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Human Molecular Genetics Pages 1215-1219  

Importance of the glycosylation and polyadenylation variants in metachromatic leukodystrophy pseudodeficiency phenotype
Introduction
Results
Discussion
Materials And Methods
   Ribonuclease protection assays
   Mutagenesis of the ASA cDNA
   Expression of the ASA cDNA
   Lysosomal targeting of the N350S ASA
Acknowledgements
References

Importance of the glycosylation and polyadenylation variants in metachromatic leukodystrophy pseudodeficiency phenotype

Importance of the glycosylation and polyadenylation variants in metachromatic leukodystrophy pseudodeficiency phenotype

John S. Harvey, William F. Carey, C. Phillip Morris1,*

Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, 5006, Australia and 1CRC for Diagnostic Technologies, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4000, Australia

Received January 5, 1998; Revised and Accepted May 28, 1998

Metachromatic leukodystrophy (MLD) is an inborn error of myelin metabolism caused by a deficiency of the lysosomal hydrolase, arylsulfatase A (ASA). About 1% of the normal population have ASA activity levels approximating those of MLD patients. This non-pathogenic reduction in ASA activity is caused by homozygosity for the ASA pseudodeficiency allele (ASA-PD). Although this allele contains two sequence alterations, a polyadenylation defect and an amino acid substitution (N350S), the reduction in ASA activity previously has been attributed to the polyadenylation defect which reduces the amount of ASA mRNA and hence ASA protein by ~90%. The identification of MLD patients who are homozygous for the ASA-PD allele has brought about the need to re-evaluate the allele in light of the possible role that it may play in the development and progression of disease. Ribonuclease protection assay analysis of ASA mRNA transcripts and an investigation into the activity and lysosomal localization of protein expressed by an ASA expression construct containing the N350S variant indicated that both the N350S and polyadenylation defects play a role in biochemically defining the ASA-PD phenotype. The combined effect of the reduction in ASA mRNA due to the polyadenylation defect and the lowering of ASA activity and aberrant targeting of the expressed N350S ASA protein to the lysosome is estimated to reduce ASA activity in pseudodeficiency homozygotes to ~8% of normal.

INTRODUCTION

Metachromatic leukodystrophy (MLD) is an autosomal recessive inborn error of metabolism caused by a deficiency of the enzyme, arylsulfatase A (ASA). A deficiency of ASA leads to the lysosomal accumulation of the natural substrate, cerebroside sulfate, within nervous tissue and the onset of the characteristic symptoms of MLD (1). Following the isolation of the ASA gene (2), a large number of MLD-causing mutations have been characterized in the ASA gene (3). However, not all deficiencies of ASA lead to the presentation of MLD. The existence of healthy individuals with ASA activity levels close to those seen in MLD patients has long been recognized (4-6). Such individuals are said to have ASA pseudodeficiency (ASA-PD). Wenger and Louie (7) generally defined enzymatic pseudodeficiencies as `the in vitro measurement of low activity of an enzyme in a healthy person such that pseudodeficient individuals may be hard to distinguish from presymptomatic adult onset patients'.

It was soon discovered that the normal and ASA-PD gene sequences differed by two A->G transitions (8). The first causes the substitution of a serine residue for a glycosylated asparagine at amino acid 350 (N350S). The loss of this glycosylation site accounts for observed structural differences in the ASA enzyme in normal and pseudodeficient individuals (9,10). The introduction of the N350S mutation into the normal ASA cDNA was reported to have no effect on the rate of synthesis, stability or specific activity of the ASA enzyme (8).

The ASA gene produces three different mRNA species of 2.1, 3.7 and 4.8 kb in length. All transcripts apparently encode an identical 507 amino acid polypeptide and vary only in the amount of their 3[prime]-untranslated sequence. The reduction in ASA activity caused by ASA-PD was attributed to a second A->G change which altered the sequence of the major 2.1 kb polyadenylation signal (AATAAC to AGTAAC). Gieselmann et al. (8) estimated from northern analysis that the 2.1 kb mRNA species accounted for ~90% of the total ASA mRNA in normal individuals and that this transcript was absent in individuals who were homozygous for the ASA-PD allele. This reduction in available ASA mRNA was reported to account for the 90% reduction in the level of ASA activity seen in ASA-PD individuals (8).

Nelson et al. (11), Chabás et al. (12) and Barth et al. (13) determined that the frequency of the ASA-PD allele was 9.4, 12.7 and 12.3% in the Australian, Spanish and British populations, respectively. Additionally, the N350S mutation was found without the polyadenylation defect in 4.8 and 5.2% of alleles in the Australian and British populations, respectively (11,13).

The ASA-PD allele has a pronounced effect on ASA activity and is found on the same allele as known MLD mutations. It is therefore important to understand the extent to which the ASA-PD allele reduces ASA activity and the mechanisms by which this reduction occurs. This has provoked a re-evaluation of the allele by examining the effect of the N350S and polyadenylation defects on ASA activity.

A ribonuclease protection assay was used to quantify the effect of the polyadenylation defect on the levels of the three ASA mRNA species. The effect of the N350S variant was also evaluated by expressing recombinant ASA containing N350S and measuring its activity and lysosomal targeting compared with that of the normal recombinant ASA.

RESULTS

The level of in vitro expression of the normal and N350S ASA expression constructs was compared to gauge the effect that the presence of the N350S mutation had on the activity of the ASA enzyme. The amino acid substitution was found to reduce the activity of the enzyme to 57 ± 15% (n = 3) of normal (Fig. 1). The background level of ASA activity in untransformed Chinese hamster ovary (CHO) cells was undetectable, i.e. <0.01% of the normal ASA expression construct (data not shown).


Figure 1. Expression of the N350S construct. An ASA cDNA construct containing the N350S variant was assayed in three separate experiments (columns 1-3). The measured level of ASA activity from each experiment is expressed as a percentage of that measured in the normal construct. Column four (shaded) shows the mean level of expression. The error bar represents one standard deviation.

Since the N350S mutation removes a glycosylated asparagine residue from ASA (14), it may remove a lysosomal targeting signal (15) and thereby reduce the in vivo amount of ASA available for substrate degradation. In order to investigate the targeting of the N350S ASA protein to the lysosome, organelles from CHO cells expressing normal and N350S ASA expression constructs were fractionated on Percoll/sucrose density gradients (Fig. 2) (16). The twentieth fraction of both gradients was found to contain the highest level of [beta]-hexosaminidase ([beta]-hex) activity and hence were identified as the lysosome-containing fractions. The ASA activity in the normal gradient was also confined to the twentieth fraction, indicating that all measurable ASA activity within the gradient was contained within the lysosomes (Fig. 2a). In contrast, only 45% of the ASA containing N350S was found in the lysosomes; the remaining 55% was found in non-lysosomal fractions (Fig. 2b) and would therefore be unavailable for the in vivo hydrolysis of sulfatide.


Figure 2. Lysosomal targeting of the normal and N350S ASA enzymes. Analysis of the Percoll/sucrose gradient fractions from CHO cell lines expressing the normal (a) and N350S (b) ASA expression constructs. Solid and dashed lines show ASA and [beta]-hexosaminidase activity, respectively. Units on the left and right y-axes of each graph represent [beta]-hexosaminidase and ASA activity, respectively (expressed as nmol/min/ml).

These results strongly suggest that the N350S amino acid substitution not only affects the activity of the ASA enzyme but also its localization to the lysosome. Together, these two factors are expected to reduce the in vivo level of lysosomal ASA activity to ~25% of normal. The presence of this variant must therefore substantially contribute to reducing ASA activity in ASA-PD homozygotes.

A ribonuclease protection assay was used to measure the proportional loss of the 2.1 kb ASA mRNA caused by the presence of the ASA-PD polyadenylation sequence variant. Normal and pseudodeficient individuals were analysed using both total RNA and cell lysates as targets for the hybridization of antisense ASA and glyceraldehyde phosphate dehydrogenase (GAPDH) control antisense RNA probes. Figure 3 shows that the 2.1 kb ASA mRNA transcript was undetectable in individuals who were homozygous for the ASA-PD allele.


Figure 3. Ribonuclease protection assay analysis of the ASA mRNA species in normal and pseudodeficient cells. The ASA (300 bp) and GAPDH (164 bp) antisense probes were hybridized to total RNA (Gel 1) and cell lysate (Gel 2). Gel 1: lanes A and G, contain the transcribed ASA and GAPDH antisense probes respectively, the third unmarked lane is blank; tracks 1-3 are of normal control individuals, tracks 4 and 5 are from individuals homozygous for the ASA-PD allele; Gel 2: tracks 1 and 2 are from normal controls and tracks 3 and 4 are from ASA-PD homozygotes. The ASA probe protects 264 bases of the 3.7 and 4.8 kb ASA mRNAs (P1) and 192 bases of the 2.1 kb ASA mRNA transcript (P2). The GAPDH control probe protects a 120 base fragment of the GAPDH mRNA (P3).

The relative contribution of the 2.1 kb and the combined 3.7 + 4.8 kb transcripts to the total amount of ASA mRNA was calculated for each normal and pseudodeficient individual. In normal individuals (n = 5), the 2.1 kb mRNA species accounted for 71 ± 2.5% of the total available ASA mRNA. The remaining 29% was made up of the two longer transcripts (Fig. 4a). This figure contrasts with the estimate of Gieselmann et al. (8) that the longer transcript made up only 10% of ASA mRNA. Conversely, in ASA-PD homozygotes (n = 4), the frequency of the major 2.1 kb transcript was reduced to undetectable levels. In these individuals, the combined 3.7 and 4.8 kb ASA mRNA species was >3-fold increased over normal, from 29% of total ASA mRNA to 96.3 ± 3.0% in ASA-PD homozygotes (Fig. 4b).


Figure 4. Contribution of the 2.1 kb and the combined 3.7 + 4.8 kb ASA transcripts to total ASA mRNA. The relative frequency of each ASA transcript has been calculated as a percentage of the total amount of ASA mRNA produced by the 2.1 kb and the combined 3.7 + 4.8 kb polyadenylation sites in normal (a) and pseudodeficient (b) individuals. Open and shaded bars represent the relative contribution of the 2.1 kb and the combined 3.7 + 4.8 kb polyadenylated mRNAs, respectively. The final column on each figure presents the mean contribution of each mRNA species. Error bars represent one standard deviation from the mean.

The question of whether the 3.7 and 4.8 kb polyadenylation sites are utilized with an increased frequency in pseudodeficient individuals is an important one. Any compensation for the loss of the 2.1 kb site by increased utilization of downstream termination signals could act to reduce the deleterious biochemical effects of the polyadenylation defect, provided that all species are translated with the same efficiency.

Like ASA, GAPDH is expressed as a house-keeping gene and is therefore suitable for use as an internal ribonuclease protection assay control (17). In order to standardize the amount of the 2.1 kb and the combined 3.7 + 4.8 kb ASA mRNAs in each assay, the frequency of the transcripts was calculated relative to that of GAPDH for each normal and pseudodeficient individual, the pooled results of this analysis are presented in Table 1.

The average usage of the downstream termination signals, relative to GAPDH, is almost identical in pseudodeficient and normal individuals (Fig. 5), suggesting that there is no increased use of the 3.7 and 4.8 kb polyadenylation sites in the absence of the 2.1 kb site. The mechanisms which regulate this relative usage are unknown.

DISCUSSION

The N350S and polyadenylation defects, which together make up the ASA-PD allele, are both important to the biochemical definition of ASA-PD. The N350S mutation is known to remove a glycosylated asparagine residue from the ASA polypeptide (14) which may be involved in targeting of ASA to the lysosome as well as affecting the catalytic activity of the enzyme. We have shown that the N350S ASA protein has reduced activity in comparison with the normal clone, and only ~45% reaches its normal functional site within the cell, i.e. the lysosome. The combination of these two effects of the N350S variant is expected to reduce the in vivo lysosomal ASA activity by ~75%, while we have shown that the polyadenylation defect reduces the total amount of available ASA mRNA to ~31% of normal. Together on the same pseudodeficiency allele, these two mutations are expected to reduce ASA activity to ~8% of normal, a value slightly lower than the original estimate of 10% of normal by Gieselmann et al. (8).

Table 1. Levels of the 2.1 kb and combined 3.7 + 4.8 kb ASA mRNAs in normal and ASA-PD individuals, expressed as a proportion of the GAPDH control and as a percentage of the total ASA mRNA in normal cells
ASA mRNA species Proportion of transcript relative to the GAPDH control mRNA Percent of total ASA mRNA in normal controls (%)
Normal (n = 5) ASA-PD (n = 4) Normal (n = 5) ASA-PD (n = 4)
2.1 kb 0.0946 0.00144 71 1.1
3.7 + 4.8 kb 0.038 0.0398 29 30
Total 0.1326 0.04124 100 31.1


Figure 5. Mean use of the 2.1 kb and the combined 3.7 + 4.8 kb ASA mRNAs in pseudodeficient and normal control individuals normalized relative to the GAPDH control. ASA levels are expressed as a proportion of the GAPDH mRNA level. Error bars represent one standard deviation from the mean. Open bars represent the frequency of the 2.1 kb ASA transcripts and shaded bars represent the frequency of the combined 3.7 + 4.8 kb transcripts.

The hypothesis that the N350S mutation had no effect on the activity of the ASA enzyme has been questioned previously. Francis et al. (18) identified an individual who carried the N350S allele without the polyadenylation defect. This individual was also an obligate MLD heterozygote (N350S/MLD). The level of ASA activity measured in this individual was significantly below that expected of a `classical' MLD heterozygote, suggesting that N350S played a role in defining the biochemical phenotype of the ASA-PD allele. This idea was supported further by a similar observation made by Shen et al. (19), who reported lower than expected levels of ASA activity in a compound heterozygote for the N350S variant and an MLD mutation. Shen et al. (19) estimated that the N350S variant reduced ASA activity to 12-21% of normal. Barth et al. (13) also suggested that N350S may slightly reduce ASA activity after identifying an individual with ASA activity below the normal range who was an N350S homozygote but who did not carry the ASA-PD polyadenylation defect.

The data presented here, suggesting that the N350S allele does contribute to the biochemical phenotype of the ASA-PD allele, do not necessarily contradict previous observations which were used to support the hypothesis that N350S was benign. A recent analysis by Leistner et al. (20) for example, of an N350S heterozygote who did not carry the polyadenylation defect, supported the hypothesis that the N350S mutation was benign, since the measured level of ASA activity in total cell lysate of this individual was within the normal range. In view of the present study, an N350S heterozygote would be expected to have ~79% of normal ASA activity in the total cell lysate assay used by Leistner et al. (20). Therefore, the presence of a single heterozygous copy of the N350S sequence variant would not be expected to alter the classification of this individual within the normal range.

The observations of Barth et al. (13) and Shen et al. (19) have significant clinical consequences and can now be explained by our observation that the N350S mutation does reduce ASA activity in the absence of the ASA-PD polyadenylation defect. Furthermore, given the frequency with which the N350S alteration occurs within the Australian MLD patient population, it is also possible that the presence of N350S may also play a role in defining disease severity when it is found in combination with disease-causing MLD mutations by acting to reduce ASA activity additively.

Our evaluation of the overall effect of the ASA-PD allele on ASA activity in pseudodeficient individuals is in close agreement with the original estimates made from northern analysis of ASA transcripts by Gieselmann et al. (8); however, the results presented here provide an alternative explanation of the cause of this reduction.

MATERIALS AND METHODS

Ribonuclease protection assays

A 264 bp ASA PCR product amplified using the forward, Q486 (5[prime]-CGCCCTGCAGATCTGCTG-3[prime]) (21), and reverse, ON5 (5[prime]-TTCCTCATTCGTACCACAGG-3[prime]) (22), primers, which span the polyadenylation site of the 2.1 kb ASA mRNA species, was cloned into the pGemT PCR cloning vector (Promega), according to the manufacturer's instructions. A control template containing a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene fragment was supplied by Dr Greg Goodall (Hanson Research Centre, Adelaide).

The ASA and GAPDH templates were linearized with the restriction endonucleases NotI and DdeI (Boehringer Mannheim), respectively, under conditions recommended by the manufacturer. Antisense RNA probes were synthesized from the restricted ASA and GAPDH clones using a Riboprobe T7 System transcription kit (Promega) as recommended by the supplier. Ribonuclease protection assays were performed on total RNA and cell lysates using a Lysate Ribonuclease Protection Kit (US Biochemicals) according to the manufacturer's instructions.

Ribonuclease protection assay gels were visualized using a Molecular Dynamics phosphorimager, and protected bands, representing the 2.1 kb and the combined 3.7 + 4.8 kb ASA mRNAs as well as the GAPDH control, were quantified using the integral volume functions of Molecular Dynamics, ImageQuant version 3.3, as described by the supplier.

Mutagenesis of the ASA cDNA

The ASA cDNA HT14/CP8 (2) was cloned directionally into M13mp19 by restricting with KpnI and SacI, the vector and insert bands were purified on an agarose gel, then ligated with the insert band in 3-fold molar excess. The ASA cDNA was mutagenized in M13 using an oligonucleotide encoding the mutant sequence of N350S (5[prime]-AAGGTGACACTGGGCAGTGG-3[prime] (8) as described by Sambrook et al. (23).

Expression of the ASA cDNA

NcoI-NotI-restricted ASA cDNA, HT14/CP8 and the rat pre-proinsulin 5[prime]-untranslated sequence (HindIII-NcoI) was ligated into the HindIII-NotI-restricted expression vector pRSVN.07 in a single reaction. The pre-proinsulin sequence was encoded by two homologous oligonucleotides, 5[prime]-AGCTTCTAGACCAGCTACGTCGAAACCATCAGCAAGCAGGTCATTGTTCCAAAC-3[prime] and 5[prime]-CATGGTTTGGAACAATGACCTGCTTGCTGATGGTTTCGACTGTAGCTGGTCTAGA-3[prime], synthesized on an Applied Biosystems DNA synthesizer. Two micrograms of each oligonucleotide were annealed in a 500 ml, 80°C water bath, which was allowed to cool to room temperature prior to the ligation reaction. The ASA cDNA and 5[prime]-untranslated sequences were present in equimolar concentrations and in 3-fold molar excess over the vector DNA. An NcoI-NotI restriction fragment containing the N350S mutagenized sequence was subcloned into the ASA expression vector. The mutagenized vector construct was sequenced to ensure that no unwanted changes had been introduced.

All ASA expression constructs were transfected into CHO cells using DOTAP transfection reagent under conditions recommended by the supplier (Boehringer Mannheim). The ASA activity of the normal and N350S expression constructs was measured in mass cultures of G418-resistant cells. The two cell lines were subcultured and grown synchronously, and assayed when the cells had reached a quiescent stage of growth, 4 days post-confluence. The expression of the N350S cell line was calculated as a percentage of normal in three separate experiments using the artificial substrate p-nitrocatechol-sulfate as described by Baum et al. (24).

Lysosomal targeting of the N350S ASA

Confluent flasks of CHO cells expressing normal and N350S ASA constructs were subjected to subcellular fractionation on Percoll/sucrose gradients as described by Brooks et al. (16). Each fraction was assayed for [beta]-hexosaminidase activity to identify lysosome-containing fractions (2). The fractionated organelles subsequently were assayed for ASA activity.

ACKNOWLEDGEMENTS

The authors are grateful to Dr Volkmar Gieselmann (Christian Albrechts University, Kiel) for supplying the ASA cDNA clone, to Dr Alan Robins (Department of Biochemistry, University of Adelaide) for supplying the pRSVN.07 expression vector, and to Dr Greg Goodall (Hanson Cancer Research Centre, Adelaide) for advice with the ribonuclease protection assay and for providing the GAPDH control template. This work was supported by the Women's and Children's Hospital Research Foundation and the National Health and Medical Research Council of Australia.

REFERENCES

1. Kolodny, E.H. and Fluharty, A.L. (1995) Metachromatic leukodystrophy and multiple sulfatase deficiency: sulfatide lipidosis. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic and Molecular Bases of Inherited Disease, 7th edn. McGraw-Hill, New York, pp. 2693-2739.

2. Stein, C., Gieselmann, V., Kreysing, J., Schmidt, B., Pohlmann, R., Waheed, A., Meyer, H.E., O'Brien, J.S. and von Figura, K. (1989) Cloning and expression of human arylsulfatase A. J. Biol. Chem., 264, 1252-1259. MEDLINE Abstract

3. Gieselmann, V., Zlotogora, J., Harris, A., Wenger, D.A. and Morris, C.P. (1994) Molecular genetics of metachromatic leukodystrophy. Hum. Mutat., 4, 233-242. MEDLINE Abstract

4. Dubois, G., Turpin, J.-C. and Baumann, N. (1975) Absence of ASA activity in healthy father of a patient with metachromatic leukodystrophy. N. Engl. J. Med., 293, 302.

5. Dubois, G., Harzer, K. and Baumann, N. (1977) Very low arylsulfatase A and cerebroside sulfatase activities in leukocytes of health members of metachromatic leukodystrophy family. Am. J. Hum. Genet., 29, 191-194. MEDLINE Abstract

6. Lott, I.T., Dulaney, J.T., Milunsky, A., Hoefnagel, D. and Moser, H.W. (1976) Apparent biochemical homozygosity in two obligate heterozygotes for metachromatic leukodystrophy. J. Pediatr., 89, 438-440. MEDLINE Abstract

7. Wenger, D.A. and Louie, E. (1991) Pseudodeficiencies of arylsulfatase A and galactocerebrosidase activities. Dev. Neurosci., 13, 216-222. MEDLINE Abstract

8. Gieselmann, V., Polten, A., Kreysing, J. and von Figura, K. (1989) Arylsulfatase A in pseudodeficiency: loss of a polyadenylylation signal and N-glycosylation site. Proc. Natl Acad. Sci. USA, 86, 9436-9440. MEDLINE Abstract

9. Fluharty, A.L., Meek, W.E. and Kihara, H. (1983) Pseudo arylsulfatase A deficiency: evidence for a structurally altered enzyme. Biochem. Biophys. Res. Commun., 112, 191-197. MEDLINE Abstract

10. Chang, P.L. and Davidson, R.G. (1983) Pseudo arylsulfatase A deficiency in healthy individuals: genetic and biochemical relationship to metachromatic leukodystrophy. Proc. Natl Acad. Sci. USA, 80, 7323-7327. MEDLINE Abstract

11. Nelson, P.V., Carey, W.F. and Morris, C.P. (1991) Population frequency of the arylsulfatase A pseudo-deficiency allele. Hum. Genet., 87, 87-88. MEDLINE Abstract

12. Chabás, A., Castellvi, S., Bayés, M., Balcells, S., Grinberg, D., Vilageliu, L., Marfany, G., Lissens, W. and Gonzalez-Duarte, R. (1993) Frequency of the arylsulphatase A pseudodeficiency allele in the Spanish population. Clin. Genet., 44, 320-323. MEDLINE Abstract

13. Barth, M.L., Fensom, A. and Harris, A. (1994) The arylsulphatase A gene and molecular genetics of metachromatic leucodystrophy. J. Med. Genet., 31, 663-666. MEDLINE Abstract

14. Herz, B. and Bach, G. (1984) Arylsulfatase A in pseudodeficiency. Hum. Genet., 66, 147-150. MEDLINE Abstract

15. Kornfeld, S. (1992) Structure and function of the mannose 6-phosphate/insulin like growth factor II receptors. Annu. Rev. Biochem., 61, 307-330. MEDLINE Abstract

16. Brooks, D.A., Harper, G.S., Gibson, G.J., Ashton, L.J., Taylor, J.A., McCourt, P.A.G., Freeman, C., Clements, P.R., Hoffmann, J.W. and Hopwood, J.J. (1992) Hurler syndrome: a patient with abnormally high levels of [alpha]-l-iduronidase protein. Biochem. Med. Metab. Biol., 47, 211-220.

17. Lagnado, C., Brown, S. and Goodall, G. (1194) AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A). Mol. Cell. Biol., 14, 7984-7995.

18. Francis, G.S., Bonni, A., Shen, N., Hechtman, P., Yamut, B., Carpenter, S., Karpati, G. and Chang, P.L. (1993) Metachromatic leukodystrophy: multiple non-functional and pseudodeficiency alleles in a pedigree: problems with diagnosis and counselling. Ann. Neurol., 34, 212-218. MEDLINE Abstract

19. Shen, N., Li, Z.-G., Waye, J.S., Francis, G. and Chang, P.L. (1993) Complications in the genotypic molecular diagnosis of pseudo arylsulfatase A deficiency. Am. J. Med. Genet., 45, 631-637. MEDLINE Abstract

20. Leistner, S., Young, E., Meaney, C. and Winchester, B. (1995) Pseudodeficiency of arylsulphatase A: strategy for clarification of genotype in families of subjects with low ASA activity and neurological symptoms. J. Inherited Metab. Dis., 18, 710-716. MEDLINE Abstract

21. Harvey, J.S., Carey, W.F., Nelson, P.V. and Morris, C.P. (1994) Metachromatic leukodystrophy: a nonsense mutation (Q486X) in the arylsulphatase A gene. Hum. Mol. Genet., 3, 207. MEDLINE Abstract

22. Gieselmann, V., Fluharty, A.L., Tonnesen, T. and Von Figura, K. (1991) Mutations in the arylsulfatase A pseudodeficiency allele causing metachromatic leukodystrophy. Am. J. Hum. Genet., 49, 407-413. MEDLINE Abstract

23. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

24. Baum, H., Dodgson, K.S. and Spencer, B. (1959) The assay of arylsulfatases A and B in human urine. Clin. Chim. Acta, 4, 453-458.

*To whom correspondence should be addressed. Tel: +61 7 3864 1427; Fax: +61 7 3864 1534; Email: p.morris{at}qut.edu.au

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