The laboratory diagnosis of sanfilippo disease

139

76 (1977)

Clinica Chimica Acta,

139-147 Biomedical Press

@ Elsevier/North-Holland

CGA 8453

THE

LABORATORY

PAUL

WHITEMAN

DIAGNOSIS

* and ELISABETH

Department of Chemical WClN 1EH (U.K.) (Received

November

OF SANFILIPPO

Pathology,

22nd,

DISEASE

YOUNG

Institute

of Child Health,

30 Guilford

Street,

London

1976)

Summary The biochemical findings in 29 patients with Sanfilippo disease are reported and a scheme for laboratory diagnosis is outlined. A grossly elevated urinary excretion of heparan sulphate was a consistent and diagnostic finding, even at birth. The excretion of heparan sulphate and chondroitin sulphate was quantitatively similar in types A and B of the condition. Modifications of previously described methods for the determination of heparin sulphamidase in leucocytes or skin fibroblasts and N-acetyla-D-glucosaminidase in plasma or fibroblasts facilitated the measurement of specific activities. Sanfilippo A disease appeared to be the commonest mucopolysaccharidosis occurring in England and Sanfilippo B disease, one of the rarest forms. Introduction Sanfilippo disease is an autosomal recessive inherited disorder of glycosaminoglycan metabolism characterised by progressive mental retardation and the excretion of excess heparan sulphate in the urine [ 1,2,3]. Studies on cultured fibroblasts have demonstrated the existence of at least two distinct genotypes, designated type A and type B, characterised by deficiencies of specific “corrective factors” [ 41. Th e corrective factor deficient in Sanfilippo A disease has been identified as the enzyme heparan sulphate sulphamidase (EC 3.1.6.?) and in Sanfilippo B disease, N-acetyl-cu-D-glucosaminidase (EC 3.2.1.?) [5,6]. These discoveries have made possible the accurate laboratory diagnosis of these sub-types which now permits at-risk pregnancies to be monitored reliably

171. This report describes the laboratory findings in 29 patients with Sanfilippo disease using improved analytical techniques and outlines a useful scheme for the diagnosis of this condition. * To

whom

all corrrspondence

should

be

addressed.

1 40

Materials

and methods

Urine specimens Urine, preserved with mcrthiolate (1 in 10 000 w/v) and stored at -3O”C, was collected from 27 patients with Sanfilippo disease. Either 24-h collections or random specimens collected between mid-morning and mid-afternoon were accbcpted for analysis. Determination of totul urinary glycosanlirzoglycan (GAG) cxcrctim The total GAG content of urine was determined by a m&hod involving complex-formation with Alcian Blue 8GX [ 8,9,10]. Chondroitin 4-sulphatr was used as a reference standard. GAG excretion was expressed as a GAG : crcatininc ratio (rng GAG escretcd per gram of crclatinine). Quafztitation of individual Gtl G c’omponcnts in urine Glycosaminoglycans were isolated from urine using a modification of a procedure involving complex-formation with Alcian Blue 8GX [lo]. Urine (1-2 ml) was equilibrated for 2 h at room temperature with a reagent containing Alcian Blue (0.5 g/l; ICI Ltd., Blackley, Manchester, U.K.), 50 mM MgCl, and 50 mM sodium acetate adjusted to pH 5.8 with acetic acid. Aft,er isolation by centrifugation, the Alcian Blue-GAG complex was dissociatt>cl by vigorous shaking in a mixture of 4 M NaCl (0.2 ml) and methanol (0.1 ml). Free Alcian Blue was precipitated by tht> addition of 0.1 M Na,CO., (0.1 ml) and water (0.4 ml). The mixture was left for 30 min at room trmpcraturc after which the denatured Alcian Blue was removed by centrifugation. Glycosaminoglycans were precipitated from the clear supernatant (1 vol.) by the addition of ethanol (3 ~01s.). After centrifugation the precipitate was dried and dissolved in water (25-50 ~1). Samples (0.5-2.0 ~1) of the resulting clear solution were applied to sheets (Celagram; Shandon Southern Instruments Ltd., cellulose acrtattl Camberlry, Surrey, U.K.) and the glycosaminoglycans separated by elcctrophoresis in 0.1 M barium acetate for 3-4 h at an applird potential gradient of 7.5 V/cm [ 111. The sheets went stained for 1 h in a solution containing Alcian Blue 8GX (0.5 g/l) and 50 mM MgCl, in a 50 mM sodium acetate buffer (pII 5.8) and then washed three times in a solution of 50 mM MgCl? in 50 mM sodium acetate buffer (pH 5.8). The individual GAG component,s were estimated by elution of the Alcian Blur complexes into a dimrthyl sulphoxide reagent as described elsewhere [ 121. Prsparation of cell homogenates Buffy coat was isolated from heparinised blood (10 ml) by ccntrifugation at 1000 X g for 10 min, and washed twice with cold 0.155 M NaCl (1 ml). After resuspension of the leucocyte pellet in cold water (0.75 ml) for 90 seconds, isotonic conditions were restored by the addition of 0.62 M NaCl (0.25 ml). The leucocytr pellet was finally homogenised in cold 0.2 M KC1 (0.3 ml), frozen and thawed once and the supernatant used in the enzyme assay. Skin fibroblasts were grown and harvested as described by Young et al. 1131, homogcnised in water and frozcan and thawed once before enzyme' assay. The protein content of fibroblast homogenates and supcrnatant of lcucocyte homogenates was determined by the method of Lowry et al. [ 141.

741

Heparin sulphamidase Heparin sulphamidase Kresse [15].

assay was determined

using

a modification

of the method

of

[N-suIphonate-35S]Heparin (Radiochemical Centre, Amersham, Bucks, U.K.) was diluted with non-radioactive heparin (“Pularin”; Evans Medical, Liverpool, U.K.) to give a specific activity of 15 pCi/mg and adjusted to a final concentration of 1.73 g/l. The preparation was dialysed at 2°C for 48 h against distilled water to remove free inorganic [““Slsulphate. Assay mixtures contained sodium acetate buffer (0.1 M; pH 5.0), sodium azide (3.1 mM), KC1 (66 mM in leucocyte assays; 33 mM in fibroblast assays), [N-sulphonate-““Slheparin (34.6 pg in leucocyte assays; 17.3 pg in fibroblast assays) and an aliquot of leucocyte extract (containing 40-70 pg protein) or fibroblast homogenate (containing 20-25 pg protein). The final total volumes of the incubation mixtures were 150 ~1 for leucocyte assays and 60 ~1 for fibroblast assays. Enzyme preparations were omitted from control experiments. After incubation at 45°C for 18 h (leucocyte extract) or 4 h (fibroblast homogenate) the mixture was centrifuged and the released sulphate in the supernatant separated by electrophoresis. Supernatant (50 ~1) was applied (5 cm from the cathode) to strips of Whatman No. 1 chromatography paper (20 cm X 3.0 cm) and electrophoresis carried out at room temperature in 0.1 M barbitone/HCl buffer (pH 8.6) containing cetylpyridinium chloride (2.5 g/l) for 90 min at an applied potential gradient of 7.7 V/cm. After drying, strips were cut into 1.5-cm sections and [35S]sulphate determined by counting the six sections nearest the anode in toluene scintillator containing 2,5-diphenyloxazole (8 g/l) and 1,4-di [2-(5-phenyloxazolyl)] benzene (0.1 g/l). The remainder of the control strip was also counted to determine the amount of [N-sulphonate“Slheparin present. Sulphamidase activity was calculated on the basis of 1.46 pmol N-sulphate/mg [N-sulphonate] heparin [ 151. The sulphamidase activity of normal leucocyte preparations was barely detectable at 37°C but showed a marked increase at higher temperatures (Fig. 1). A four-fold linear increase in activity occurred between 45 and 55°C; an

Temperature Fig.

1.

Effect

were

otherwise

of

incubation

those

(‘Cl temperature

of the standard

on

procedure

Ieucocyte-heparin (see

text).

sulphamidase

activity.

Assay

conditions

142

Fig. 2. Effect of protein content of the assay mixture on leucocyte-heparin sulphamidase activity. Apart from thr addition of different amounts of leucocyte extract, assay conditions were those of the standard procedure (SC? text). Fig. 3. Effect of substrate concentration on heparin-sulphamidasz activity. Apart, from the presence of different concentrations of [n’-.~ulpl~~~~rof~~-“Slheparin. assay conditions were those of the standard leucocyte extract. I-, fibroblast homogenate. Proccclure (see text). @-A,

incubation temperature of 45°C was chosen for routine assays. This interesting temperature effect, which does not appear to he a simple thermal activation of sulphamidase, has not been investigated further. Sulphamidase activity was proportional to the amount of protein in the leucocyte assay mixture up to a concentration of approximately 0.5 g/l (Fig. 2). A volume of leucocyte extract containing 40-70 pg protein was used in the standard assay. The effect of substrate concentration on enzyme activity is shown in Fig. 3. The substrate concentrations used routinely were 0.23 g/l in leucocyte assays and 0.29 g/l in fihroblast assays. N-Acetyl-a-U-glucosaminiclase assay Plasma N-acetyl-a-D-glucosaminidase activity was determined using a modification of the method of von Figura et al. [16]. The concentration of p-nitrophenyl-2-acetamido-2-deoxyiy-D-glucopyranoside (Koch-Light Laboratories, Colnbrook, Bucks, U.K.) was reduced to 5 mmol/l and the assay mixture incubated for 4 h at 37°C. The absorbance of released p-nitrophenol was read at 400 nm. No difference in specific activity was found if serum was used instead of plasma. Fibroblast homogenate (containing approximately 100 1.18protein) was incubated for 6 h at 37°C in a mixture (final volume, 100 ~1) containing 0.05 M sodium acetate buffer (pH 4.5) and 5 mM p-nitrophenyl-2-acetamido-2-deoxya-D-glucopyranoside. The reaction was stopped by addition of 0.825 M perchloric acid (50 ~1). After centrifugation, a portion of the supernatant (100 ~1) was mixed with 0.4 M glycine/NaOH buffer (pH 10.4; 300 ~1) and the absorbance of the released p-nitrophenol measured at 400 nm. Results Studies on urinary glycosaminoglycans Patients with Sanfilippo disease,

whose

ages ranged

from

1 day to 20 years,

TABLE

I

BIOCHEMICAL GAG,

FINDINGS

glycosaminoglycan:

keratan

sulphate:

Patients

leuc..

PATIENTS

WITH

chondroitin

leucocytes; Sex

Age

IN 29 CS.

Urinary GAG:

fibro., GAG

creatinine

with

Sanfilippo

4 (Sib. No.

of

Heparin

N-Acetyl-a-D-

sulphamidase

glucosaminidase

pm01

Plasma

Fibro-

(nmol/

blasts

ml/

(nmol/mg

min)

protein/h)

of total

Control

(mg/g)

range

GAG) _

for

CS

HS

*

LlSlC.

Fibro.

disease 182-395

34

66


414

41-261

40

60


2Y

M

345

82-186

43

57


0.96

3 y

M

274

51-140

39

61


0.92

5

3Y

M

462

51-140

36

64

3Y

M

452

51-140

40

60

I

4Y

M

310

51-l

37

63


4 y

F

390

51-140

37

63


No.

sulphate/

mg protein/h

0.94 <150

1.72

40

1.28 2.25 <150

1.03 1.44

1)

9

4Y

F

570

51-140

35

65

10

4Y

F

486

51-140

44

56

F

356

51-109

39

61


1.05 1.22

11

(Sib.

of

No.

10)

5

Y

<150

18.1

12

5Y

F

485

51-109

44

56


13

5Y

F

384

51-109

33

67


14

5Y

M

230

51-109

33

67

15

7Y

M

361

34-107

40

60


1.1

16

7Y

F

382

34-107

33

67


2.13

17

8~

M

356

34-107

40

60


1.59

18

9Y

F

286

30-109

36

64


1.2

9 y

F

345

30-109

32

68

a0

2.93

255

30-109

33

67


19

(Sib. No.

of

1.5 <150

14.2

16)

20

9Y

F

21

9Y

M

22

9Y

M

23

(Sib.

of

No.

22)

11

Y

M

0.82 <150

267

30-109

38

62

21.3


2.16


0.22

24

12

Y

1’

270

22.--lo4

39

61

25

13

Y

F

331

19-84

39

61

26

13

Y

M

36

64


1.09

14

y

M

354

19-84

34

66


1.21

20

Y

M

139

13-38

24

76

F

288

34

66

27

(Sib.

of

No.

26)

28 29 Patients

with

30

other

mucopolysaccharidoses

0.89


0

910

1.0 <150

9.5

cs

DS

39

57

4

184

0.88

14

58

28

104

0.36

62

8

189

0.45

278

0.27

_

9m

M

871

41-261

3Y

M

910

51-l

4Y

M

898

51-140

30

8~

M

197

34-107

**

HS

(Maroteaux-

Lamy) 31

40

(Hunter) 32 (MaroteauxLamy) 33 (Morquio) * Control **

15.4

3)

of

ranges

CS = 67%:

taken

KS = 32%

from by

Whitrman

2-dimensional

KS,

Individual

1017

m

sulphate:

components

6 8 (Sib.

heperan

F

10

3

HS,

F

1 day

2

sulphate:

Enzymology

Patient

age

DISEASE

dermatan

excretion

(B

1

DS.

fibroblasts.

ratio

Patients

SANFILIPPO

sulphate;

[171. electrophoresis.

Whiteman

1171.

144

cs

cs

DS HS

DS HS . 0

*a

..

STD

a.

.br

N

A

l I

B

ST;’

Fig. 4. Elelectrophoretic pattern of urinary glycosaminoglycans in Sanfilippo disease. Solutions (2 ~1) uf standard and urinary GAGS were applied to cellulose acetate membranes and the components separated by electrophoresis in 0.1 M barium acetate for 3 h at an applied potential gradient of 7.5 V/cm. (InternatiOnal reference standard GAGS were kindly given by Dr. M.B. Mathews. Dr. J.A. Cifonelli and Dr. 1,. Rod&. Department of Pediatrics, University of Chicago). STD. standard GAGS; CS, chondroitin sulphate; DS. dermatan sulphate; HS. heparan sulphate; N. normal child: A, Sanfilippo A disease; B. Sanfilippo B disease.

had GAG: creatinine ratios which were grossly elevated above an age-matched control range (Table I). Previous studies, described in detail elsewhere [17] demonstrated no significant sex difference in the GAG : creatinine ratios of age-matched control groups. Qualitative analysis indicated that, irrespective of age or sub-type, heparan sulphate was consistently the predominant component and chondroitin sulphate a minor component in the urine of these patients. Dermatan sulphate was present only in trace amounts. This particular pattern of mucopolysacchariduria is diagnostic of Sanfilippo disease and does not occur in any other type of mucopolysaccharidosis [ 171. Chondroitin sulphate accounted for more than 80% of the total urinary GAG excreted by 8 healthy children with ages ranging from 2 months to 12 years. Using the methods outlined in this report, chondroitin sulphate, dermatan sulphate and heparan sulphate had similar chromogenicities. This is not necessarily the case with some other methods and care must be exercised when values reported by different workers are compared [ 171. Fig. 4 shows the electrophoretic patterns given by urinary GAGS from patients with Sanfilippo disease. The patterns given by electrophoresis in 0.1 LM barium acetate do not allow differentiation of sub-types A and B although a difference in the mobility of a heparan sulphate sub-fraction can be detected using 2-dimensional electrophoresis (method of Whiteman [17,18]). Enzyme studies A marked sulphamidase deficiency was demonstrated in the leucocytes or fibroblasts of 25 patients with Sanfilippo disease, all of whom had normal N-acetyl-cu-D-glucosaminidase activity in their plasma or fibroblasts (Table I). In addition, three other patients probably had Sanfilippo A disease; one patient

145

TABLE

II

ENZYME

ACTIVITIES

IN CONTROL

Subjects

SUBJECTS

Heparin pm01

AND

UNAFFECTED

sulphamidase

sulphate

released/mg

protein/h

RELATIVES

OF

PATIENTS

N-Acetyl-ol-D-gluc(,saminidasr

Plasma

Fibroblasts

(nmol/ml/

(nmol/mg

protein/h)

min) Leucocytes

Fibroblasts

172

2757

Controls Mean

value

Gross

range

52-458

Number Relatives

42

5164460 14

0.58 0.14-1.37 58

19.5 6.4-39.0 19

of:

Patients

No.

18

Father

134

0.57

Mother

53

Sister

98

0.43

185

0.37

Father

203

0.52

Mother

92

0.30

Sister

42

0.27

Brother Patients

Patient

Nos.

No.

22

and

23

21

Mother Patient

No. Nos.

Father

850

20.0

610

14.5

2

Mother Patients

0.43

1 and

8

-

101

0.63

(No. 10) was the sibling of a patient (No. 11) with known sulphamidase deficiency and two other patients (Nos. 5 and 28) had normal plasma N-acetyl-a-Dglucosaminidase activity. One patient (No. 23) with Sanfilippo A disease, who was in a terminal state, had a plasma N-acetyl-cu-D-glucosaminidase activity in the low normal range; the others showed increased or high normal activities (including the sibling; patient No. 22). In control groups, enzyme activities had non-Gaussian frequency distributions and for this reason only the gross ranges have been given (Table II). Although none of the control subjects had mucopolysaccharidosis, some were investigated for other types of storage disorder. When fibroblast or leucocyte preparations from control subjects were mixed with similar preparations from patients with Sanfilippo A disease the expected intermediate sulphamidase activities were obtained. Patients with other types of mucopolysaccharidosis had normal heparin sulphamidase activity in leucocytes and normal N-acetyl-Q-Dglucosaminidase activity in plasma (Table I). The assay of heparin sulphamidase in lcucocytes was unsuitable for the detection of carriers of Sanfilippo A disease, although estimation of the enzyme in cultured fibroblasts gave better discrimination (Table II). Using fibroblasts, only one of the control subjects (out of 14) had a sulphamidase activity lower than those given by the two obligate heterozygotes. Cultured skin fibroblasts from one patient (No. 25; from Warsaw, Poland) had normal sulphamidase activity but showed a marked deficiency of N-acetyl-

146

a-D-glucosaminidase activity. This patient, who had Sanfilippo B disease, was clinically indistinguishable from patients with Sanfilippo A disease. Unfortunately, plasma from this patient was not available for study. Discussion At the Hospital for Sick Children, London, a diagnosis of mucopolysaccharidosis has been established in 70 patients during the last 5 years. Patients with Sanfilippo A disease, most of whom were indigenous to the United Kingdom, formed the largest single group. Only one patient, who was from Poland, had Sanfilippo B disease. Thus, in England, Sanfilippo A disease appeared to be the commonest mucopolysaccharidosis and Sanfilippo B disease one of the rarest forms. Now that prenatal diagnosis is possible in this condition [ 71, it is particularly important that an accurate laboratory diagnosis is made in the propositus. Initial diagnosis of Sanfilippo disease requires a high index of suspicion on the part of the clinician since some children with this condition may appear relatively normal during the first year of life. The present study shows that abnormal mucopolysacchariduria is a consistently reliable finding in this condition, even at birth, provided care is exercised in the choice of analytical technique and in the method of expressing the results [ 10,171. Excessive heparan sulphate excretion is also evident in the foetus affected with Sanfilippo disease [7,18]. The diagnosis of Sanfilippo disease should first be established by urinary GAG studies since a characteristic pattern of mucopolysacchariduria occurs in this condition. Wessler’s simple method of clectrophoresis in 0.1 M barium acetate is suitable for the qualitative analysis of urinary GAGS [ll]. The specific sub-type may then be determined by the diligent use of the more costly and time-consuming enzyme studies. Singh et al. [19] were able to differentiate Sanfilippo A and B diseases by the fractionation characteristics of the urinary GAGS on gel-filtration chromatography. Since there may be more than two sub-types of Sanfilippo disease it would still necessary to undertake specific enzyme studies to confirm the diagnosis. Lewis et al. [20] claimed to be able to differentiate sub-types of Sanfilippo disease by the presence or absence of the urinary chondroitin sulphate component but this is questionable on theoretical grounds. In our experience, disappearance of the chondroitin sulphate component is likely to be due to bacterial contamination of the urine specimen [17]. The present study shows that it is not possible to differentiate Sanfilippo A and B diseases using Wesschondroitin sulphate and heparan sulphate ler’s method of electrophoresis, being excreted in both sub-types. Acknowledgement We wish to express our gratitude for the support given by Professor Barbara E. Clayton and for the helpful advice given by Dr. A.D. Patrick. Thanks are also due to Mrs. Jean Gurney (supported by Action Research for the Crippled Child), who undertook the cell-culture work and to Dr. Rosemary Stephens, Professor O.H. Wolff and other members of the Staff of the Hospital for Sick

147

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