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Elevated sulfatide excretion in compound heterozygotes of metachromatic leukodystrophy and ASA-pseudodeficiency allele
Clinical Biochemistry,Vol, 30, No. 4, 325-331, 1997 Copyright © 1997 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/97 $17.00 + .00 ELSEVIER
PII S0009-9120(97)00033-7
Elevated Sulfatide Excretion in Compound Heterozygotes of Metachromatic Leukodystrophy and ASA-Pseudodeficiency Allele AGNIESZKA EUGOWSKA, 1 ANNA TYLKI-SZYMANSKA, 1 JOHANNES BERGER,2 and BRUNHILDE MOLZER2 1The Children's Memorial Health Institute, Department of Metabolic Diseases, Warsaw, Poland, and 2Clinical Institute of Neurology, Department of Neuropathology and Neurochemistry, University of Vienna, Austria Objective: Use of sulfatide excretion in differentiating MLD/PDheterozygotes from MLD-patients and PD/PD-homozygotes. Design and Methods: Sulfatide was extracted from urine sediment with chloroform/methanol (2:1, v/v). The quantity of sulfatide was measured densitometrically (h = 580 nm) after thin-layer chromatography. ASA and ~-galactosidase activities were assayed enzymatically. Results: MLD/PD-heterozygotes excreted sulfatide in the range of 4.8-36.3 nmol/mg lipid (mean -+ SD = 17.8 _+ 10.7), whereas sulfatide in MLD-patients ranged from 74.3-411.6 nmol/mg lipid (mean -+ SD = 184.5 +- 130.8) and in PD/PD-homozygotes sulfatide excretion remained in normal range of 0.0-5.9 nmol/mg lipid (mean + SD = 1.64 _+ 2.12). ASA activities in these groups were very low or lowered. Conclusions: The quantitative measurement of sulfatide in urine allows differentiation between MLD/PD-heterozygotes and MLDheterozygotes, as well as between MLD/PD-heterozygotes with very low ASA activity and MLD-patients or PD/PD-homozygotes. The quantitative measurement of sulfatide in urine differs between MLD-carriers and controls.
KEY WORDS: metachromatic leukodystrophy; sulfatide; arylsulfatase A; arylsulfatase A-pseudodeficiency; lysosomal storage disease. Introduction
rylsulfatase A (ASA, EC 3.1.6.1) is a lysosomal A enzyme essential for degradation of sulfatide, an important glycosphingolipid component of myelin. Inability of ASA to decompose sulfatide leads to accumulation of this lipid in the myelin of the central and peripheral nervous system, causing metachromatic leukodystrophy (MLD, McKusick 250100)--a recessively inherited, fatal, demyelinating disorder. Sulfatide also accumulates in some M a n u s c r i p t r e c e i v e d S e p t e m b e r 30, 1996; r e v i s e d a n d a c c e p t e d J a n u a r y 5, 1997.
Correspondence: Agnieszka Lugowska, M.Sc., The Children's Memorial Health Institute, ZDL, Department of Metabolic Diseases, A1. Dzieci Polskich 20, 04-736 Warsaw, Poland. CLINICAL BIOCHEMISTRY, VOLUME 30, J U N E 1997
visceral organs of affected patients, e.g., kidneys or gallbladder, and are excreted in high amounts in urine and bile (1). So far, at least 60 MLD-specific mutations in the ASA gene have been disclosed (2-8). MLD-related mutations can be divided into two groups: alleles associated with no residual enzyme activity (0 alleles), and alleles encoding for low residual enzyme activity (R alleles) (9). Patients homozygous for 0 alleles suffer from the severe, late-infantile MLD. Compound heterozygosity for 0 and R alleles results in the juvenile MLD, whereas presence of two R alleles leads predominantly to adult MLD (10). There are also two mutations in the ASA gene associated with so called ASA-pseudodeficiency (PD). Particularly the loss of the polyadenylation signal leads to reduced synthesis of ASA polypeptides. However, the enzyme activity is still sufficient for normal sulfatide degradation. The second mutation, loss of the glycosylation site is of minor importance. It is thought that homozygotes of ASA-PD (0.5-2% of the population) neither show clinical symptoms of MLD, nor do they excrete elevated amounts of sulfatide in urine. Seven to 15% of the population bear the ASA-PD allele. The PD mutations are detectable by PCR. In diagnosing of metachromatic leukodystrophy, measurement of ASA activity in leukocytes is commonly used. It is hardly possible to reliably differentiate MLD/PD-heterozygotes from ASA-pseudodeficiency homozygotes or MLD patients with high residual ASA activity by means of artificial substrates (11,12). High frequency of pseudodeficiency allele in general population (7-15%), so among relatives of probands as well, demands a particular procedure in pre- and postnatal diagnosing of MLD. Our objective was to use the measurement of sulfatide excretion in differentiating MLD/PD-heterozygotes from MLD patients and PD/PD-homozygotes, as well as to compare the biochemical findings 325
LUGOWSKA ET AL.
and to find the differences between genetically defined MLD/PD compound heterozygotes, MLD-heterozygotes, and other groups of subjects. Materials
and methods
Biological material consisted of leukocytes isolated from venous blood and 24 h urine collections sediments obtained from subjects investigated in this study: • MLD/PD compound heterozygotes (n = 6): 3 females and 3 males, age 3 0 - 4 4 y, parents of MLD patients additionally carrying arylsulfatase A-pseudodeficiency allele; • MLD-heterozygotes (n -- 23): 12 females and 11 males, age 23-55 y, parents of MLD patients not carrying arylsulfatase A-pseudodeficiency allele; • MLD patients (n = 8): 3 females and 5 males, age 2-17 y, children at different ages affected with MLD; • PD/PD-homozygotes (n = 7): 2 females and 5 males, age 1-9 y, children homozygous for ASA pseudodeficiency allele, not affected with MLD; • NO/PD-heterozygotes (n = 5): 3 females and 2 males, age 2 7 - 4 0 y, unrelated healthy persons heterozygous for ASA pseudodeficiency allele; • Controls (n = 30): 18 females and 12 males, age 2 6 - 5 0 y, 10 persons of staff of the Children's Memorial Health Institute in Warsaw and 20 parents of children with cerebral palsy not carrying the ASA-pseudodeficiency allele. Bovine serum albumine, p-nitrocatechol, p-nitrocatechol sulfate, dimethylsulfoxide (DMSO), 4-methylumbelliferon, were purchased from the Sigma Chemical Co. (St. Louis, MO, USA); 4-methylumbelliferyl-~-D-galactopyranoside was purchased from Koch-Light Laboratories Ltd.; methanol, from Riedel de Hahn AG; florisil 6 0 - 1 0 0 mesh, from Fluka Chemika; bovine brain sulfatide, from Supelco; Tris and ethidium bromide, from United States Biochemical Co.; SDS and mineral oil, from Serva; agarose, from Gibco BRL & Life Technologies; glass fiber filters GF/A 15.0 cm, from W h a t m a n International Ltd.; proteinase K, was from Boehringer Mannheim Biochemica; Taq-DNA polimerase, from Biomedica; and XDNA HindIII/EcoRI Digest, from proMega. All other chemicals were purchased from Merck. Leukocytes were isolated from 10 mL of heparinized venous blood, contaminating erythrocytes were removed by hypotonic shock (13,14). Leukocytes were suspended in 1.0 mL distilled water and sonified (30 s, 40 cycles). The homogenate was centrifuged 30 min, 4500 rpm. The pellet consisting of cellular debris was used for isolation of DNA. Enzymatic assays were carried out in supernatants. Protein content in supernatants was determined by the use of Folin-Ciocalteau reagent according to the method of Lowry et al. (15). ASA activity was determined in supernatants with 0.01 mol/L p-nitrocatechol sulfate in 0.5 mol/L 326
acetate buffer pH 5.0 as substrate. The reaction mixture contained also sodium pirophosphate in 0.5 mol/L acetate buffer pH 5.0 (11.2 mg/50 mL). Probes were incubated 30 min in 37°C. The enzymatic reaction was stopped with 1 mol/L N a O H as previously described by Bass et al. (16). The absorbance of assayed samples was measured at X = 510 nm in Hitachi U-2000 spectrofotometer. ASA activity was expressed in nmoles ofp-nitrocatechol/mg protein/h. Beta-galactosidase activity was determined in homogenized leukocytes supernatants as control enzyme with 1 mmol]L 4-methylumbelliferyl-~-D-galactopyranoside in 0.1 mol/L citrate buffer pH 4.5, as previously described by Suzuki et al. (17). After 1 h incubation in 37°C, the reaction was stopped by adding of 0.2 mol/L glycine-NaOH buffer pH 10.5. Fluorescence was measured in type FP777 Jasco spectrofluorimeter at excitation X = 364 nm and emission X = 446 nm. Beta-galactosidase activity was expressed in nmoles of 4-methylumbelliferon/mg protein/h. Sulfatides were determined in 24 h urine collections. Urine samples were kept at - 2 0 ° C until processing. Urine was centrifuged at 16.000 g for 30 min. The sediment was extracted in a glass homogenizer with 30 mL of chloroform/methanol mixture (2:1; v/v; C/M). After Folch partition the lipid fraction was applied on a glass column (0.5 z 15 cm) filled with Florisil in chloroform. After removal of neutral lipids with 15 mL of chloroform, glycolipids were eluted with 40 mL C/M. Aliquots of eluate were chromatographed on silica gel G-60 precoated plates in C/M/water (70:30:5, v/v/v). As standards 2-20 nmol of bovine brain sulfatides were applied (linearity preserved between 0 and 10 nmol). Sulfatides were visualized with orcinol/sulfuric acid/water (0.2 g/75 mL/25 mL), and quantified densitometrically in type CS9000 Shimadzu densitometer at X = 580 nm (limit of detection was 0.5 nmol) (18). Results are given as nmoles sulfatides/mg lipid. DNA was isolated from cellular debris according to the method of Maniatis with proteinase K (20 mg/mL) overnight digestion at 55°C. DNA was extracted by phenol/chloroform (1:1, v/v), precipitated with ice-cold 96% ethanol and 3 mol/L sodium acetate, washed with 70% ethanol, and redissolved in 40 ~L1of water (19). ASA pseudodeficiency allele was detected in genomic DNA using a PCR-method with a pair of specific primers as previously described by Gieselmann (20). Samples of investigated DNA (approximately 0.1 ~g) were amplified with 1.25 U/sample Taq polymerase in OmniGene-Hybaid thermocycler. Thirty-five cycles of 1 min denaturation at 93.7°C, 1 min annealing at 58°C, and 1 min elongation at 72°C were performed. Amplification products were electrophoresed in 1.2% agarose gels. DNA fragments were visualized by ethidium bromide staining; XDNA HindIII/EcoRI Digest was applied as molecular weight standard. CLINICAL BIOCHEMISTRY, VOLUME 30, JUNE 1997
SULFATIDE IN MLD/PD-HETEROZYGOTES TABLE 1 ASA Activity, ASA/~-Gal Ratio, and Sulfatide Excretion in MLD/PD-Heterozygotes, MLD-Heterozygotes, MLD Patients, and Controls
Subjects MLD/PD-het. (n = 6) MLD-het. (n = 23) MLD patients (n = 8) NO/PD (n = 5) PD/PD (n = 7) Controls (n = 30)
ASA activity a [nmol/mg p/h] mean - SD (range) median
ASA/[3-Gal mean _+ SD (range) median
Gal-Sulfatide b [nmol/mg lipid] mean -+ SD (range) median
42.00 + 31.52 (14-89) med = 33.0 67.26 -+ 22.44 (31-107) med = 74.0 15.63 _ 6.32 (9-25) med = 13.5 81.20 _+ 26.42 (57-117) med = 70.0 47.71 _+ 18.56 (25-74) med = 53.0 140.57 _+ 55.75 (75-288) med = 133.5
0.12 _+ 0.06 (0.06-0.21) med = 0.11 0.20 +_ 0.05 (0.10-0.32) med = 0.20 0.05 _+ 0.02 (0.03-0.08) med = 0.05 0.27 _+ 0.05 (0.22-0.34) med = 0.28 0.23 _+ 0.14 (0.11-0.50) med = 0.18 0.43 _+ 0.12 (0.23-0.87) med = 0.40
17.8 -+ 10.7 (4.8-36.3) med = 19.3 7.4 -+ 4.6 (1.3-16.1) med = 5.5 184.5 _+ 130.8 (n = 5) (74.3-411.6) med = 145.5 1.7 _+ 2.1 (n = 3) (0.0-4.1) reed = 1.0 1.64 _+ 2.12 c (0.0-5.9) med = 1.4 2.2 _+ 1.7 (n = 29) (0.0-5.9) med = 1.9
"Estimated in leukocytes. bEstimated in 24 hrs urine sediment. tin 3 of 7 PD/PD homozygotes sulfatide excretion was not detectable; this seems to be reason that SD is higher than mean value. Results R e s u l t s of ASA activity, A S A / ~ - g a l a c t o s i d a s e ratio, a n d s u l f a t i d e e x c r e t i o n in i n d i v i d u a l g r o u p s a r e c o m p i l e d in T a b l e 1. I n t h e g r o u p of six M L D / P D - h e t e r o z y g o t e s ASA a c t i v i t y r e v e a l e d v a l u e s b e t w e e n 14 a n d 89 n m o l / m g p r o t e i n / h w i t h m e a n v a l u e _+ SD = 42.00 _+ 31.52 a n d m e d i a n = 33.0. T h e s e v a l u e s a r e located bet w e e n v a l u e s c h a r a c t e r i s t i c of M L D - p a t i e n t s a n d M L D - h e t e r o z y g o t e s (Table 1). T h e r a n g e s of ASA a c t i v i t i e s in t h e g r o u p s of M L D / P D - , M L D - h e t e r o z y gotes, M L D p a t i e n t s , a n d P D / P D - h o m o z y g o t e s m o r e or less o v e r l a p p e d ( F i g u r e 1). N o n p a r a m e t r i c M a n n W h i t n e y t e s t of m e d i a n s revealed insignificant differences "(Z >- 0.05) for: MLD/PD- a n d MLD-heterozygotes, MLD/PD-heterozygotes a n d M L D patients, MLD/PD-heterozygotes a n d PD/PD-homozygotes. T h e r a n g e of ASA/[3-galactosidase r a t i o s in M L D / P D g r o u p did not o v e r l a p w i t h t h e r a n g e c h a r a c t e r i s t i c of controls a n d e q u a l l e d 0 . 0 6 - 0 . 2 1 ; m e a n __ SD = 0.12 __ 0.06 a n d m e d i a n = 0.11 ( F i g u r e 2). M e a n a n d m e d i a n v a l u e s in M L D / P D , M L D - h e t e r o z y g o t e s a n d M L D p a t i e n t s w e r e well d i s c r i m i n a t e d (Table 1). ASA/[3-galactosidase r a t i o s o v e r l a p p e d (ranges, m e a n s , a n d m e d i a n s ) in MLDheterozygotes a n d PD/PD-homozygotes. M a n n - W h i t n e y t e s t of m e d i a n s revealed significant differences (Z < 0.05) for: MLD/PD- a n d MLD-heterozygotes, MLD/PD-heterozygotes a n d M L D patients, MLD/PDheterozygotes a n d PD/PD-homozygotes. CLINICAL BIOCHEMISTRY, VOLUME 30, JUNE 1997
S u l f a t i d e excretion in M L D / P D - h e t e r o z y g o t e s w a s e l e v a t e d a n d v a r i e d f r o m 4 . 8 - 3 8 . 3 n m o l / m g lipid; m e a n -+ SD = 22.2 _+ 13.0, m e d i a n = 19.3. T h e r a n g e of s u l f a t i d e e x c r e t i o n in M L D / P D - a n d M L D - h e t erozygotes overlapped, although mean value and m e d i a n w e r e m u c h l o w e r in M L D - h e t e r o z y g o t e s g r o u p (7.4 _+ 4.6; 5.5, r e s p e c t i v e l y ) (Figure 3) (Table 1). S u l f a t i d e levels o b t a i n e d in M L D p a t i e n t s w e r e e x t r e m e l y e l e v a t e d a n d did n o t o v e r l a p w i t h r e s u l t s of a n y o t h e r group; range: 7 4 . 3 - 4 1 1 . 6 n m o l / m g lipid, m e a n _+ SD = 184.5 _+ 130.8, m e d i a n = 145.5. P D / P D - h o m o z y g o t e s did not e x c r e t e e l e v a t e d a m o u n t s of s u l f a t i d e (Table 1). M a n n - W h i t n e y t e s t of m e d i a n s r e v e a l e d s i g n i f i c a n t differences (Z < 0.05) for M L D / P D - a n d M L D - h e t e r o z y g o t e s , M L D / PD-heterozygotes and MLD patients, MLD/PD-heterozygotes and PD/PD-homozygotes. We ha ve found an inverse relationship between ASA/[3-galactosidase r a t i o s a n d s u l f a t i d e excretion. Low A S A / ~ - g a l a c t o s i d a s e r a t i o s u s u a l l y c o r r e l a t e d with high sulfatide excretion and conversely (Figure 4). T h i s is w h y t h e u p p e r a r m of t h e c u r v e is m a d e u p of t h e r e s u l t s of M L D p a t i e n t s a n d t h e l o w e r a r m of r e s u l t s in controls. R e s u l t s f o u n d in M L D - h e t erozygotes and MLDfPD compound heterozygotes c o m p o s e d t h e t r a n s i t i o n s e g m e n t of t h e c u r v e . T h e r e s u l t s of e a c h g r o u p of s u b j e c t s c u m u l a t e d tog e t h e r on t h e c u r v e , d e l i n e a t i n g specific r a n g e s . T h i s p e r m i t s good d i f f e r e n t i a t i o n b e t w e e n s t u d i e d groups. 327
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Discussion
In this study we have found that comparison of ASA/~-galactosidase ratios gave better discrimination between investigated groups than ASA activities. ASA/~-galactosidase ratios mean and median values characteristic of MLD/PD-heterozygotes were about twice so high as for MLD patients and about twice lower than in MLD-heterozygotes or PDfPD-homozygotes (Table 1). This p a r a m e t e r was underestimated so far, and was mentioned in literature only by Harzer (21), who had observed that a quotient between ASA activity and another control enzyme (lysosomal) with normal activity leads to better discrimination between MLD-heterozygotes and normozygotes. It is interesting that ASA activities in MLD/PDheterozygotes are divided into two groups (Figure 1). The first group of ASA activities is in the range characteristic of MLD patients, and the second group of enzyme activities is in the range for MLD-heterozygotes. The same heterogeneity in MLD/PD-heterozygotes described Francis et al. (22) and Leistner et al. (23). After carrying out of initial biochemical and genetic investigations among members of one fam328
ily, Francis et al. constructed polymerase chain reactions enabling detection of two mutations in ASA gene related to pseudodeficiency allele. Persons heterozygous for MLD and the loss of polyadenylation signal showed ASA activities very low, similar to these found in MLD patients. Persons heterozygous for MLD and the loss of N-glicosylation site had ASA activities in the ranges characteristic of MLDheterozygotes or NO/PD-heterozygotes. However, in our study, we have detected the ASA-pseudodeficiency allele without looking for these two particular mutations, and we have also observed heterogeneity among MLD/PD-compound heterozygotes. The main "biochemical" feature of MLD patients is excretion of large amounts of sulfatide in urine and very low ASA activity in leukocytes. The mean value of excreted sulfatide in MLD patients (184.5 -+ 130.8 nmol/mg lipid) was nearly 100 times higher as in controls (2.1 -+ 1.7 nmol/mg lipid). Molzer et al. (18) and Natowicz et al. (24) described also so high amounts of sulfatide in urine from MLD patients, irrespective of how high was the residual ASA activity. The mean sulfatide excretion value in the CLINICAL BIOCHEMISTRY, VOLUME 30, JUNE 1997
SULFATIDE IN MLD/PD-HETEROZYGOTES
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Figure 3 -- Sulfatide excretion in investigated groups. MLD/PD group (17.8 _+ 10.7 nmol/mg lipid) was lower than in MLD patients, but it was higher than in the MLD-heterozygotes (7.4 _+ 4.6). The combination of an MLD-related mutation and the ASA-PD allele seems to increase sulfatide steady state concentration (25), e.g., in kidney cells (tubuli) thus elevating sulfatide excretion generally more than in MLD-heterozygotes. The higher sulfatide excretion in MLD/PD-heterozygotes than in MLD-heterozygotes is corroborated by sulfatide loading studies in fibroblasts of those subjects (25), where the lower sulfatide turnover (corresponding with higher sulfatide excretion) was found in MLD/PD group. On the other hand our results differ from those obtained by Penzien et al. (26) and Kappler et al. (12), where among 13 investigated MLD/PD subjects only 1 person revealed elevated sulfatide excretion, 2 other displayed about 10% of the mean value found in MLD-patients, and in the remaining persons sulfatide was not detectable or was found in traces. The sulfatide level found in 7 PD/PD-homozygotes remained in normal range (0.0-5.9 nmol/mg lipid), although the ASA activity was lowered. It seems CLINICAL BIOCHEMISTRY, VOLUME 30, J U N E 1997
Figure 4 -- Relation between ASA/~-Gal ratios and sulfatide excretions in investigated groups. that estimating of sulfatide in urine may be an excellent biochemical parameter differentiating between PD/PD-homozygotes and MLD/PD-heterozygotes or MLD patients (with genotypes MLD/MLD as well as MLDpD/MLD or MLDpD/MLDpD). The problem of misdiagnosing MLDpD/MLDpD patients as PD/PD-homozygotes by means of only DNA analysis was described earlier by Leistner et al. (23), but unlike PD/PD-homozygotes, MLDpD/MLDpD patients excrete pathological amounts of sulfatide in urine. According to Kolodny and Fluharty (1) two types of patients specifically require an assessment of urinary sulfatide excretion: the patients with clinical signs of MLD but normal ASA activity, and the patients with ASA deficiency without clinical signs of MLD or with neurologic signs not typical of MLD. In the first class of patients, an increase in urine sulfatide suggests the diagnosis of cerebroside sulfate sulfatase activator protein deficiency. In the second type of patients, the increased excretion of sulfatide indicates a presymptomatic or atypical case of MLD, whereas normal urine sulfatide excludes the MLD genotype. Relationship between ASA]~-galactosidase ratios and sulfatide excretion formed the hiperbolic curve 329
LUGOWSKA E T
(Figure 4). This curve is ver y similar to a curve obtained by Molzer et al. (18), who compared homoand heterozygotes o f m e t a c h r o m a t i c leukodystrophy as well as persons with low ASA activities. The inverse relationship between sulfatide excretion and ASA activity r emai ns a kinetic model proposed by Conzelmann and Sandhoff(27). This model refers to the inverse relation between steady-state s ubst rat e concentration and enzyme activity in the lysosomes. In consequence to the decrease of enzyme activity, the model predicts an increase of steady-state subs t ra t e concentration, finally leading to s ubst rat e accumulation in the lysosomes. Described here method of thin-layer chromatography of sulfatide extracted from the urine sediment seems to be valuable method in the detection of individuals who are MLD-heterozygotes and MLD/ PD-compound heterozygotes and are relatives of MLD patients. The importance of this problem is emphasized by the existence of minimal organic brain damages found in compound MLD/PD-heterozygotes, which unlike in MLD patients did not lead to an y neurological lesions [18, and unpublished observation]. Various mu tatio n s in ASA gene lead to changed kinetic characteristic of the enzyme activity. Some m u t a tio n s are MLD related, but others are the cause of ASA-pseudodeficiency only. This fact makes the diagnosis of m e t a c h r o m a t i c leukodystrophy only based on ASA activity m e a s u r e m e n t s difficult. Unequivocal discrimination of MLD patients and PD/PD-homozygotes is feasable by d e t e r m i n a t i o n of sulfatide excretion. Differentiation of MLD/PD-heterozygotes, MLD-heterozygotes, as well as PD/PDhomozygotes or NO/PD-heterozygotes needs, however, the triad of ASA activity (or ratio of ASA and a lysosomal enzyme not affected, e.g., [3-galactosidase), sulfatide excretion in 24 h urine sediment (or loading test in fibroblasts) and PCR test to detect ASA-pseudodeficiency allele. Acknowledgements
This study was supported by a grant from the Austrian Ministery of Science and Research (GZ.45 264/2-4 6a/93). The authors would like to thank Mrs. Marta Zobel for her excellent technical assistance and the great warm heartedness, which she showed us during realization of this study. References
1. Kolodny EH, Fluharty AL. Metachromatic leukodystrophy and multiple sulfatase deficiency: Sulfatide lipidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds): "The Metabolic Basis of Inherited Disease", 6th Ed. New York: Mc Graw-Hill, pp 1721-50, 1989. 2. Gieselmann V, Zlotogora J, Harris A, Wenger DA, Morris CP. Molecular Genetics of Metachromatic Leukodystrophy. H u m Mutat 1994; 4: 233-42. 3. Luyten JA, Wenink PW, Steenbergen-Spanjers GC, et al. Metachromatic leukodystrophy: a 12-bp deletion in exon 2 of the arylsulfatase A gene in a late infantile variant. H u m Genet 1995; 96: 357-60. 330
AL.
4. Regis S, Carrozzo R, Filocamo M, Serra G, Mastropaolo C, Gatti R. An AT-deletion causing a frameshiff in the arylsulfatase A gene of a late infantile metachromatic leukodystrophy patient. Hum Genet 1995; 96: 233-5. 5. Zlotogora J, Bach G, B6senberg C, Barak Y, von Figura K, Gieselmann V. Molecular basis of late infantile metachromatic leukodystrophy in the Habbanite Jews. Hum Mutat 1995; 5: 137-43. 6. Pastor-Soler NM, Raft MA, Hoffman JD, Hu D, Wenger DA. Metachromatic leukodystrophy in the Navajo Indian population: a splice site mutation in intron 4 of the arylsulfatase A gene. Hum Mutat 1994; 4: 199-207. 7. Heinisch U, Zlotogora J, Kafert S, Gieselmann V. Multiple mutations are responsible for the high frequency of metachromatic leukodystrophy in a small geographic area. A m J H u m Genet 1995; 56: 51-7. 8. Kappler J, Sommerlade HJ, von Figura K, Gieselmann V. Complex arylsulfatase A alleles causing metachromatic leukodystrophy. Hum Mutat 1994; 4: 119-27. 9. Gieselmann V, Polten A, Kreysing J, et al. Mutations in arylsulfatase A alleles causing metachromatic leukodystrophy. Brain Dysfunction 1991; 4: 235-43. 10. Polten A, Fluharty AL, Fluharty CB, et al. Molecular basis of different forms of metachromatic leukodystrophy. N Engl J Med 1991; 324: 18-22. 11. Hohenschutz C, Eich P, Friedl W, Waheed A, Conzelmann E, Propping P. Pseudodeficiency of arylsulfatase A: a common genetic polymorphism with possible disease implications. H u m Genet 1989; 82: 45-8. 12. Kappler J, Leinekugel P, Conzelmann E, et al. Genotype-phenotype relationship in various degrees of arylsulfatase A deficiency. Hum Genet 1991; 86: 463-70. 13. Kampine JP, Brady RO, Kanfer JN, Feld M, Shapiro D. Diagnosis of Gaucher's disease and Niemann-Pick disease with small samples of venous blood. Science 1966: 155: 86-8. 14. Fallon HJ, Frei E III, Davidson JD, Trier JS, Burk D. Leukocyte preparations from human blood: Evaluation of their morphologic and metabolic state. J Lab Clin Med 1962; 59: 779-91. 15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 16. Bass NH, Witmer EJ, Dreifuss FE. A pedigree study of metachromatic leukodystrophy. Neurology 1970; 20: 52-62. 17. Suzuki Y, Hayakawa T, Azaki MY, Hiratani Y. GM1gangliosidosis. A variant with high activity of hepatic neutral [3-galactosidase. Eur J P e d 1976; 122: 177-86. 18. Molzer B, Sundt-Heller R, Kainz-Korschinsky M, Zobel M. Elevated sulfatide excretion in heterozygotes of metachromatic leukodystrophy: dependence on reduction of arylsulfatase A activity. A m J Med Genet 1992; 44: 523-6. 19. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982. 20. Gieselmann V. An assay for the rapid detection of the arylsulfatase A pseudodeficiency allele facilitates diagnosis and genetic counseling for metachromatic leukodystrophy. Hum Genet 1991; 86: 251-5. 21. Harzer K. Inheritance of the enzyme deficiency in three neurolipidoses: variant 0 of Tay-Sachs disease (Sandhoffs disease), classic Tay-Sachs disease, and metachromatic leukodystrophy. Identification of the heterozygous carriers. Humangenetik 1973; 20: 9-24. CLINICAL BIOCHEMISTRY,VOLUME 30, JUNE 1997
SULFATIDE IN MLD/PD-HETEROZYGOTES 22. Francis GS, Bonni A, Shen N et al. Metachromatic leukodystrophy: multiple nonfunctional and pseudodeficiency alleles in a pedigree: problems with diagnosis and counseling. A n n Neurol 1993; 34: 212-18. 23. Leistner S, Young E, Meaney C, Winchester B. Pseudodeficiency of arylsulphatase A: Strategy for clasification of genotype in families of subjects with low ASA activity and neurological symptoms. J Inher Metab Dis 1995; 18: 710-16. 24. Natowicz MR, Prence EM, Chaturvedi P, Newburg DS. Urine sulfatides and the diagnosis of metachromatic leukodystrophy. Clin Chem 1996; 42: 232-8. 25. Leinekugel P, Michel S, Conzelmann E, Sandhoff K.
CLINICAL BIOCHEMISTRY, VOLUME 30, JUNE 1997
Quantitative correlation between the residual activity of ~-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease. H u m Genet 1992; 88: 513-23. 26. Penzien JM, Kappler J, Herschkowitz N, et al. Compound heterozygosity for metachromatic leukodystrophy and arylsulfatase A pseudodeficiency alleles is not associated with progressive neurological disease. A m J H u m Genet 1993; 52: 557-64. 27. Conzelmann E, Sandhoff K. Partial enzyme deftciences: residual activities and the development of neurological disorders. Dev Neurosci 1983/84; 6: 5 8 71.
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PII S0009-9120(97)00033-7
Elevated Sulfatide Excretion in Compound Heterozygotes of Metachromatic Leukodystrophy and ASA-Pseudodeficiency Allele AGNIESZKA EUGOWSKA, 1 ANNA TYLKI-SZYMANSKA, 1 JOHANNES BERGER,2 and BRUNHILDE MOLZER2 1The Children's Memorial Health Institute, Department of Metabolic Diseases, Warsaw, Poland, and 2Clinical Institute of Neurology, Department of Neuropathology and Neurochemistry, University of Vienna, Austria Objective: Use of sulfatide excretion in differentiating MLD/PDheterozygotes from MLD-patients and PD/PD-homozygotes. Design and Methods: Sulfatide was extracted from urine sediment with chloroform/methanol (2:1, v/v). The quantity of sulfatide was measured densitometrically (h = 580 nm) after thin-layer chromatography. ASA and ~-galactosidase activities were assayed enzymatically. Results: MLD/PD-heterozygotes excreted sulfatide in the range of 4.8-36.3 nmol/mg lipid (mean -+ SD = 17.8 _+ 10.7), whereas sulfatide in MLD-patients ranged from 74.3-411.6 nmol/mg lipid (mean -+ SD = 184.5 +- 130.8) and in PD/PD-homozygotes sulfatide excretion remained in normal range of 0.0-5.9 nmol/mg lipid (mean + SD = 1.64 _+ 2.12). ASA activities in these groups were very low or lowered. Conclusions: The quantitative measurement of sulfatide in urine allows differentiation between MLD/PD-heterozygotes and MLDheterozygotes, as well as between MLD/PD-heterozygotes with very low ASA activity and MLD-patients or PD/PD-homozygotes. The quantitative measurement of sulfatide in urine differs between MLD-carriers and controls.
KEY WORDS: metachromatic leukodystrophy; sulfatide; arylsulfatase A; arylsulfatase A-pseudodeficiency; lysosomal storage disease. Introduction
rylsulfatase A (ASA, EC 3.1.6.1) is a lysosomal A enzyme essential for degradation of sulfatide, an important glycosphingolipid component of myelin. Inability of ASA to decompose sulfatide leads to accumulation of this lipid in the myelin of the central and peripheral nervous system, causing metachromatic leukodystrophy (MLD, McKusick 250100)--a recessively inherited, fatal, demyelinating disorder. Sulfatide also accumulates in some M a n u s c r i p t r e c e i v e d S e p t e m b e r 30, 1996; r e v i s e d a n d a c c e p t e d J a n u a r y 5, 1997.
Correspondence: Agnieszka Lugowska, M.Sc., The Children's Memorial Health Institute, ZDL, Department of Metabolic Diseases, A1. Dzieci Polskich 20, 04-736 Warsaw, Poland. CLINICAL BIOCHEMISTRY, VOLUME 30, J U N E 1997
visceral organs of affected patients, e.g., kidneys or gallbladder, and are excreted in high amounts in urine and bile (1). So far, at least 60 MLD-specific mutations in the ASA gene have been disclosed (2-8). MLD-related mutations can be divided into two groups: alleles associated with no residual enzyme activity (0 alleles), and alleles encoding for low residual enzyme activity (R alleles) (9). Patients homozygous for 0 alleles suffer from the severe, late-infantile MLD. Compound heterozygosity for 0 and R alleles results in the juvenile MLD, whereas presence of two R alleles leads predominantly to adult MLD (10). There are also two mutations in the ASA gene associated with so called ASA-pseudodeficiency (PD). Particularly the loss of the polyadenylation signal leads to reduced synthesis of ASA polypeptides. However, the enzyme activity is still sufficient for normal sulfatide degradation. The second mutation, loss of the glycosylation site is of minor importance. It is thought that homozygotes of ASA-PD (0.5-2% of the population) neither show clinical symptoms of MLD, nor do they excrete elevated amounts of sulfatide in urine. Seven to 15% of the population bear the ASA-PD allele. The PD mutations are detectable by PCR. In diagnosing of metachromatic leukodystrophy, measurement of ASA activity in leukocytes is commonly used. It is hardly possible to reliably differentiate MLD/PD-heterozygotes from ASA-pseudodeficiency homozygotes or MLD patients with high residual ASA activity by means of artificial substrates (11,12). High frequency of pseudodeficiency allele in general population (7-15%), so among relatives of probands as well, demands a particular procedure in pre- and postnatal diagnosing of MLD. Our objective was to use the measurement of sulfatide excretion in differentiating MLD/PD-heterozygotes from MLD patients and PD/PD-homozygotes, as well as to compare the biochemical findings 325
LUGOWSKA ET AL.
and to find the differences between genetically defined MLD/PD compound heterozygotes, MLD-heterozygotes, and other groups of subjects. Materials
and methods
Biological material consisted of leukocytes isolated from venous blood and 24 h urine collections sediments obtained from subjects investigated in this study: • MLD/PD compound heterozygotes (n = 6): 3 females and 3 males, age 3 0 - 4 4 y, parents of MLD patients additionally carrying arylsulfatase A-pseudodeficiency allele; • MLD-heterozygotes (n -- 23): 12 females and 11 males, age 23-55 y, parents of MLD patients not carrying arylsulfatase A-pseudodeficiency allele; • MLD patients (n = 8): 3 females and 5 males, age 2-17 y, children at different ages affected with MLD; • PD/PD-homozygotes (n = 7): 2 females and 5 males, age 1-9 y, children homozygous for ASA pseudodeficiency allele, not affected with MLD; • NO/PD-heterozygotes (n = 5): 3 females and 2 males, age 2 7 - 4 0 y, unrelated healthy persons heterozygous for ASA pseudodeficiency allele; • Controls (n = 30): 18 females and 12 males, age 2 6 - 5 0 y, 10 persons of staff of the Children's Memorial Health Institute in Warsaw and 20 parents of children with cerebral palsy not carrying the ASA-pseudodeficiency allele. Bovine serum albumine, p-nitrocatechol, p-nitrocatechol sulfate, dimethylsulfoxide (DMSO), 4-methylumbelliferon, were purchased from the Sigma Chemical Co. (St. Louis, MO, USA); 4-methylumbelliferyl-~-D-galactopyranoside was purchased from Koch-Light Laboratories Ltd.; methanol, from Riedel de Hahn AG; florisil 6 0 - 1 0 0 mesh, from Fluka Chemika; bovine brain sulfatide, from Supelco; Tris and ethidium bromide, from United States Biochemical Co.; SDS and mineral oil, from Serva; agarose, from Gibco BRL & Life Technologies; glass fiber filters GF/A 15.0 cm, from W h a t m a n International Ltd.; proteinase K, was from Boehringer Mannheim Biochemica; Taq-DNA polimerase, from Biomedica; and XDNA HindIII/EcoRI Digest, from proMega. All other chemicals were purchased from Merck. Leukocytes were isolated from 10 mL of heparinized venous blood, contaminating erythrocytes were removed by hypotonic shock (13,14). Leukocytes were suspended in 1.0 mL distilled water and sonified (30 s, 40 cycles). The homogenate was centrifuged 30 min, 4500 rpm. The pellet consisting of cellular debris was used for isolation of DNA. Enzymatic assays were carried out in supernatants. Protein content in supernatants was determined by the use of Folin-Ciocalteau reagent according to the method of Lowry et al. (15). ASA activity was determined in supernatants with 0.01 mol/L p-nitrocatechol sulfate in 0.5 mol/L 326
acetate buffer pH 5.0 as substrate. The reaction mixture contained also sodium pirophosphate in 0.5 mol/L acetate buffer pH 5.0 (11.2 mg/50 mL). Probes were incubated 30 min in 37°C. The enzymatic reaction was stopped with 1 mol/L N a O H as previously described by Bass et al. (16). The absorbance of assayed samples was measured at X = 510 nm in Hitachi U-2000 spectrofotometer. ASA activity was expressed in nmoles ofp-nitrocatechol/mg protein/h. Beta-galactosidase activity was determined in homogenized leukocytes supernatants as control enzyme with 1 mmol]L 4-methylumbelliferyl-~-D-galactopyranoside in 0.1 mol/L citrate buffer pH 4.5, as previously described by Suzuki et al. (17). After 1 h incubation in 37°C, the reaction was stopped by adding of 0.2 mol/L glycine-NaOH buffer pH 10.5. Fluorescence was measured in type FP777 Jasco spectrofluorimeter at excitation X = 364 nm and emission X = 446 nm. Beta-galactosidase activity was expressed in nmoles of 4-methylumbelliferon/mg protein/h. Sulfatides were determined in 24 h urine collections. Urine samples were kept at - 2 0 ° C until processing. Urine was centrifuged at 16.000 g for 30 min. The sediment was extracted in a glass homogenizer with 30 mL of chloroform/methanol mixture (2:1; v/v; C/M). After Folch partition the lipid fraction was applied on a glass column (0.5 z 15 cm) filled with Florisil in chloroform. After removal of neutral lipids with 15 mL of chloroform, glycolipids were eluted with 40 mL C/M. Aliquots of eluate were chromatographed on silica gel G-60 precoated plates in C/M/water (70:30:5, v/v/v). As standards 2-20 nmol of bovine brain sulfatides were applied (linearity preserved between 0 and 10 nmol). Sulfatides were visualized with orcinol/sulfuric acid/water (0.2 g/75 mL/25 mL), and quantified densitometrically in type CS9000 Shimadzu densitometer at X = 580 nm (limit of detection was 0.5 nmol) (18). Results are given as nmoles sulfatides/mg lipid. DNA was isolated from cellular debris according to the method of Maniatis with proteinase K (20 mg/mL) overnight digestion at 55°C. DNA was extracted by phenol/chloroform (1:1, v/v), precipitated with ice-cold 96% ethanol and 3 mol/L sodium acetate, washed with 70% ethanol, and redissolved in 40 ~L1of water (19). ASA pseudodeficiency allele was detected in genomic DNA using a PCR-method with a pair of specific primers as previously described by Gieselmann (20). Samples of investigated DNA (approximately 0.1 ~g) were amplified with 1.25 U/sample Taq polymerase in OmniGene-Hybaid thermocycler. Thirty-five cycles of 1 min denaturation at 93.7°C, 1 min annealing at 58°C, and 1 min elongation at 72°C were performed. Amplification products were electrophoresed in 1.2% agarose gels. DNA fragments were visualized by ethidium bromide staining; XDNA HindIII/EcoRI Digest was applied as molecular weight standard. CLINICAL BIOCHEMISTRY, VOLUME 30, JUNE 1997
SULFATIDE IN MLD/PD-HETEROZYGOTES TABLE 1 ASA Activity, ASA/~-Gal Ratio, and Sulfatide Excretion in MLD/PD-Heterozygotes, MLD-Heterozygotes, MLD Patients, and Controls
Subjects MLD/PD-het. (n = 6) MLD-het. (n = 23) MLD patients (n = 8) NO/PD (n = 5) PD/PD (n = 7) Controls (n = 30)
ASA activity a [nmol/mg p/h] mean - SD (range) median
ASA/[3-Gal mean _+ SD (range) median
Gal-Sulfatide b [nmol/mg lipid] mean -+ SD (range) median
42.00 + 31.52 (14-89) med = 33.0 67.26 -+ 22.44 (31-107) med = 74.0 15.63 _ 6.32 (9-25) med = 13.5 81.20 _+ 26.42 (57-117) med = 70.0 47.71 _+ 18.56 (25-74) med = 53.0 140.57 _+ 55.75 (75-288) med = 133.5
0.12 _+ 0.06 (0.06-0.21) med = 0.11 0.20 +_ 0.05 (0.10-0.32) med = 0.20 0.05 _+ 0.02 (0.03-0.08) med = 0.05 0.27 _+ 0.05 (0.22-0.34) med = 0.28 0.23 _+ 0.14 (0.11-0.50) med = 0.18 0.43 _+ 0.12 (0.23-0.87) med = 0.40
17.8 -+ 10.7 (4.8-36.3) med = 19.3 7.4 -+ 4.6 (1.3-16.1) med = 5.5 184.5 _+ 130.8 (n = 5) (74.3-411.6) med = 145.5 1.7 _+ 2.1 (n = 3) (0.0-4.1) reed = 1.0 1.64 _+ 2.12 c (0.0-5.9) med = 1.4 2.2 _+ 1.7 (n = 29) (0.0-5.9) med = 1.9
"Estimated in leukocytes. bEstimated in 24 hrs urine sediment. tin 3 of 7 PD/PD homozygotes sulfatide excretion was not detectable; this seems to be reason that SD is higher than mean value. Results R e s u l t s of ASA activity, A S A / ~ - g a l a c t o s i d a s e ratio, a n d s u l f a t i d e e x c r e t i o n in i n d i v i d u a l g r o u p s a r e c o m p i l e d in T a b l e 1. I n t h e g r o u p of six M L D / P D - h e t e r o z y g o t e s ASA a c t i v i t y r e v e a l e d v a l u e s b e t w e e n 14 a n d 89 n m o l / m g p r o t e i n / h w i t h m e a n v a l u e _+ SD = 42.00 _+ 31.52 a n d m e d i a n = 33.0. T h e s e v a l u e s a r e located bet w e e n v a l u e s c h a r a c t e r i s t i c of M L D - p a t i e n t s a n d M L D - h e t e r o z y g o t e s (Table 1). T h e r a n g e s of ASA a c t i v i t i e s in t h e g r o u p s of M L D / P D - , M L D - h e t e r o z y gotes, M L D p a t i e n t s , a n d P D / P D - h o m o z y g o t e s m o r e or less o v e r l a p p e d ( F i g u r e 1). N o n p a r a m e t r i c M a n n W h i t n e y t e s t of m e d i a n s revealed insignificant differences "(Z >- 0.05) for: MLD/PD- a n d MLD-heterozygotes, MLD/PD-heterozygotes a n d M L D patients, MLD/PD-heterozygotes a n d PD/PD-homozygotes. T h e r a n g e of ASA/[3-galactosidase r a t i o s in M L D / P D g r o u p did not o v e r l a p w i t h t h e r a n g e c h a r a c t e r i s t i c of controls a n d e q u a l l e d 0 . 0 6 - 0 . 2 1 ; m e a n __ SD = 0.12 __ 0.06 a n d m e d i a n = 0.11 ( F i g u r e 2). M e a n a n d m e d i a n v a l u e s in M L D / P D , M L D - h e t e r o z y g o t e s a n d M L D p a t i e n t s w e r e well d i s c r i m i n a t e d (Table 1). ASA/[3-galactosidase r a t i o s o v e r l a p p e d (ranges, m e a n s , a n d m e d i a n s ) in MLDheterozygotes a n d PD/PD-homozygotes. M a n n - W h i t n e y t e s t of m e d i a n s revealed significant differences (Z < 0.05) for: MLD/PD- a n d MLD-heterozygotes, MLD/PD-heterozygotes a n d M L D patients, MLD/PDheterozygotes a n d PD/PD-homozygotes. CLINICAL BIOCHEMISTRY, VOLUME 30, JUNE 1997
S u l f a t i d e excretion in M L D / P D - h e t e r o z y g o t e s w a s e l e v a t e d a n d v a r i e d f r o m 4 . 8 - 3 8 . 3 n m o l / m g lipid; m e a n -+ SD = 22.2 _+ 13.0, m e d i a n = 19.3. T h e r a n g e of s u l f a t i d e e x c r e t i o n in M L D / P D - a n d M L D - h e t erozygotes overlapped, although mean value and m e d i a n w e r e m u c h l o w e r in M L D - h e t e r o z y g o t e s g r o u p (7.4 _+ 4.6; 5.5, r e s p e c t i v e l y ) (Figure 3) (Table 1). S u l f a t i d e levels o b t a i n e d in M L D p a t i e n t s w e r e e x t r e m e l y e l e v a t e d a n d did n o t o v e r l a p w i t h r e s u l t s of a n y o t h e r group; range: 7 4 . 3 - 4 1 1 . 6 n m o l / m g lipid, m e a n _+ SD = 184.5 _+ 130.8, m e d i a n = 145.5. P D / P D - h o m o z y g o t e s did not e x c r e t e e l e v a t e d a m o u n t s of s u l f a t i d e (Table 1). M a n n - W h i t n e y t e s t of m e d i a n s r e v e a l e d s i g n i f i c a n t differences (Z < 0.05) for M L D / P D - a n d M L D - h e t e r o z y g o t e s , M L D / PD-heterozygotes and MLD patients, MLD/PD-heterozygotes and PD/PD-homozygotes. We ha ve found an inverse relationship between ASA/[3-galactosidase r a t i o s a n d s u l f a t i d e excretion. Low A S A / ~ - g a l a c t o s i d a s e r a t i o s u s u a l l y c o r r e l a t e d with high sulfatide excretion and conversely (Figure 4). T h i s is w h y t h e u p p e r a r m of t h e c u r v e is m a d e u p of t h e r e s u l t s of M L D p a t i e n t s a n d t h e l o w e r a r m of r e s u l t s in controls. R e s u l t s f o u n d in M L D - h e t erozygotes and MLDfPD compound heterozygotes c o m p o s e d t h e t r a n s i t i o n s e g m e n t of t h e c u r v e . T h e r e s u l t s of e a c h g r o u p of s u b j e c t s c u m u l a t e d tog e t h e r on t h e c u r v e , d e l i n e a t i n g specific r a n g e s . T h i s p e r m i t s good d i f f e r e n t i a t i o n b e t w e e n s t u d i e d groups. 327
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Discussion
In this study we have found that comparison of ASA/~-galactosidase ratios gave better discrimination between investigated groups than ASA activities. ASA/~-galactosidase ratios mean and median values characteristic of MLD/PD-heterozygotes were about twice so high as for MLD patients and about twice lower than in MLD-heterozygotes or PDfPD-homozygotes (Table 1). This p a r a m e t e r was underestimated so far, and was mentioned in literature only by Harzer (21), who had observed that a quotient between ASA activity and another control enzyme (lysosomal) with normal activity leads to better discrimination between MLD-heterozygotes and normozygotes. It is interesting that ASA activities in MLD/PDheterozygotes are divided into two groups (Figure 1). The first group of ASA activities is in the range characteristic of MLD patients, and the second group of enzyme activities is in the range for MLD-heterozygotes. The same heterogeneity in MLD/PD-heterozygotes described Francis et al. (22) and Leistner et al. (23). After carrying out of initial biochemical and genetic investigations among members of one fam328
ily, Francis et al. constructed polymerase chain reactions enabling detection of two mutations in ASA gene related to pseudodeficiency allele. Persons heterozygous for MLD and the loss of polyadenylation signal showed ASA activities very low, similar to these found in MLD patients. Persons heterozygous for MLD and the loss of N-glicosylation site had ASA activities in the ranges characteristic of MLDheterozygotes or NO/PD-heterozygotes. However, in our study, we have detected the ASA-pseudodeficiency allele without looking for these two particular mutations, and we have also observed heterogeneity among MLD/PD-compound heterozygotes. The main "biochemical" feature of MLD patients is excretion of large amounts of sulfatide in urine and very low ASA activity in leukocytes. The mean value of excreted sulfatide in MLD patients (184.5 -+ 130.8 nmol/mg lipid) was nearly 100 times higher as in controls (2.1 -+ 1.7 nmol/mg lipid). Molzer et al. (18) and Natowicz et al. (24) described also so high amounts of sulfatide in urine from MLD patients, irrespective of how high was the residual ASA activity. The mean sulfatide excretion value in the CLINICAL BIOCHEMISTRY, VOLUME 30, JUNE 1997
SULFATIDE IN MLD/PD-HETEROZYGOTES
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NO/PI)-
hetero zygotes
Figure 3 -- Sulfatide excretion in investigated groups. MLD/PD group (17.8 _+ 10.7 nmol/mg lipid) was lower than in MLD patients, but it was higher than in the MLD-heterozygotes (7.4 _+ 4.6). The combination of an MLD-related mutation and the ASA-PD allele seems to increase sulfatide steady state concentration (25), e.g., in kidney cells (tubuli) thus elevating sulfatide excretion generally more than in MLD-heterozygotes. The higher sulfatide excretion in MLD/PD-heterozygotes than in MLD-heterozygotes is corroborated by sulfatide loading studies in fibroblasts of those subjects (25), where the lower sulfatide turnover (corresponding with higher sulfatide excretion) was found in MLD/PD group. On the other hand our results differ from those obtained by Penzien et al. (26) and Kappler et al. (12), where among 13 investigated MLD/PD subjects only 1 person revealed elevated sulfatide excretion, 2 other displayed about 10% of the mean value found in MLD-patients, and in the remaining persons sulfatide was not detectable or was found in traces. The sulfatide level found in 7 PD/PD-homozygotes remained in normal range (0.0-5.9 nmol/mg lipid), although the ASA activity was lowered. It seems CLINICAL BIOCHEMISTRY, VOLUME 30, J U N E 1997
Figure 4 -- Relation between ASA/~-Gal ratios and sulfatide excretions in investigated groups. that estimating of sulfatide in urine may be an excellent biochemical parameter differentiating between PD/PD-homozygotes and MLD/PD-heterozygotes or MLD patients (with genotypes MLD/MLD as well as MLDpD/MLD or MLDpD/MLDpD). The problem of misdiagnosing MLDpD/MLDpD patients as PD/PD-homozygotes by means of only DNA analysis was described earlier by Leistner et al. (23), but unlike PD/PD-homozygotes, MLDpD/MLDpD patients excrete pathological amounts of sulfatide in urine. According to Kolodny and Fluharty (1) two types of patients specifically require an assessment of urinary sulfatide excretion: the patients with clinical signs of MLD but normal ASA activity, and the patients with ASA deficiency without clinical signs of MLD or with neurologic signs not typical of MLD. In the first class of patients, an increase in urine sulfatide suggests the diagnosis of cerebroside sulfate sulfatase activator protein deficiency. In the second type of patients, the increased excretion of sulfatide indicates a presymptomatic or atypical case of MLD, whereas normal urine sulfatide excludes the MLD genotype. Relationship between ASA]~-galactosidase ratios and sulfatide excretion formed the hiperbolic curve 329
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(Figure 4). This curve is ver y similar to a curve obtained by Molzer et al. (18), who compared homoand heterozygotes o f m e t a c h r o m a t i c leukodystrophy as well as persons with low ASA activities. The inverse relationship between sulfatide excretion and ASA activity r emai ns a kinetic model proposed by Conzelmann and Sandhoff(27). This model refers to the inverse relation between steady-state s ubst rat e concentration and enzyme activity in the lysosomes. In consequence to the decrease of enzyme activity, the model predicts an increase of steady-state subs t ra t e concentration, finally leading to s ubst rat e accumulation in the lysosomes. Described here method of thin-layer chromatography of sulfatide extracted from the urine sediment seems to be valuable method in the detection of individuals who are MLD-heterozygotes and MLD/ PD-compound heterozygotes and are relatives of MLD patients. The importance of this problem is emphasized by the existence of minimal organic brain damages found in compound MLD/PD-heterozygotes, which unlike in MLD patients did not lead to an y neurological lesions [18, and unpublished observation]. Various mu tatio n s in ASA gene lead to changed kinetic characteristic of the enzyme activity. Some m u t a tio n s are MLD related, but others are the cause of ASA-pseudodeficiency only. This fact makes the diagnosis of m e t a c h r o m a t i c leukodystrophy only based on ASA activity m e a s u r e m e n t s difficult. Unequivocal discrimination of MLD patients and PD/PD-homozygotes is feasable by d e t e r m i n a t i o n of sulfatide excretion. Differentiation of MLD/PD-heterozygotes, MLD-heterozygotes, as well as PD/PDhomozygotes or NO/PD-heterozygotes needs, however, the triad of ASA activity (or ratio of ASA and a lysosomal enzyme not affected, e.g., [3-galactosidase), sulfatide excretion in 24 h urine sediment (or loading test in fibroblasts) and PCR test to detect ASA-pseudodeficiency allele. Acknowledgements
This study was supported by a grant from the Austrian Ministery of Science and Research (GZ.45 264/2-4 6a/93). The authors would like to thank Mrs. Marta Zobel for her excellent technical assistance and the great warm heartedness, which she showed us during realization of this study. References
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