Z. Kinderheflk. 108, 17--31 (1970)

Biochemical Definition of the Mucopolysaccharidoscs* J. W. Srl~A~G~ Department of Pediatrics (Prof. Dr. H.-R. Wiedemann), University of K i d Medical School Received August 29, 1969

Abstract. The excretion pattern of urinary acid mucopolysaccharides was determined in 72 patients with different clinical types of mucopolysaccharidoses and mucolipidoses. Using a chromatographic fractionation method, characteristic excretion patterns were found in the six classical types of mucopolysaecharidoses. Patients with Hunter's disease excreted relatively more heparansulfate and less dermatansulfate than patients with Httrler's disease. I n Sanfilippo's disease the excretion of heparansulfate only was increased. I n Morquio's disease abnormal amounts of substances with characteristics of chondroitin-4(6)-sulfate were found in addition to keratansulfate. In one patient with Ullrich-Scheie's disease and six patients with Maroteaux's disease relatively large amounts of substances with characteristics of dermatansulfate were present. There was very little overlap between the excretion patterns of different types of mucopolysaccharidoses. I n the types of mucolipidoses investigated, the urinary excretion of acid mucopolysaccharides was normal. The intrafamilial variability in eight pairs of related children was small. Our data suggest that the excretion pattern of urinary acid mucopolysaccharides, as determined by the Dowex Column Chromatography Method, is a valuable aid in the definition of the mucopolysaccharidoses. Key-Words: Mucopolysaecharidoses - - Mucolipidoses - - Acid Mucopolysaccharides - - I-Iurler's Disease. Zusammen/assung. Bei 71 Patienten mit verschiedenen Formen der Mucopolysaccharidosen and Mueolipidosen wurde das Ausscheidungsmuster saurer Mucopolysaccharide sEulenchromatographisch bestimmt. Im H a m yon Patienten mit Mb. Pfaundler-l~urler land sich relativ mehr Dermatansulfat und weniger Heparansulfat als bei Patienten mit Mb. Hunter. Beim M b Sanfilippo war allein die Ausscheidung von Heparansulfat erh6ht. Abnorme Mengen yon Substanzen mit Charakteristika yon Chondroitin-4(6)-sulfat and Keratansulfat fanden sich beim b~b. Morquio. Ein Patient mit Mb. ~llrich-Scheie (sogenarmter ,,Spi~t-Hurler") and 6 Patienten mit Mb. Maroteaux hatten in ihrem Urin vorzugsweise Substanzen mit Charakteristika yon Dermatansulfat. Die Ausscheidungsmuster der verschiedenen Biotypen iiberlappten kaum. Patienten mit verschiedenen Formen der Mucolipidosen hatten eine normale Ausscheidung saurer Mucopolysaccharide. Die intrafamilii@e Variabflitiit der Ausscheidungsmuster bei 8 Paaren verwandter Kinder war gering. Nach diesen Ergebnissen ist die s/~ulenchromatographisehe Bcstimmung der sauren Mucopolysaccharide im H a m ein wertvolles I-Iilfsmittel zur Differentialdiagnose der Mucopolysaccharidosen und Mucolipidosen. Diese Differenzierung ist aus prognostischen, genetisehen und pathogenetischen Griinden unerl~Blich. * Supported by Grants No. W I 80 of the Deutsche Forschungsgemeinschaft. 2 Z. Kinderheilk., Bd. 108

18

J.W. Spranger:

Mucopolysaccharidoses are systemic disorders of mueopolysaeeharide metabolism resulting in abnormal intra- and extracellular accumulation of acid mucopolysaeeharides (AMPS). Clinically, the patients are characterized b y a syndrome which typically includes coarse facial features, small stature, skeletal deformities, mental retardation, hepatosplenomegaly, corneal clouding and impaired hearing in varying combination. On the basis of different phenotypic expression several groups of patients have been delineated which presumably correspond to different genotypes. The morphological features of these types have been summarized in recent reviews [18, 23, 28, 42]. The laboratory diagnosis of affected individuals has been based on the increased urinary excretion of AMPS. Substances identified in the urine have biochemical characteristics of dermatansuffate, heparansulfate, ehondroitin-4(6)-sulfate, keratansulfate and hyaluronie acid 1. Although definite excretion patterns have helped to establish the identity of several mucopolysaccharidoses, especially Sanfilippo's disease [38], Morqnio's disease [22], Maroteaux's disease [25], the issue of biochemical differentiation of the mucopolysaceharidoses has remained controversial [18, 28, 39]. I t is the purpose of this paper to present evidence t h a t the urinary excretion p a t t e r n of the AMPS m a y be correlated with the morphological patterns of the different mueopolysaecharidoses. Patients and Classification Clinical, radiological information and urine specimens were obtained from 72 patients with different types of mucopolysaccharide storage diseases. Many of the patients were personally examined by the author. In other cases information was kindly provided by physicans sending urine from patients suspected of having Hurler's diseases. On the bases of clinical and radiologic criteria, 51 of the patients 1 Nomenclature of acid mucopolysaccharides [11]: Former name

. ~ e w TbaT/?,e

Chondroitin-4-Sulfate (CH-4-S) Chondroitinsulfate A Chondroitin-6-Sulfate (CH-6-S) Chondroitinsulfate C Dermatansulfate (DS) Chondroitinsulfate B Heparansulfate (HS) Heparitinsulfate Keratansulfate (KS) Keratosulfate Hyaluronic acid (HYS) Hyaluronie Acid 2 The cases listed in Table 3 are patients from: Prof. Bierich, Tiibingen (ease 2), Dozent Dehnert, Konstanz (case 19), Dr. Eckert, EckernfSrde (case 29), Dr. Eekert, Hamburg (case 8), Dr. Greinacher, Mainz (cases 5, 40), Dozent Hoen, Darmstadt (case 4), Prof. Hungerland, Bonn (case 15), Dr. Klimt, Berlin (eases 9, 18), Prof. Koch, Gieflen (cases 13, 46, 47), Prof. Kosenow, Krefeld (cases 30, 31), Dr. Murken, l~Iiinchen (case 27), Dozent Natzsehka, Berlin (case 48), Dr. v. Rohden, Magdeburg (case 24), Prof. Sehreier, Niirnberg (cases 34, 35, 36, 39), Dozent Schuster, Erlangen (cases 12, 37, 38), Dr. Seller, Osnabriick (cases 20, 21, 32), Dozent Simon, Oldenburg (case 3), Dr. Weirich, Homburg (case 6), Prof. Wiedemann, Kiel (cases 50, 51).

Biochemical Definition of the Mucopolysaccharidoses

19

Table 1. Major clinical criteria used in the di]erentiation o/mucopolysaccharldoses Type

Sex

Mental Skeletal retardation dysplasia

Dwarfisma Corneal opacities

Genetic b evidence

I II III IV V VI

m,f m m, f m, f m,f m, f

q-qqq-q----

q-qq-q-+ (-}-) q-

a.r. X.r. a.r. a.r. a.r. a.r.

q-qq(q-) q-qq+ +

q--q-~ q-

- - not present, (q-) mildly present, q- present, + q - markedly present, a N o t in first year of life. b Geneti6 evidence for autosomal recessive (a. r.) mode of inheritance: male and female siblings involved, parental consanguinity, abnormal bloodcell or fibroblast granulations in father and mother. Genetic evidence for X-chromosomal recessive (X. r.) mode of inheritance: males only, similar cases in maternal family, abnormal blood-cell or fibroblast granulations in mother only. Urine ~""~Determine

creatinine content

Dialyze againl 0.9o/o NaCI Precipitate at ~ ,H 5.0 with CTAB* 48h

!

,4~

Centrifuge, wa~sh precipitate four times with NaCl-saturated 96O/o alcohol Dissolve precipitate in 1Oo~ Na-Acetate, adjust to p i t 9.0, centrifuge

|

Adjust super:atant to pH 5.0, precipitate with 4 volumes abs. alcohol 48h ! t 4~

1

Dissolve precipitate in 2.0 ml aq. dest. ~ o 2 e : 2 : ~ a t o g r ~ x u r o n i c

acid content

0.5m - - 1.25m - - 1.5m - - 2.0m - - 3.0fia NaC1 ~rbaz~ole ~ t i o~n' ' ' ' " Anthron~e reaction Fig. 1. Purification and isolation of urinary acid mucopolysaccharides, according to [23, 44, 47] * CTAB = Cetyltrimethylammoniumbromide 2"

20

J . W . Spranger:

were assigned to one of the six known types of mucopolysaccharidoses, using clinical criteria set forth by Maroteaux and Lamy [23], Leroy and Crocker [18] and MeKusick [28]. Prominent diagnostic criteria used to classify the patients are summarized in Table I. These patients form the basis of the present report. Ten patients with increased mucopolysaccharide excretion could not be assorted to one of the better known mueopolysaecharidoses and were omitted from ~his

study. In additional 11 children with clinical symptoms of the mucopolysaeeharidoses the urinary mucopolysaccharide excretion was normal. Further investigation permitted their diagnostic assignment to one of the "mucolipidoses" [43] i.e. a group of condition such as pseudopolydystrophy [24], I-cell-disease [19], Fucosidosis [51] and Lipomucopolysaecharidosis [45]. Their clinical diagnosis was established following criteria set forth in the original publications. B i o c h e m i c a l Methods Urinary acid mucopolysaccharides were determined according to methods previously described [41, 44, 47]. The principal steps of purification and separation of individuM A ~ P S are outlined in Fig. 1. Uronic acid was determined by the carbazole method [5] and the naphtoresorcinol reaction [46]. /~eutral sugar was determined with anthrone [37]. Digestion with hyaluronidase was performed according to 1Katthews and Inouye [27] using the disappearance of stainibility with toluidin blue [2] as parameter. Creatinine was determined according to Langley and Evans [17] in a modification for autoanalyzer. Results A . Quantitative Excretion

I n n o r m a l c h i l d r e n t h e t o t a l e x c r e t i o n of A M P S , as e x p r e s s e d b y t h e u r o n i c a c i d / c r e a t i n i n e r a t i o , fell w i t h i n c r e a s i n g age (Tabtc 2). This r a t i o was e l e v a t e d in 50 o f 62 classifiable p a t i e n t s w i t h different t y p e s o f m u c o p o l y s a c e h a r i d e s t o r a g e diseases. One o f t h e r e m a i n i n g 12 p a t i e n t s h a d t h e a d u l t t y p e o f m u c o p o l y s a c c h a r i d o s i s (Ullrieh-Seheie's disease [40, 49]) a n d 11 b e l o n g e d t o t h e so-called " m u c o l i p i d o s e s " , exh i b i t i n g s y m p t o m s of b o t h t h e m u c o p o l y s a c e h a r i d o s e s a n d t h e sphingolipidoses. N o correlation was f o u n d b e t w e e n t h e uronic a c i d / c r e a t i n i n e r a t i o a n d t h e t y p e o f m u e o p o l y s a e c h a r i d o s i s (Table 3). Table 2. Quantitative excretion o/acid mucopolysaccharides in the urine o/ normal individuals, expressed as mg AMPS-hexu~vnic acid per g creatinine Age in years

No. of indiv. Mean tested

Range

0.1--0.2 0.3--1.0 1.1--4.0 4.1--10.0 10.1--15.0

6 8 8 18 15

(58.9--92.8) (13.5--55.1) (7.5--27.2) (2.8--15.2) (2.2--14.6)

72.2 34.1 14.3 S.4 7.2

Biochemical Definition of the Mucopolysaccharidoses % 100-

I

T;

21

TrY

50

1,25 1.50

2.0

"3.0 1.25 1.50

9r v

0

],25

LS0

2.0

3.0

1.25 1.50

2.0

3.0 mNoCt

2.0

3,0

].20

2.0

3.0

~

2.0

3.0

[,25

LS0

].50

mNoC[

L~= CHARACTERISTICS OF DERMATANSULFATE [ ] = NEUTRAL SUGAR [PRES. KERATANSULFATE] I = RANGE

Fig. 2. mean and range of percent distribution of carbazol equivalents in 6 clinical types of mucopolysaccharidoses

B. Chromatographic Fractionation Pattern The fraetionation pattern of AMPS, as expressed by the percent distribution of the carbazol equivalents, differed according to the type of mueopolysaccharidosis present (Table 3). As seen in Fig. 2 there was minimal overlap between the excretion patterns of types I, I I and III. Most of the AMPS excreted in 2 patients with Morquio's disease (type IV) appeared in fraction 1.5 m NaC1. In addition, there was a small but significant amount of neutral-sugar containing and carbazol-negative material in fraction 3.0 m NaCI. The total excretion of AMPS was normal in 1 patient with Ullrich-Scheie's disease (type V). Most of the material appeared in fraction 1.5 m NaC1 and had characteristics of dermatansulfate (see section C). 6 patients with Maroteaux's disease (type VI) excreted AMPS that mostly appeared in fraction 1.5 m and 2.0 m l~aC1. Again no overlap was noted with the distribution pattern of any of the other high-excreting types. In 11 patients classified as belonging to the "mucolipidoses" the distribution pattern of urinary AMPS corresponded to that of normal individuals [44]. I n 9 patients with types I, I I I and VI the excretion of urinary AMPS was re-evahiated after periods ranging from 2 days to 4 years. Though some variation of the excretion pattern was observed, in no

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

J.P. A. lVI. M.L. B.B. F.M. S.L. IV[. M. K.K. K.E. J.J. I~. K. It. K. C.S. J.H. M. )5. J.C. ] A.C. f

0.2 1 2 2 2 3 3 4 4 4 4 4 5 5 6 6 8

Type I (Hurler's Disease)

Age in years

m m f f m f f m f m m f m m m m f

Sex

0 0.8 0 0 0 0 0 0 3.2 0 0.2 0 0 2.3 1.0 0 0 0.4 0--3.2

128.0 185.0 93.4 59.2 39.7 187.2 63.1 125.9 33.8 73.0 80.4 107.9 59.9 52.5 179.0 330.0 268.0 Mean Range

47.8 34.7--59.0

49.0 47.8 51.5 40.1 53.7 49.2 48.8 34.7 39.9 52.6 59.0 51.6 47.8 46.1 47.6 44.2 50.4 33.0* 11.1~9.9

39.2* 36.9* 33.0* 49.9* 11.1" 26.0* 34.1" 31.0" 44.9* 20.2* 23.7* 31.5" 40.2* 38.5* 37.8* 35.1" 28.9*

18.9" 11.8--35.1

11.8" 14.4" 16.9" 15.3" 35.1" 24.9* 17.0" 34.2* 12.1" 27.2* 17.5" 16.8" 12.0" 12.6" 13.6' 20.5* 20.5*

DS*

0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

3.0 m KS

Ch-4-S Ch-6-S DS*

in NaC1 fraction

HS

containing predominantly HyS

mg uronic acid g creatinine

mg neutral sugar g creatinine

~ carbazole equivalents in NaC1 fraction 0.5 m 1.25 m 1.5 m 2.0 m

Total AMPSexcreted as

Quantitative and qualitative urinary excretion o/acid mucopolysaccharides in 62 patients with di~erent /orms o/mucopolysaccharidoses and mucolipidoses

Name of patient

Table 3.

5~

Biochemical Definition of the Mucopolysacch~ridoses

O

23

Age in years

K.S. M.S.

8 13

f f

Sex

W.W.

31

2 3 6 19 21 22

"lYlucolipidoses"

K.T. A.B. A.V. H. ~ . K.L. E.L.

f f m m f f

m

19.9 5.8--30.2

30.2 26.5 15.7 26.8 14.2 5.8

27.6

30.5 35.3

52.9* 44.2--75.9

44.2* 50.5* 46.4* 44.7* 75.9* 55.9*

58.0*

50.6 54.1

26.9* 8.2--37.9

25.5* 23.1" 37.9* 28.6* 8.2* 37.9*

14.4"

19.0 10.6

KS

in NaC1 fraction 3.0 m

mg neutral sugar g creatinine

O 0 0 0 0 0

0

39.8 2.2

Normal excretion of urinary acid mucopolysaccharides

0.4 0--1.8

l~ean Range 11 patients:

0 0.3 0 0 1.8 0.4

0

0 0

~ carbazole equivalents in NaC1 fraction 0.5 m 1.25 m 1.5 m 2.0 m containing predominantly HyS HS CL-4-S DS* Ch-6-S DS*

140.1 61.0 229.7 39.0 11.4 9.4

2.6

88.0 14.5

mg uronie acid g creatinine

excreted as

Total AMPS-

}patients related. * Substances with characteristics of dermatansulfate (c~rbazole/naphtoresorcinol quotient below 0.7, not digestible by testicular hyaluronidase, digestible by hyaluronidase from dermatansulfate-adapted bacteria).

46] 47] 48 49 50) 51~

Type VI (~aroteanx's disease)

45

TyTe V (Sp~t-Hurler or Scheie's Disease)

41~ 44]

Type IV (Morquio's disease)

patient

Name of

Table 3 (Continued)

~

Biochemical Definition of the Mucopolysaccharidoses

25

instance would the results of the repeated examination have led to a different classification of the patient. Among our patients there were 7 pairs of siblings with types I IV and VI, 2 cousins with type VI, and 5 siblings from 2 families with mucohpidoses. Their excretion pattern showed but little variation, and in no instance it would have led to a classification of blood related patients in different groups.

C. Types o[ A M P S Excreted ~ o efforts were made to characterize fully the AMPS appearing in individual NaC1 fractions. From studies with purified material [41, 44] it is known that hyahironic acid and glycoproteins appear in fraction 0.5 m NaC1. Only minute amounts of these substances were found in this fraction throughout the present study. In previous studies heparansuffate was shown to appear in fraction 1.25 m [41, 44, 47]. Kneeht et al. [13] eluted from the urine of patients with Hurler's disease two fractions of heparansulfate with 1.0 m NaC1 and 1.3 m NaC1 respectively, using a similar chromatographic system. From these observations it is inferred that fraction 1.25 m NaC1 contained mostly heparansulfate or degradation products thereof. In this study the relative amounts of heparansulfate excreted was the most valuable criterion to differentiate types I, II, I I I and VI. In types I, II, V and VI material appearing in fractions 1.5 m and 2.0 m had a carbazol/naphtoresorcinol ratio below 0.7 and was not digested b y testicular hyaluronidase. In contrast to this, it was readily digested by hyaluronidase from dermatansulfate-adapted bacteria a. These observations are consistent with the assumption that patients with types I, II, V and VI excreted large amounts of dermatansulfate in addition to heparansulfate, material in fractions 1.5 m and 2.0 m from patients with types III, IV and the "mucolipidoses" was digestible by testicular hyaluronidase and had carbazol/naphtoresorcinol ratios above 3.0. From this and from the previous observation that C-4(6)-S appears in fractions 1.5 m and 2.0 m [41, 44, 47], it is concluded that patients with Sanfilippo's disease, Morquio's disease and the "mucolipidoses" excreted varying amounts of these substances in addition to heparansulfate. Our patients with Morquio's disease excreted material that was eluted with 3.0 m ~aC1, gave a strong reaction with anthrone and contained little or no uronic acid. Since purified keratansulfate appears in fraction 3.0 m NaC1 [23, 44], this material is considered to represent keratansulfate [3, 12]. Though the total excretion of ~MPS was normal in our patient with Ullrich-Scheie's disease (type V), much of the material appearing in 3 Courtesy Dr. 1VIaroteaux,Paris.

26

J . W . Spranger:

Table 4. Excretion Tattern o/urinary acid mucopolysaccharides in diUsrent types o]

mucopolysaccharidoses Type

Total Substances with characteristics of amount Heparan- Dermatan- Chon- Keratanof AMPS sulfate sulfate droitin- sulfate excreted 4(6)sulfate

I Hurler's Disease I I Hunter's Disease I I I Sanfilippo's Disease IV biorquio's Disease V Ullrich-Scheie's Disease VI Maroteaux's Disease ,,Mucolipidoses"

+ + + + +/-+ --

+ ++ -4-+ -+/--. .

+4-+ + --+ + + .

---+ ---

---+ ---

.

-4- increased, +-t- markedly increased, - - not increased, + / - - sometimes increased. fractions 1.5 m a n d 2.0 m h a d characteristics of dermatansulfate. Large a m o u n t s of d e r m a t a n s u l f a t e are n o t f o u n d in n o r m a l individuals [44, 47].

D. Diagnostic Scheme The m a j o r diagnostic criteria derived from the above d a t a are summarized in Table 4. P a t i e n t s with t t u r l e r ' s disease (type I) excreted relatively less heparansnlfate a n d more dermatansulfate t h a n patients with H u n t e r ' s disease (type I I ) . P a t i e n t s with Sanfilippo's disease (type I I I ) excreted large a m o u n t s of heparansulfatc only. I n Morquio's disease (type IV) increased a m o u n t s of ehondroitin-4(6)-sulfate a n d keratansulfate were excreted. Our p a t i e n t with Ullrich-Scheie's disease (type V) excreted small, t h o u g h a b n o r m a l a m o u n t s of material having characteristics of dermatansulfate. P a t i e n t reported in the literature [21, 48] h a d an a b n o r m a l l y high excretion of heparansulfate as well. P a t i e n t s with M a r o t e a u x ' s disease (type VI) excreted mainly substances with characteristics of dermatansulfate. P a t i e n t s classified in the heterogenous group of mucolipidoses excreted n o r m a l a m o u n t s of acid mucopolysaccharides, showing a n o r m a l c h r o m a t o g r a p h i c distribution pattern.

Discussion Our findings confirm a n d e x t e n d previous reports t h a t some of the mucopolysaccharidoses differ in their u r i n a r y excretion p a t t e r n of mucopolysaeeharides [1, 9, 20, 23, 28, 48]. There is general consent on the excretion p a t t e r n s of types I I I , I V a n d VI. I n t y p e I I I excessive a m o u n t s of heparansulfate only h a v e been f o u n d [1, 9, 30, 38, 48]. I n t y p e I V

Biochemical Definition of the Mucopolysaccharidoses

27

increased amounts of keratansulfate and chondroitin-6-su]fate were described [1, 3, 12, 22, 35]. Excessive amounts of dcrmatansulfate are thought to be typical for type VI [25, 28]. As far as types I and I I are concerned, Terry and Linker [48] and, more recently, Manley and Williams [20] presented data that sex-linked eases excreted significantly more heparansulfate in proportion to the total amount of AMPS excreted as did autosomal cases. Their data confirmed earlier results by Meyer et al. [30, 31], but were disputed by other authors [15, 23, 28]. Our own data are in accordance with the findings of Terry and Linker as well as Manley and Williams [20, 48] and seem to indicate that patients with type I excrete relatively more dermatansulfate and less heparansulfate than patients with type II. I t should be kept in mind that Campbell and Fried [4] to whose work McKusick et al. [29] referred did not qualitatively separate the urinary mucopolysaccharides of their patients with type I I and that the patient presented by McKusiek [29] probably had type V and not type II. Lagunoff et al. [15] conceded that their findings might have been influenced by technical difficulties. In type V abnormal amounts of AMPS consisting mainly of dermatansulfate and heparansulfate were found by various groups [9, 21, 28, 48]. Normal amounts were present in our patient a n d in the case reported by Langer el al. [16]. I t seems possible that on fractionation these authors would have found an abnormally high proportion of dermatansulfate as was the case in our patient. More studies are necessary to establish the identity of type V patients with and without excessive mucopolysacehariduria. Children with clinical and radiologie features of the mueopolysaccharidoses but without abnormal mucopolysacchariduria belong to a heterogenous group of diseases [19, 24, 45, 51]. The determination of urinary AMPS is critical to differentiate these diseases from the classical mucopolysaccharidoses, but obviously does not contribute to their individualization. Several points of caution need to be mentioned. The method delineated above is not suited to fully characterize individual AMPS excreted in a given patient with a mucopolysaccharidosis. Rather it is a method of the clinical laboratory, offering a chromatographic distribution pattern of AMPS that empirically correlates with one or the other clinical type of mucopolysaccharidosis. As such it will add another dimension to the spectrum of secondary diagnostic criteria. I t is not clear how much variability of mucopolysaccharide excretion is to be accepted in a given genotype. Acid mucopolysaeeharides of normal tissue show a wide range of heterogeneity, differing in molecular weight, sulfation and structural composition [36]. In mucopolysaceharide storage diseases these

28

J.W. Spranger:

substances obviously are subjected to varying degrees of degradation before they are extruded from the cell and excreted in the urine [10]. Thus a wide range of degradation products is to be expected [6, 14, 26]. Heparansuifate occurs in the urine of tturler patients in at least two different forms [13]. Our finding of substances with characteristics of dermatansulfate in fractions of such different ionic strength as 1.5 m and 2.0 m NaCI possibly reflects a similar heterogeneity of dermatansuffate degradation products [7, 26]. I n view of the chemical heterogeneity to be expected in mucopolysaccharide degradation defects, the homogeneity of the excretion patterns in related patients with different forms of mucopolysaecharidoses seems quite surprising but obviously adds to the diagnostic usefulness of the proposed method. Since a wide intrafamilial variability would have to be expected if different clinical types of mucopolysaccharidoses were only variants of a single gene defect, as has been proposed on the basis of enzyme studies [50] our data as well as those of others [1, 30, 48] would not support such an assumption. Recent reports on patients with type V having considerably higher excretion of heparansulfate and dermatansulfate than found in our patient [21, 48], on patients with assumed Morqnio's disease and high excretion of dermatansulfate [8], and finally on patients with type VI of the mucopolysaccharidoses and high excretion of heparansulfate in addition to dermatansulfate [9], would favor a rather high variability of mucopolysaccharide excretion, not reflected in our data. Before accepting a high degree of variability, however, rigid diagnostic criteria should be applied to avoid confusion resulting from the investigation of different, though phenotypically similar genotypes. I t is obvious t h a t the final classification of the genetic mueopolysaccharidoses will depend on the definition of the underlying abnormal primary gene products, probably enzymes. Low beta-galaetosidase activities have been found in the skin and liver of patients with clinical features of Hurler's disease, Hunter's disease and Sanfilippo's disease [32, 33, 50]. Beta-galactosidase was absent in all examined tissues of 5 patients with generalized gangliosidosis [50]. No specific enzyme deficiency has been detected in patients with Sanfilippo's disease and Morqnio's disease. The clinical and biochemical characteristics in these diseases and in Maroteaux's disease (type VI), and their predictable natural course seem evidence enough to suggest t h a t they are related to specific enzyme defects as well. References 1. Berggard, J., Beam, A. G.: The Hurler syndrome. A biochemical and clinical study. Amer. J. l~ed. 89, 221 (1965). 2. Berry, H. K., Spinanger, J. : A paper spot test useful in the study of Hurler's syndrome. J. Lab. clin. Med. 55, 136 (1960).

Biochemical Definition of the Mucopolysaccharidoses

29

3. Brovelli, A., Laterza, L., Lenzi, L., Castellani, A . A . : Characterization of urinary peptoglycans in Morquio's disease. Calcff. Tissue Res. 1, 324 (1968). 4. Campbell, T. N., Fried, M. : Urinary mucopolysaecharide excretion in the sexlinked form of the Hurler syndrome. Proc. Soc. exp. Biol. (N.Y.) 108, 529 (1961). 5. Dische, Z. : A new specific color reaction of hexuronic acids, g. biol. Chem. 167, 189 (1947). 6. Dorfman, A. : Metabolism of acid mucopolysaccharides. Biophys. J. 4, Suppl. 155 (1964). 7. - - Personal communication. 8. Durand, P., Borrone, C., Della Cella, G.: Studying the Morquio syndrome. Pedlar. Res. 1, 408 (1967). 9. Fallis, N., Barnes, F. L., Di Ferrante, N. : A case of polydystrophic dwarfism with urinary excretion of dermatansulfate and heparansulfate. J. clin. Endocr. 28, 26 (1968). 10. Fratantoni, J. C., Hall, C. W., Neufeld, E. F. : The defect in Hurler's and Hunter's syndromes: faulty degradation of mucopolysaccharide. Proc. Nat. Acad. Sei. (Wash.) 60, 699 (1968). 11. geanloz, R. W.: The nomenclature of mucopolysaccharides. Arthr. and Rheum. 8, 233 (1960). 12. Kaplan, D., Mc Kusick, V.A.,Trebach, S., Lazarus, 1~.: Keratosulfate-ehondroitinsulfate peptide from normal urine and from urine of patients with tVforquio syndrome (mucopolysaceharidosis IV). J. Lab. clin. Med. 71, 48 (1968). 13. Knecht, J., Cifonelli, J . A . , Dorfman, A.: Structural studies on heparitin sulfate of normal and Hurler tissues. J. biol. Chem. 242, 4652 (1967). 14. - - Dorfman, A. : Structure of heparitin sulfate in tissues of the Hurler syndrome Biochem. biophys. Res. Commun. 21, 509 (1965). 15. Lagunoff, D., Pritz, P., Scott, C . R . : Urinary N-sulfate glycosaminoglycan excretion in children: normal and abnormal values. Proc. Soc. exp. Biol. (N.Y.) 126, 34 (1967). 16. Langer, L.O., Kronenberg, R.S., Gorlin, R . J . : A case simulating Hurler syndrome of unusual longevity, without abnormal mucopolysacchariduria. Amer. J. 1Vfed. 40, 448 (1966). 17. Langley, W., Evans, M.: Determination of creatinine with sodium-3,5-dinitrobenzoate. J. biol. Chem. 115, 333 (1936). 18. Leroy, J. C., Crocker, A. C. : Clinical definition of the Hurler-Hunter phenotypes. Amer. J. Dis. Child. 112, 518 (1966). 19. - - DelVIars, R . L . , Opitz, J. IV[.: I-cell-disease. Proc. :First Conf. on the Clin. Delineation of Birth Defects. Baltimore, 1V[ay20--25, 1968 (in press). 20. )/Ianley, G., Williams, K. : Urinary excretion of glycosaminoglycans in the various forms of gargoylism. J. clin. Path. 22, 67 (1969). 21. ~aroteaux, P.: Die Sp~it-I{urlersche Krankheit. In: Wiedemarm, H.-R.: Dysostosen. Stuttgart: :Fischer 1965. 22. - - Lamy, 1VI.: Opacit6s corn6ennes et troubles m6taboliques dans la maladie de iVforquio. Rev. fran 9. ]~tud. clin. biol. 6, 481 (1961). 23. ~ - - Hurler's Disease, 1V1orquio's Disease and related mucopolysaccharidoses. J. Pedlar. 67, 312 (1965). 24. - - - - La pseudodystrophie de Hurler. Presse m6d. 74, 2889 (1966). 25. - - Leveque, B., Marie, J., Lamy, 1VI.: Une nouvelle dysostose avec 61imination urinaire de chondroitine-sulfate B. Presse m6d. 71, 1849 (1963). 26. Matalon, R., Dorfman, A. : The structure of acid mucopolysaccharides produced by Hurler fibroblasts in tissue culture. Proc. Nat. Acad. Sci. (Wash.) 60, 179 (1968).

30

J . W . Spranger:

27. Mathews, M. B., Inouye, M. : The determination of chondroitinsulfate C-type polysaccharides in mixtures with other acid mucopolysaccharides. Biochim. biophys. Acta(Amst.) 53, 509 (1961). 28. McKusick, V. A.: Heritable Disorders of Connective Tissue, Ed. 2, St. Louis: Mosby 1966. 2 9 . - Kaplan, D., Wise, D., Hanley, W. B., Suddarth, S. B., Sevick, M.E., Maumanee, A.E.: The genetic mucopolysaccharidoses. Medicine (Baltimore) 44, 445 (1965). 30. Meyer, K., Grumbach, M. M., Linker, A., Hoffman, P.: Excretion of sulfated mueopolysaeeharides in gargoylism (Hurler's syndrome). Proe. Soc. exp. Biol. (N.Y.) 97, 275 (1958). 31. Excretion of sulfated mucopolysaccharides in gargoylism (tturlet's syndrome) II. Additional studies. Proc. Soc. exp. Biol. (N.Y.) 102, 587 (1959). 32. 0ckermann, P. A. : Acid hydrolases in skin and plasma in gargoylism. Deficiency of beta-galactosidase in skin. Clin. chim. Acta 20, 1 (1968). 33. - - KShlin, P. : Glycosidases in skin and plasma in Hunter's syndrome. Acta paediat, scand. 57, 281 (1968). 34. Pedrini, V., Lenuzzi, L., Zambotti, V. : Isolation and identification of keratosulfate in urine of patients affected by Morquio-Ullrich's disease. Proc. Soc. exp. Biol. (N.Y.) 110, 847 (1962). 35. Robins, IV[.M., Stevens, It. F., Linker, A.: Morquio's disease: an abnormality of mucopolysaceharide metabolism. J. Pediat. 62, 881 (i963). 36. Roden, L.: Linkage of acid mucopolysaccharides to protein. 4th int. Conf. on Cystic Fibrosis of the Pancreas (Mucoviscidosis), Berne/Grindelwald 1966, Part II, pp. 185--202. Basel-New York: Karger 1968. 37. Roe, J. H. : The determination of sugar in blood and spinal fluid with anthron reagent. J. biol. Chem. 212, 335 (1955). 38. Sanfilippo, S.J., Podosin, R., Langer, L., Good, R.A.: Mental retardation associated with acid mucopolysacchariduria (heparitin sulfate type). J. Pediat. 63, 837 (1963). 39. Schafer, I.A., Sullivan, J.C., Svejcar, J., Kofoed, J., Robertson, W. V. B.: Study of the Hurler syndrome using cell culture; definition of the biochemical phenotype and the effect of ascorbie acid on the mutant cell. J. clin. Invest. 47, 321 (1968). 40. Seheie, H., Hambriek, G. G. W., B~rness, L.A.: A newly recognized forme fruste of Hurler's disease (gargoylism). Amer. J. OphthM. 53, 753 (1961). 41. Schiller, S., Slover, G.A., Dorfman, A. : A method for the separation of acid mucopolysaccharides: Its application to the isolation of heparin from the skin of rats. J. biol. Chem. 236, 983 (1961). 42. Spranger, J.: Neue klinische und biochemisehe Erkenntnisse bei generalisierten Dysostosen. I n Wiedemarm, H. R.: Dysostosen. Stuttgart: Fischer 1965. 43. - - The genetic mucopolysaccharidoses. Proc. First Conf. on the Clin. Delineation of Birth Defects. Baltimore, May 20--25, 1968 (in Press). 44. - - Wiedemann, H.-R. : Untersuchungen zur Zusammensetzung der Urin-Mucopolysaccharide bei Kindern nnd Erwachsenen. Clin. china. Acta 17, 142 (1967). 45.-Tolksdorf, M., Graucob, E., Caesar, R.: Lipomucopolysaccharidose. Z. Kinderheilk. 103, 285 (1968). 46. Teller, W.: Kolorimetrischer Naehweis yon Chondroitinsulfat B im Gemisch mit anderen sauren Mucopolysacchariden. Z. klin. Chem. 5, 29 (1967). 4 7 . - Ziemann, A.: Die s~ulenchromatographische Fraktionierung der sauren Mucopolysacch~ride im Ham. Klin. Wschr. 44, 1141 (1966).

Biochemical Definition of the Mucopolysaceharidoses

31

48. Terry, K., Linker, A.: Distinction among four forms of tturler's syndrome. Prec. See. exp. Biol. (N.Y.) 115, 394 (1964). 49. Ullrich, O. : Die Pfaundler-tIurlersehe Krankheit. Ergebn. inn. IVied.Kinderheflk. 68, 929 (1943). 50. Van Hoof, F., Hers, H. G. : The abnormalities of lysosomal enzymes in mueopolysaccharidoses. Europ. J. Bioehem. 7, 34 (1968). 51. - - - - Mucopolysaccharidosis by absence of alpha-fucosidase. Lancet 1968 I, 1198. Priv.-Doz. Dr. J. Spranger Universitgts-Kinderklinik 2300 Kiel, Fr6belstra•e 15--17