exit the lysosome via a transport system for cationic amino acids, which is not affected in cystinosis. Therefore renal function in the patients can be preserved by the oral application of cysteamine [272].

Gaucher disease type I is the first lysosomal storage disorder that has been treated successfully by enzyme replacement [233]. Compared to other lysosomal storage diseases, the prerequisites for enzyme replacement have been particularly favourable in type I Gaucher disease. Accumulation of the lipid in this disease is restricted to macrophages [348]. These cells express mannose receptors, which can endocytose polypeptides bearing terminal mannose residues on their oligosaccharide side chains and deliver them to lysosomes [372]. /3-D-glucocerebrosidase


can be modified enzymatically to increase the number of terminal mannose residues on oligosaccharide side chains and the modified enzyme is targeted 40-70 times more efficiently to macrophages than the unmodified enzyme [403]. fl-D-glucocerebrosidase purified from human pla- centa has been used in a number of patients with the nonneuronopathic form of Gaucher disease. Enzyme replacement increases hemoglobin and platelet counts and leads to a regression of hepatosplenomegaly within 6 to 12 months of treatment [232,233]. Gaucher patients frequently suffer from skeletal abnormalities and it seems that after the start of enzyme replacement, no new skeletal abnormalities will develop and bone abnormalities may also be reversed [373]. The idea that lysosomal storage disease may be cured by enzyme replacement therapy was fueled by the observation that in cultured cells of patients storage material could be degraded when the missing enzyme was exogenously supplied in the culture medium [374]. At that time, enzymes were not available in amounts sufficient for continuous enzyme replacement in clinical trials. Recombi- nant DNA technology nowadays allows to produce lysosomal enzymes in amounts that will make clinical trials for various lysosomal storage diseases feasible in the near future. Since it can be expected that exogenously supplied enzyme does not penetrate the blood brain barrier, suitable candidates for enzyme replacement are disorders without neurological involvement (e.g., Maroteaux Lamy, Morquio B, Fabry, late onset Pompe disease). However, leaving economical consideration aside, those disease where extra- neural complication is major source of discomfort, pain or death, should not be exempted. This may for example be MPS I, in which respiratory infections, ear infections and obstructive airway disease are common complications [381] or MPS IV, in which subluxation of the odontoid process [381] is a frequent lethal complication.

The prime candidates for enzyme replacement are patients with adult Pompe disease and with the cardiac variant of Fabry disease. In these disorders the target cells are muscle cells, heart muscle cells and endothelial cells, which are readily accessible by the blood stream. In mucopolysaccharidoses it may be more difficult to provide some cells with sufficient enzyme due to poor vasculariza- tion of some target tissues. Thus, hepatosplenomegaly is likely to be treatable, but skeletal abnormalities may be more difficult to influence. Enzyme replacement has been tried with recombinant /3-D-glucuronidase in newborn MPS VII mice [375]. The distribution of the injected enzyme resembled the pattern of expression of the mannose-6- phosphate receptors [376] through which lysosomal enzymes are endocytosed. Most of the substituted enzyme was found in liver and heart, but significant amounts were also found in brain. Closer examination revealed that the brain enzyme is mainly found in blood vessel and meninges but not in neurons [375]. To what extent the pathology and symptoms of the infused animals will be altered has not yet been examined.

The potential use of bone marrow transplantation has been documented in several animal models of lysosomal storage disease [238-240]. Since bone marrow derived macrophages are able to cross the blood brain barrier and reside as microglial cells in the brain, they may turn out to be a useful vector for enzyme delivery into the brain [390] and therefore bone marrow transplantation is more likely to influence neural pathology than enzyme replacement. Improvement of neurologic symptoms and partial reversal of neural lesions after bone marrow transplantation has been seen in several animal models of lysosomal storage disease [167,168,239]. In twitcher mice, this has led to an extended life span, but failed to cure the animals, indicating that the defect was only partially complemented [238]. In the fucosidosis dog, too, improvements of neurologic symptoms were found [240]. In both animal models increased enzyme activities were found in the brain. In the fucosidosis dog, the increased enzyme activities in the brain were not detectable before 6 months after transplantation, indicating that bone marrow derived macrophages infiltrated the brain only slowly. This may explain why the results of bone marrow transplantation were better when animals were transplanted before the development of symptoms [240].

A dramatic improvement of neurologic symptoms and neural pathology has been found in bone marrow transplanted cats, suffering from a-mannosidosis. Treated animals showed little neurologic symptoms up to 2 years after transplantation, while untreated animals deceased after about 6 months of age [169]. Histologically lysosomal storage in neurons was prevented and also reversed. Whether the differences in the effectiveness of bone marrow transplantation in diseases with involvement of the nervous system are due to disease or species related factors is not yet clear.

In humans, bone marrow transplantation has been tried for various lysosomal storage disorders (for review see [401]). Reversal of visceral pathology has been demonstrated in several diseases. In bone marrow transplanted MPS VI patients for instance hepatosplenomegaly decreased within 2 years after transplantation [402]. Improve- ments of neurological symptoms have been documented in several cases [391,392], however, the benefits remain con- troversial. Although in some cases, the course of the disease after bone marrow transplantation has been compared to that of affected untreated siblings, it cannot be excluded that at least parts of the effects that were seen are due to intrafamilial phenotypic variations rather than bone marrow transplantation. Since it has so far only been possible to follow patients for some years it remains to be seen whether the effects of the therapy are satisfying or whether a severe disease has just been converted into a milder but still lethal form. More clinical studies are necessary for evaluation of the therapy.

The combination of gene transfer techniques into hematopoietic stem cells and autologous bone marrow


transplantation will lead to novel approaches for the treatment of lysosomal storage diseases in the near future.

Autologous bone marrow transplantation with retrovi- rally modified cells has been performed in /3-D-glucuronidase deficient mice [242]. Mice expressing different levels of /3-o-glucuronidase from the inserted retrovirus have been obtained. When compared to normal animals up to 2%, 6%, and 26% of enzyme activity was found in liver, spleen and bone marrow of transplanted mice, respectively. There seemed to be a correlation between the amount of enzyme expressed in individual animals and the reduction of storage material, such that high expressers had a more complete reversal of organ pathology. However, in none of the mice was the /3-D-glucuronidase able to correct the skeletal and neuronal abnormalities and no increase in enzyme activity in the central nervous system was found. With the same animal model a different approach was taken by Moullier et al. [262]. Fibroblasts were removed from mutant mice and were infected with a fl-D-glucuronidase cDNA containing retrovirus. These /3-D-glucuronidase expressing fibroblasts were then imbedded into a collagen lattice on plastic fibres. When cells had grown to high density on this support, it was transplanted into the peritoneal cavity of the mutant animals. These lattices were rapidly vascularized and have been termed neoorgans. These neoorgans were kept in the peritoneal cavity for up to 155 days. No uncontrolled growth, fibrosis or immunological reaction was observed. When compared to control animals the transplanted animals showed increased enzyme activities in spleen, liver, lung and other organs. The enzyme activity varied between 0.5% and 6% of normal littermates. The therapeutic effect was mainly seen in liver and spleen, which showed a drastic reduction of storage material. No effect was seen on the bone and brain lesions and this approach may therefore be more suitable for nonneuronopathic diseases.

To achieve an adequate therapy for diseases with neurological symptoms, the development of vectors which directly target neurons or glial cells has to be considered. Two have attracted particular attention, namely the herpes simplex virus and adenovirus derived vectors. In /3-o- glucuronidase-deficient mice, transient in vivo expression in a limited number of neurons has been achieved with herpes simplex-derived vectors [243]. However, the complexity of the herpes virus genome, its tendency to recom- bine and the fact that herpes simplex encephalitis is a life-threatening disease, make it unlikely that this vector will be used in clinical trials in the foreseeable future.

5. Concluding remarks

Work on lysosomal storage diseases during the last years has focused on the cloning of genes and the analysis of mutations. The results of these efforts have shed some light on genotype-phenotype correlations in lysosomal

storage diseases. Three facts became apparent: First, in most lysosomal storage disorders the residual enzyme activity is an important determinant of clinical severity. Second, low residual enzyme activities are sufficient to sustain a normal phenotype and, third, among patients with identical genotype the clinical variability is still remarkable. Other genetic or epigenetic factors have to be postu- lated and only their identification will allow a complete understanding of phenotypic variability. The expectation that the elucidation of phenotypic variability at the molecular level would allow for precise prediction of the clinical course has not been met. In many cases, molecular analysis yielded little practical consequences, because frequently the phenotypic variation occurring on the same genotype makes a prediction of the outcome of the disease on an individual basis impossible. Furthermore, we have learned that apparently small differences in the mutant enzyme can cause different clinical phenotypes. The same codon can be affected by mutations causing different amino acid substitutions. Although in some instances the effects of two mutations on the enzyme seemed to be identical when analysed in heterologous cells, either mutation cause different clinical phenotypes (e.g., Tay Sachs disease and MPS II). Furthermore, the expression of the defect may be tissue-specific or differ in vivo and in vitro. In metachromatic leukodystrophy and Pompe disease the clinical phenotype did not correlate with the residual enzyme activity found in fibroblasts of some patients. Our current knowledge is yet incomplete and our in vitro methods are too crude to understand the complexity of factors that influence the patients phenotype.

Knowledge of mutations has only improved the genetic counselling for diseases which occur at high frequency in a particular ethnic group and where only few alleles account for the majority of all mutant alleles (e.g., Tay Sachs or Gaucher disease in the Ashkenazi Jews). This has made population based screening for particular alleles feasible. In contrast, in most storage diseases heterogeneity of mutant alleles has made the diagnosis based on the detection of mutations unpractical. Simple functional tests like enzyme activity determinations and analysis of metabolites in urine are still the first step in establishing the diagnosis. With particular importance for genetic counselling improvements due the knowledge of specific mutations have been seen with the genetic differentiation of pseudodeficiencies like in Tay Sachs disease and metachromatic leukodystrophy.

Aside from the genotype-phenotype correlations and some improvements of diagnostic tests, the analysis of mutations was expected to reveal aspects of the biological function of lysosomal enzymes. Unfortunately, most of the amino acid substitutions have not been examined with respect to their influence on the particular enzyme. In cases where this has been done, the mutations frequently do not interfere with specific functions of the enzymes. A notable exception is the a-subunit of /3-hexosaminidase A


where several mutations affecting the substrate specificity in different ways cluster in a particular region of the enzyme. The mutations occurring in the saposin genes have allowed to conclude which saposin is involved in the degradation of a particular lipid in vivo. Most of the mutations cause rather unspecific effects like ER retention and rapid degradation and have not contributed to the understanding of specific biological functions of polypeptides. Research on lysosomal storage diseases is now focussing on the development of transgenic mouse models. These are expected to facilitate studies on the molecular mechanism of pathogenesis and in particular trials on the suitability of gene replacement therapies.

Acknowledgements

The author thanks the following colleagues for helpful suggestions, comments and corrections: Hans Andersson, Christoph Peters, Kurt von Figura, Roscoe Brady, John Hopwood, Joel Zlotogora, Yoshiyuki Suzuki, Allessandra D'Azzo, Detlev Schindler. I apologise to all colleagues whose articles I missed or who feel that they have not been cited appropiately. Many articles published in 1994 could not be included.

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