Prenatal diagnosis of sanfilippo disease type C using a simple fluorometric enzyme assay

PRENATAL DIAGNOSIS, VOL. 1 4 17-22 (1 994)

PRENATAL DIAGNOSIS OF SANFILIPPO DISEASE TYPE C USING A SIMPLE FLUOROMETRIC ENZYME ASSAY

WANG HE*, YA. V. VOZNYIt, J. G. M. JXJLIMANS*, G. C. GEEEN*, E. A. KARPOVAS, T. V. DUDUKINAS, J. ZAREMBA~, 0. P. VAN DIGGELEN* AND w. J. KLEIJER*

*Department of Clinical Genetics, Erasmus University, Rotterdam, The Netherlandr; ?Institute of Biochemistry, Academy of Sciences, Yerevan, Armenian Republic: $Institute of Biological and Medical Chemistry, Moscow,

Russia; $Department of Genetics, Institute of Psychiatry and Neurology, Warsaw, Poland

Received 2 April 1993 Accepted 18 June 1993

SUMMARY A new fluorogenic substrate, Cmethylumbelliferyl &D-glucosaminide, was used for the assay of acetyl CoA:glu-

cosaminide N-acetyltransferase in chorionic villi, cultured villus cells, and amniocytes. Optimal conditions for the assay and the ranges of enzyme activity were established for the various types of fetal cells. This simple fluorometric assay provides a reliable method for early prenatal diagnosis of Sanfilippo disease type C which is more convenient than current methods using radiolabelled substrates. The method was applied to amniotic fluid cells and fetal fibroblasts from an at-risk pregnancy in which an affected fetus was diagnosed by two-dimensional electrophoresis of glycosaminoglycans in the amniotic fluid.

KEY worn,+-Prenatal diagnosis, chorionic villi, Sanfilippo disease type C, mucopolysaccharidosis type I11 C acetyl CoAglucosaminide N-acetyltransferase.

INTRODUCTION

Mucopolysaccharidosis type I11 C (MPS I11 C) is one of four known types of Santilippo disease, each of which is characterized by severe and pro- gressive neurological and behavioural disturbances but relatively mild somatic involvement (for a review see Neufeld and Muenzer, 1989). In each type, a different enzymatic defect in the degrada- tion of heparan sulphate is responsible for the accumulation of this mucopolysaccharide in lyso- somes of various tissues. The defective enzyme in MPS I11 C is acetyl CoA:a-glucosaminide N- acetyltransferase; its activity has so far been assayed by using radiolabelled oligosaccharides (Klein et al., 1988) or ['4C]glucosamine as substrates (Hopwood and Elliott, 1981; Pallini et al., 1984). Recently we have reported the synthesis and use of two fluorogenic substrates,

Addressee for correspondence: Dr W. J. Kleijer, Department of Clinical Genetics, Erasmus University, P.O. Box 1738,3000 DR, Rotterdam, The Netherlands.

0 1994 by John Wiley & Sons, Ltd. 01 97-385 1/94/0 100 1 7-O6$08 .OO

the a- and 8-anomers of 4-methylumbelliferyl- D-ghcosaminide (Voznyi et aZ., 1993). Both substrates appeared to be acetylated by N- acetyltransferase and were used for the enzyme assay in skin fibroblasts and leucocytes to diagnose MPS I11 C.

Prenatal diagnosis of MPS I11 C using the radiochemical enzyme assay on chorionic villi has been reported in one case (Di Natale et al., 1987). Here we describe the use of the fluorogenic enzyme assay in chorionic villi and amniotic fluid cells. The new method was applied to amniotic fluid cells from a pregnancy in which we had diagnosed a fetus affected with MPS I11 by two-dimensional electrophoresis of glycosaminoglycans in the fluid.

PATIENTS AND METHODS

Prenatal analysis was requested in the pregnancy of a mother of a girl with Sanfilippo disease. The diagnosis in the proband was based on clinical manifestations and an increased level of heparan

18 WANG HE ET AL.

sulphate in urine. The patient died at 9 years of age shortly before prenatal diagnosis was requested and without enzymatic establishment of the type of Sanfilippo disease.

Amniotic fluid from the pregnancy at risk was obtained in the 17th week in Warsaw and sent to Rotterdam. Two-dimensional electrophoresis of glycosaminoglycans in the amniotic fluid was performed as described elsewhere (Abeling et a l , 1974; Mossman and Patrick, 1982; Kleijer et al., 1984). Amniotic fluid cells and skin fibroblasts were cultured in Ham’s F10 medium with 20 and 15 per cent fetal calf serum, respectively, and homogenized by sonication in 0.25 per cent (w/v) Triton X-100 for the assay of N-acetyltransferase or in water for other enzymes. Chorionic villi were obtained after elective termination of pregnancies in the eighth to 11 th weeks. The villi were selected, washed in saline and stored at - 70°C. For enzyme assay, the villi were homogenized in a Potter tube before sonication.

The standard procedure for the measurement of N-acetyltransferase activity with 4- methylumbelliferyl P-D-glucosaminide (MU- PGlcN) as a substrate was as previously described (Voznyi et al., 1993). Cell or villus homogenate containing 20 pg protein was incubated for 17 h at 37°C in a volume of 30p1 with acetyl CoA (2 mM) and MU-PGlcN (1 mM) in McIlvain’s phosphate/ citrate buffer, pH 5.6. Apparent activities were established by measurement of the amount of free MU which was present directly after the 17 h incubation period. True activity was established after a second incubation period of 17 h at 37’C in the presence of P-hexosaminidase during which any remaining N-acetylated substrate (MU- PGlcNAc) was hydrolysed quantitatively; the co-substrate, acetyl CoA, was destroyed after the first incubation period to stop the acetylation reaction (Voznyi et al., 1993).

For the assessment of heparan sulphamidase activity, [35S]heparan sulphate (Amersham) was used (Kleijer et al., 1986), whereas for a-N-acetylglucosaminidase a MU substrate (Calbiochem) was used (Marsh and Fensom, 1985). Protein was determined using the BCA method (Smith et al., 1985).

RESULTS Enzyme assay conditions

Various conditions for the assay of N-acetyltransferase activity in normal chorionic

villi (CV) and in cultured amniocytes were investigated and compared with the previously established standard procedure for skin fibroblasts (see Methods). The results are shown in Fig. 1. The pH optimum and apparent X, for N-acetyltransferase in villi and amniocytes was established as pH 5.6 and 0 - 2 m , which are not significantly different from the previously established data with skin fibroblasts from control individuals (pH 5-7; K, 0.1 m; Voznyi et af., 1993). The acetylation reaction proceeded linearly with time up to 15 and 20 h in amniocytes and villi, respectively, and with the amount of cellular protein up to 40pg. Figure 1D also shows that there was no difference between the apparent and true transferase activities. This means that all the N-acetylated MU substrate (MU-PGlcNAc) formed is hydrolysed by en- dogenous P-hexosaminidase during the first 17 h incubation period. Under these conditions, a second incubation with added P-hexosaminidase is not necessary.

Based on the above results, standard assay conditions for N-acetyltransferase in villi and amniocytes similar to those previously established for fibroblasts were chosen (see Methods). The ranges of N-acetyltransferase were established for CV, cultured CV cells, and amniocytes, and were compared with the ranges for fibroblasts of control individuals and of MPS I11 C patients (Table I). Both the apparent and the true activity were measured in all samples; there appeared to be no significant difference between the apparent and true activities either in the ranges and means or in any of the individual samples. N- Acetyltransferase appeared to be more active in CV than in cultured skin fibroblasts; the level in cultured CV and amniotic fluid cells was lower than that in fibroblasts from normal individuals. Activity in the fibroblasts of MPS I11 C patients was practically absent (i.e., less than 1 per cent of control values).

Prenatal diagnosis Immediately after receipt of the amniotic fluid,

two-dimensional electrophoresis of glycosaminoglycans was performed on the supernatant. The resulting pattern showed an excessive amount of heparan sulphate (Fig. 2), as we have previously demonstrated in several pregnancies with fetuses affected with MPS I11 A (n=6) and MPS I11 B (n=3). Based on this unequivocal result, it was concluded that the fetus was affected with MPS type 111, although the subtype could not be

SANFILIPPO DISEASE TYPE C 19

c "1

40-

0 5 10 15 20 0 5 10 15 20 Incubation time (h) PH

I - 4 - 2 0 2 4 6 8

h S

> > c .- .- 4-

8

D 4-

3-

, , , , , , , l 0 10 20 30 40

Protein Mug) 1/S (l/mM)

Figure I-Kinetic properties of acetyl CoAglucosaminide N-acetyltransferase in uncultured chorionic villi ( A ,A) and cultured amniotic 5uid cells ( ,El). (A) pH dependence; (B) time course; (C) Lineweaver-Burk plot, S= substrate concentraton (4MU$GlcN), V=specific activity (nmoV 17 h per mg protein); (D) protein dependence, open symbols=true activity, closed symbols= apparent activity

assigned. The pregnancy was terminated without awaiting the results of enzyme studies.

Cultured amniocytes were tested for heparan sulphamidase and a-N-acetylglucosaminidase activity after 3-4 weeks of cultivation; both activities were normal. Later, reduced activity of N-acetyltransferase was demonstrated using radiolabelled monosaccharide substrate (at risk, 0-5nmoYh per mg; controls 1.8-2-7; Dr B. Poorthuis, Leiden). In retrospect, these amniotic fluid cells and the fetal skin fibroblasts cultured after termination of the pregnancy were subjected to investigation of N-acetyltransferase using the fluorometric assay with MU-PGlcN substrate. Complete enzyme deficiency was demonstrated in both samples, which confirmed MPS I11 C in the fetus (Table 11).

DISCUSSION

We have investigated the use of a newly designed fluorometric assay of N-acetyltransferase, using MU-P-glucosaminide as a substrate, for the prenatal diagnosis of MPS I11 C. Although the enzyme is known as an a-glucosaminide N-acetyltransferase, it has apparently no preferen- tial specificity towards the a- or /?-anomer of MU-glucosaminide (Voznyi et al., 1993). The P-anomer is effectively acetylated in normal cells, whereas activity is completely absent in MPS 111 C patients. The /3-anomer was chosen for the present study because of the abundance in most types of cells of highly active P-hexosaminidase. This guar- antees immediate and rapid progress of the second reaction step: the release of fluorescent MU. A

20 WANG HE ET AL.

Table I- True and apparent activities of acetyl CoA:glucosaminide N-acetyltransferase in normal chorionic villi and cultured fetal cells using 4MU-~-glucosaminide as a substrate

Apparent activity* True activity*

Chorionic villi (CV) 43-125 44-142 n=24t 86 f 23 96 f 30

Cultured CV cells 11-52 21-56 n=lOt 3 5 f 1 1 3 9 f 10

Amniotic fluid cells

Skin fibroblasts

n=17t

Controls n= 107

n = l l t MPS 111 C patients

12-59 34f 10

40-89 61 !C 13

0.2-0.7 0.4

18-40 28 f 7

44-98 59 f 19 -

*nmoi/17 h per mg protein. Activities are measured in the presence of 2 m~ acetyl CoA; in the absence of acetyl CoA, the activities are less than 2 nmou17 h per mg in all samples of all types of cells (blank value).

tNumber of samples from different individuals.

Figure 2-Two-dimensional electrophoresis of glycosaminoglycans in amniotic fluid from (A) a pregnancy with a MPS I11 C affected fetus and (B) a normal pregnancy. ha=hyaluronic acid; cs=chondroitin sulphate; hs= heparan sulphate

second incubation with added P-hexosaminidase to complete the hydrolysis of the acetylated product MU-BGlcNAc is not needed. Optimal assay conditions, such as pH, substrate concentration (K,,.,) and protein and incubation time dependences, were established for CV, cultured CV cells, and amniocytes; these conditions were not significantly different from those for cultured skin fibroblasts and leucocytes (Voznyi et al., 1993). This allows the use of one standard assay procedure for the usual types of cells in pre- and postnatal diagnosis. The activity of N-acetyltransferase in CV is higher (1.5 times) than it is in skin fibroblasts; the level in cultured CV cells and amniocytes is some-

what lower (0.5 times). A deficiency of N-acetyltransferase activity in the CV of an affected fetus has been reported (Di Natale et al., 1987) and a similar result would be expected for amniotic fluid cells. Indeed, we have shown a complete N-acetyltransferase deficiency in the amniotic fluid cells from a pregnancy in which MPS I11 had already been indicated by the results of electrophoresis of glycosaminoglycans in the fluid. The latter approach has proven to be a reliable method for most or probably all types of MPS (Mossman and Patrick, 1982; Young, 1992; Zhao et al., 1990; Kagie et al., 1992). This is also our experience for Sanfilippo disease. In all

SANFILIPPO DISEASE TYPE C 21

Table 11-Acetyl CoA.glucosaminide N-acetyltransferase activity in amniocytes from a fetus affected with San6lippo disease type C

~ ~ ~~

Acetyl- Heparan- a- N-Acetyl- Vansferase* sulphamidase* glucosaminidase*

Amniocytes Pregnancy at risk D 0.0 3.4 Control 28 2.9

Ski fibroblasts MPS I11 A patient - 0.4 MPS I11 B patient - - MPS I11 C patient 0.2 - Fetus D (after TOP) 0 3 - Control (postnatal) 59 5.0

29 29

- 0-8 - - 74

*Enzyme activities are expressed in nmoU17 h per mg, % sulphate released, and nmoU17 h per mg, respectively. The activity of /3-galactosidase, measured as a reference enzyme, was normal in all samples.

TOP=Termination of pregnancy.

pregnancies with a fetus affected with MPS 111 A (n=6) and MPS I11 B (n=3), a clearly abnormal spot of heparan sulphate was present (Kleijer et al. , 1984), whereas no false-positive results were obtained in at-risk pregnancies with a normal fetus. A similar electrophoretic pattern was found in the case of MPS I11 C described in detail here and in a second case which we recently mentioned briefly (Zaremba et al., 1992). In general, reliable prenatal diagnosis may be expected only if the exact (enzymatic) diagnosis in the proband has been established early enough. In the case of strong evidence for MPS, based on clinical presen- tation and urine analysis, prenatal diagnosis by electrophoresis using amniotic fluid may be suc- cessful, as it was in the present case. In well- prepared cases, first-trimester diagnosis on CV by enzyme analysis may be the method of choice for all types of MPS including MPS 111 C. The present fluorometric assay facilitates this approach for MPS I11 C.

ACKNOWLEDGEMENTS

We thank Dr B. J. H. M. Poorthuis, Leiden for performing enzyme assays with the radiolabelled substrate.

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Prenatal diagnosis of sanfilippo disease type C using a simple fluorometric enzyme assay