December 1975
The Journal o f P E D I A T R I C S
1193
Fucosidosis: Detection of the carrier state in peripheral blood leukocytes We have utilized the fluorogenic substrate 4-methylumbelliferyl-c~-L-fucoside to measure the activity of aL-fucosidase in white blood cells and serum, l,Ve have compared the findings with those using the pnitrophenyl derivative, p H activity curves showed two major peaks of activity in leukocyte lysates, with different specificities to these substrates. C~-L-Fucosidase activity was determined in peripheral leukocytes. isolated mononuclear cells (mainly lymphocytes), and granulocytes in 21 members of a family in which fucosidosis had occurred and in normal control subjects. The activity in the leukocytes, lymphocytes, and granulocytes of the normal subjects was 300. 7 +_ 79.8, 190.1 +_ 43.9, and 281.9 +_ 73.1 nmoles 4methylumbelliferone/mg protein~hour with the fluorogenic substrate, and 150.0 +_ 31.8, 154.8 + 21.0, and 148.3 +_ 48.3 nmoles p-nitrophenol/mg protein~hour with the colorigenic substrate, respectively. No activity was detected in the patients" cells with the colorigenic substrate, whereas with the fluorogenic substrate the apparent activity varied from 0.5 to 1.1. In the lymphocytes of both of the patients' parents, two grandparents, and six other potential carriers, the activity fell between the normal and patients" values. Great variation in a-t-fucosidase activity, with broad overlap between normal subjects and heterozygotes, was observed in serum and plasma. Our findings" indicate that detection of carriers for fucosidosis is possible by direct fucosidase determinations in isolated mononuclear cells.
Nicholas G. Beratis, M.D., Bryan M. Turner, Ph.D., and Knrt Hirschhorn, M.D.,* New York, N. Y.
FUCOSIDOSIS is a lysosomal storage disease characterized by an autosomal recessive mode of inheritance and accumulation in the tissues of fucose containing sphingolipids, glycoproteins, and smaller amounts of mucopolysaccharides. ' z The basic metabolic defect has been found to be severe deficiency of ~-L-fucosidase,:' an enzyme that catalyzes the cleavage of c~-L-fucoside bonds. ~ We have recently identified genetic heterogeneity in fucosidosis~ and we have designated the two distinct forms of the disease as types t and 2. 7 Patients with type 1 develop psychomotor retardation, severe neurologic signs, and bony deformities during the second year of life. They From the Division of Medical Genetics, Department of Pediatrics, Mount Sinai School o f Medicine of the City University of New York. Supported by United States Public Health Service Grant HD02552 and Genetics Center Grant GM19443. *Career Scientist of the Health Research Council of the City of New York (1-513).
also demonstrate increased concentration of sodium and chloride in the sweat; none has survived beyond the age of six years. In type 2 the psychomotor retardation and neurologic signs are less severe; patients develop skin lesions of angiokeratoma corporis diffusum, and the concentration of electrolytes in the sweat is normal, This form is compatible with survival into early adulthood. Detection of the enzymatic defect in fucosidosis has been reported in brain, lung, pancreas, and kidney, ~ liver,~. 8. ~ serum,tO cultured skin fibroblasts,2- ~1 and peripheral leukocytes} t_~.x3 However, data pertinent to the identification of the heterozygotes for fucosidosis are very limited. Matsuda and associatesz failed to reliably identify the carrier state of fucosidosis in peripheral leukocytes and cultured skin fibroblasts. More recently the same group 18 reported detection of heterozygosity for fucosidosis by measuring the ratio of a-L-fuc0sidase to a-Dmannosidase activity in the peripheral leukocytes of two obligate heterozygotes for this disorder. In this paper we report the activity of C~-L-fucosidase in
Vol. 87, No. 6, part 2, pp. 1193-1198
1 19 4
Beratis, Turner, and Hirschhorn
I
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The Journal of Pediatrics December 1975
~
9
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,~' '~
Fig. 1. Pedigree of the kindred. peripheral leukocytes, isolated mononuclear cells (mainly lymphocytes), polymorphonuclear leukocytes, and plasma or serum obtained from 21 members of a family in which two cases of fucosidosis type 2 had Occurred. We have developed a technique that utilizes the fluorogenic substrate 4-methylumbelliferyl-c~-L-fucoside and the results are compared with those obtained from the hydrolysis of the p-nitrophenyl derivative. MATERIALS
AND METHODS
Heparinized blood was obtained from two patients with fucosidosis type 2, their parents and grandparents, and 11 other family members who were at risk for carrying the fucosidosis gene (Fig. 1). Blood was also obtained from two individuals who had married into the family and were, therefore, not at risk and from 12 other normal controls. Ten milliliters of blood were mixed with 2 ml of 6% dextran, and the erythrocytes were allowed to sediment for 30 minutes. The supernatant leukocyte-rich plasma was centrifuged at 150 x g .for 15 minutes. For lysis of contaminating erythrocytes the leukocyte pellet was resuspended in 5 ml of ice cold solution of 0.83% NH4C1 containing 0.1% KHCO~ and 0.0037% sodium ethylenediamine tetra-acetic acid. The cell suspension was kept at 4~ and was agitated every minute. After six minutes it was centrifuged and leukocytes were washed twice with cold saline. Cells were stored at --80~ until enzyme determinations were performed. Separation of the mononuclear cells from the granulocytes was accomplished by differential flotation using a modification of the Ficoll-Hypaque system5 ~ Twenty milliliters of heparinized blood were carefully layered on top of an equal volume of Ficoll-Hypaque mixture, and centrifuged at 600 x g for 40 minutes. The mononuclear cells which accumulated at the interface were collected with a Pasteur pipet; Since the vast majority of the cells in the mononuclear-cell layer were lymphocytes, cells obtained in this layer will be referred to as lymphocytes
For recovery of the granulocytes the erythrocytic-granulocyte layer derived from 20 ml of blood was suspended in the NH4C1 solution described above to a final volume of 50 ml. The cell suspension was kept at 4~ and was agitated every minute for 10 minutes. Subsequently it was centrifuged and the pellet was resuspended in 10 ml of cold NH~CI solution. After ten minutes the cell suspension was centrifuged and the granulocyte pellet was washed twice with saline. Cells were lysed by eight cycles of rapid freezing and thawing. The lysates were centrifuged at 1,800 x g for five minutes and the supernatant fractions were used for enzymatic determinations. a-L-Fucosidase activity in the cell lysates was determined using 4-methylumbelliferyl-a-L-fucoside as substrate. One hundred microliters of 0.78 mM substrate in 0.1M citrate-phosphate buffer, pH 5.7, were incubated with 20/B lysate (5 to 7/~g of protein) for 30 minutes at 37~ and the reaction was stopped with 5.0 ml 0.25M glycine-carbonate buffer, pH 10.0. The amount of 4methylumbelliferone released was measured against a blank in a Beckman Model 772 ratio fluorometer at 450 nm after excitation at 360 nm. A linear relationship between fluorescence and amount of protein used per assay was observed between 4 and 8/xg of protein. Extrapolation of the line passing through these points also passed through zero. Eight micrograms of protein per assay was the highest amount used, whereas less than 4/~g resulted in a decrease of specific activity. Maximum velocity was obtained at substrate concentration of 0.5 raM. The reaction was linear for at least one hour. When the p-nitrophenyl glycoside was used as substrate, optimum activity was reached at a substrate concentration of 3.0 raM. a-L-Fucosidase activity was also measured using the pnitrophenyl substrate by the method of Zielke and associates" except that the concentration of the substrate (pnitrophenyl-a-L-fucoside) in the reaction mixture was increased to 3.0 raM, and the incubation time was reduced to 2 hours, a-o-Mannosdase activity was estimated by incubating 100 ttl of 7.2 mM 4-methylumbelliferyl-a-Dmannoside in 0.1M acetate buffer, pH 4.4, with 20 td of cell lysate (6 to 8/zg of protein). After 30 minutes at 37~ the reaction Was stopped with 4.0 ml 0.25M glycinecarbonate buffer, pH 10.0. Protein was determined according to Lowry and associates. 15 All determinations were carried out in duplicate. Activity is expressed in nmoles of 4-methylumbelliferone or nmoles of p-nitrophenol per milligram of protein per hour, when the fluorogenic or col0rigenic substrate was used. C~-L-Fucosidase activity was also measured in serum and/or plasma of the 21 family members and 50 control
Volume 87 Number 6, part 2
-
360
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Carrier detection in fucosidosis
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PERIPHERALLEUKOCYTES MONONUCLEARCELLS 4-MUI~-L-FU p-NP-oL-UFU 4-MUi-o~-L-FUp-NP-oeL-FU c FM C FM C FM C FM
I
P MN,4-MU
'=-o 2 8 0
:
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subjects. Serum was diluted tenfold with water and assayed by the method described above. Activity is expressed in nmoles of 4-methylumbelliferone per milliliter of serum per hour. RESULTS The pH-activity curve for a-L-fucosidase, when the 4methylumbelliferyl glycoside was used as substrate, had a maximum between pH 5.0 and 5.8 (Fig. 2). Within this pH range all samples showed two separate peaks, one at pH 5.0 and one at pH 5.7. A major shoulder of activity was at pH 6.0 to 7.0 and a smaller one at pH 4.5 to 4.75. There was some variation in the pattern of activity between samples, but the general shape of the profile remained the same a n d the two peaks at pH 5.0 and 5.7 were always present. It was observed that in some samples the activity of the first peak was lower than the second, whereas in other samples the two peaks were approximately equal. Granulocytes and lymphocytes obtained from the same donor gave strikingly similar pH profiles. With the pnitrophenyl glycoside as substrate, a slightly different pH profile was obtained with. optimum activity at pH 5.8 (Fig. 2). Assays performed under optimum conditions for both substrates showed a faster hydrolysis of the 4-methylumbelliferyl glycoside than the p-nitrophenyl derivative. The mean hydrolysis of the 4-methylumbelliferyl and p-nitrophenyl glycosides by the leukocyte lysates of 14 normal subjects was 300.7 and 150.0, respectively, giving a ratio of 2:1. In granulocytes from these individuals a very simitar
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Fig. 2. PH-activity profiles of polymorphonuclear (PMN) cells
and lymphocytes (L Y) using 4-methylumebellifeJ/yl-a-L-fucoside (4-MU) and p-nitrophenyl-a-L-fucoside (p-nitrophenol) substrates.
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Fig. 3. a-L-Fucosidase activity in 14 control subjects, two obligate heterozygotes for fucosidosis, two patients with fucosidosis, and 15 family members at risk for carrying the fucosidosis gene. The family members I-4 and 1-9 who are not at risk are listed as controls. 4MU-a-L-FU = 4-methylumbelliferyl-a-L-fucoside; pNP-a-FU = p-nitrophenyl-a-L-fucoside; C = Controls; FM = family members ratio (1.9:1) was obtained, but in lymphocytes the ratio was only 1.2:1. Table I lists the a-L-fucosidase and a-D-mannosidase activities in peripheral leukocytes, lymphocytes, and granulocytes of 14 normal subjects and 21 members of the family in which fucosidosis had occurred (Fig: 1). By using the fluorogenic substrate a-L-fucosidase activity in the mixed leukocytes of two control subjects overlapped with the activity in the leukocytes of an obligate heterozygote. Although there was no overlap between normal subjects and carriers in the leukocytes, when the pnitrophyenyl glycoside was used as substrate, the activity between these two groups was very close (Fig. 3). Overlap between two normal subjects and an obligate heterozygote was also present in the granulocytes. The smallest variation in enzyme activity among normal individuals was observed in the lymphocytes. With both the 4-methylumbelliferyl and the p-nitrophenyl substrates there was no overlap between normal subjects and carriers (Fig. 3). In both of the patients' parents, their
1 19 6
Beratis, Turner, and Hirschhorn
The Journal of Pediatrics December 1975
Table I. a-L-Fucosidase a n d a - D - m a n n o s i d a s e activity in p e r i p h e r a l leukocytes, lymphocytes, a n d granulocytes
Leukocytes Fucosidase Subject Normal (14)~ Range I- 1 I-2 I-3 I-4 1-5 I-6 I-7 1-8 I-9 II-1 II-2 II-3 II-4 II-5 1I-6 II-7 II-8 II-9 III- 1 Ill-2 1II-3
4-MU
p-NP
Lymphocytes* Mannosidase 4-MU
4-MU
M annosidase
Mannosidase
4-MU
p-NP
4-MU
154.8_+21.0
378.9_+76.0
281.9_+73.1
t48.3_+48.3
999.4_+221.2
94.4-217.8 556.2- 1248,4 160.l -288.4 117.0-202.2 115.5 824.0 181.2 150.8 33.l 660.4 80.0 57.9 60.1 714.6 91.6 68.1 162.3 888.0 288.4 123.6 148.0 604.4 257.8 160.0 124.6 616.0 213.0 140.5 80.5 899.0 93.6 43.1 53.4 684.8 69.2 44.4 159.4 895.6 216.2 163.7 89.0 562.4 80.4 81.7 153.3 49 t .6 214.8 132.5 96.8 841.9 150.3 124.9 69.7 666.4 103.8 61.5 72.7 1016.6 127.2 87.6 74.6 947.0 125.2 68.2 47.6 913.0 95.2 59.1 96.7 816.0 197.9 111.5 124.4 649.4 167.2 151.6 0.0 1548.8 0.6 0.0 0.0 1357.0 0.8 0.0 53.4 1005.2 90.8 44.4
296.6-544.5 735,9 497.2 631.2 325.2 515.0 370.4 624.0 459.9 330.4 558.6 292.4 314.4 367.4 526.5 543.9 345:9 869.0 432.6 1194.8 849.4 470.6
144.9-446.8 359.4 106.8 153.4 304.8 318.8 ---314.2 108.0 404.2 185.6 191.4 120.6 218.2 -242.0 0.5 0:7 --
55.0-208.1 638.2- 1549.0 121.2 1578.9 58.3 1064.5 53.2 534.3 146.5 1038.2 135.1 952.2 -----154.1 868.4 70.7 628.4 164.7 1396.6 68.3 607.0 46.2 1038.5 82.2 1512.8 57.7 907.0 --168.6 1285.0 0.0 1318.4 0.0 713.8 -
190.1_+43.9
p-NP
Fucosidase
4-M U
300.7_+ 79.8 150.0_+31.8 861.2_+179.6 169.6-422.6 230.8 110.0 | 16.5 351.0 322.6 210.3 132.7 122.2 307.1 104.6 219.5 184.2 124.4 173.9 163.4 139.7 264.2 251.9 0.8 1.1 128.5
Fucosidase
Granulocytes
Activity is expressed in nmoles/mg protein/hour. 4-MN = 4-methylumbellferone; F-NP = p-nitrophenol. *Mainly lymphocytes; also monocytes and platelets. tMean = SD.
grandmothers, a n d six other potential carriers for the fucosidosis gene, the a-L-fucosidase activity fell b e t w e e n that of the patients a n d control subjects. All p o t e n t i a l carriers who were identified as heterozygotes for fucosidosis in lymphocyte p r e p a r a t i o n s also h a d e n z y m e activities in the leukocytes that were b e t w e e n those o f the patients a n d the control subjects, a l t h o u g h the activity in some of t h e m was close to the lowest region o f the n o r m a l range. T h e fucosidase to m a n n o s i d a s e ratios o b t a i n e d in the peripheral leukocytes of some heterozygotes a n d n o r m a l donors were very close or o v e r l a p p i n g and, therefore, reliable distinction between the two groups could not b e m a d e by using this enzyme ratio. No a-L-fucosidase activity was detected in the leukocyte, lymphocyte, a n d granulocyte lysates o f b o t h p a t i e n t s by using the colorigenic substrate. W h e n the 4 - m e t h y l u m belliferyl glycoside was used some a p p a r e n t residual activity was present in the cell lysates. Hydrolysis o f the substrate failed to increase f u r t h e r by increasing the a m o u n t of cellular protein per assay.
T a b l e . l I summarizes the a-g-fucosidase activity f o u n d in the serum a n d plasma o f the 21 family m e m b e r s a n d 50 control subjects. Since n o significant differences were f o u n d between the enzymatic activity of serum a n d plasma, results from b o t h types o f s a m p l e h a v e b e e n combined. In the control g r o u p activity o f the e n z y m e varied over a wide range; two individuals were f o u n d with extremely low levels of activity. In these two samples activity was only slightly greater t h a n t h a t in s e r u m a n d plasma from the two affected children. I n m e m b e r s o f the family serum a-g-fucosidase varied over a similar r a n g e to that f o u n d in the general p o p u l a t i o n . A l t h o u g h family m e m b e r s who h a d b e e n diagnosed as carriers by assay o f the white cell enzyme had, o n average, r a t h e r lower activity t h a n noncarriers, the difference is not significant (p > 0.2). a-L-Fucosidase activity was assayed in leukocytes, lymphocytes, a n d granulocytes o f one o f the two n o r m a l individuals with low serum activity. W i t h the fluorogenic substrate values were, respectively, 218.1, 166.6, a n d 288.5. These values are within the n o r m a l range. Activity
Volume 87 Number 6, part 2
was also within the normal range when the p-nitrophenyl substrate was used.
Carrier detection in fucosidosis
1 19 7
Table II. a-L-Fucosidase activity in serum or plasma from 50 normal subjects and from 20 members of the family shown in Fig. 1
DISCUSSION Our findings demonstrate that a-L-fucosidase activity in the leukocytes of heterozygotes for fucosidosis is generally lower than in normal subjects. In a small number of normal donors, however, the enzyme activity in the peripheral leukocytes fell within the carrier range, when the fluorogenic substrate was used. Also in the leukocytes, by using the colorigenic substrate, the upper level of activity of the carriers was too close to the lower level of activity of the normal controls to allow reliable identification of the heterozygotes. There was no overlap between normal subjects and heterozygotes, when the enzyme activity was determined in the mononuclear cells (mainly lymphocytes) of the peripheral blood. It appears, however, that the colorigenic substrate may be more suitable for the identification of the carriers. Homozyg0tes, heterozygotes, and normal subjects were clearly delineated in the lymphocytes by using the p-nitrophenyl glycoside as substrate (Fig. 3). The granulocytes demonstrated the greatest variability of all types of white blood cells in a-L-fucosidase activity. In some preparations the activity was lower and in others higher than in the leukocytes and lymphocytes. This may be due to the fact that some of the enzyme was released from the granulocytes during the isolation of the cells. The apparent small amount of residual a-L-fucosidase activity present in the lysates of the two patients with fuc0sidosis, when the 4-methylumbelliferyl glycoside was utilized, appears to be due to nonspecific hydrolysis of the substrate. This is because increasing the amount of cellular protein per assay did not increase further the fluorescence of the final reaction mixture. Matsuda and associates 13 found elevated a-D-mannosidase activity in the leukocytes of a patient with fucosidosis and our findings confirm this. Contrary to this previous report, however, the ratio of fucosidase to mannosidase activity was not always diagnostic of the carrier state. The fluorometric assay is both simpler and more sensitive than the spectrophotometric assay. These properties make the fluorogenic substrate the substrate of choice for kinetic studies of the enzyme. a-L-Fucosidase has been shown to exist in a number of distinct isozymic forms. We recently described a common genetic polymorphism of the enzyme in the North American population." Each of the three different genetically determined phenotypes consists of six or more isozymes which differ in the number of sialic acid residues bound to them. TM In addition, two forms of the enzyme with differing molecular weights have been described. 1~ 2o The
Enzyme activity*
Subject (No.) Family members Normal in lymphocytes (9) Heterozygotes in lymphocytes (8) Obligate heterozygotes (2) Homozygores (2) Control subjects (50)
Pedigree No.
Mean _+SD
(21) I-1,4,5,6,9, 340.0_+ 153.3 II2,3,8,9 I-2,7,8, 276.6_+174.5 II-1,4,7, III-3 II-5,6 219.7 IlI-1,2
Range
134.5-618,5
37.0-435.0 201.0-238.5
10.7
10.2- 11.3
326.9 _+182.4~
13.2-660.0
*Activity is expressed in nmoles 4-methylumbelliferone released per ml of serum per hour. tin two control subjects a very low enzyme activity of 13.2 and 19.2 was found.
complex pH activity profile of a-L-fucosidase in lymphocytes and granulocytes (Fig. 2) is probably due to the underlying heterogeneity of the enzyme. The two different molecular weight forms have been shown to have different pH optima. 19, 20 Possible differences in the genetically determined and sialic acid containing isozymes remain to be studied. The existence of these various different isozymes is probably also responsible for the variation from one tissue to another in the ratio of activity toward the two different substrates. In leukocytes the activity toward the fluorogenic substrate was two times greater than that toward the colofigenic. In peripheral lymphocytes the ratio was only 1.2 to 1. The observed ratio of 1.9 to 1 in granulocytes, rather than the expected higher one, could possibly be attributed to changes that occurred during the isolation of the granulocytes. Ratios' of 4 to 1 and 1.3 to 1 have previously been reported for the liver and serum enzymes, respectively?~ The great variability of the a-L-fucosidase activity in sera from normal subjects indicates that this system cannot be used for assessment of carrier status or even for reliable diagnosis of fucosidosis. Two normal donors with very low serum activities were found. The a-L-fucosidase activity in the leukocytes and lymphocytes Of one of these individuals was measured and was within the normal range. This finding suggests that individuals with low serum activity can be distinguished from homozygotes
1 19 8
Beratis, Turner, and Hirschhorn
and heterozygotes for fucosidosis by studying the enzyme levels in leukocytes. Since mixed peripheral leukocyte preparations o f normal subjects only occasionally g a v e borderline or overlapping values with the heterozygotes, use o f leukQcytes for the identification of the carrier state of fucosidosis is feaSible. For more reliable identification o f the heterozygotes, howeVer, subjects with enzymatic ~ictivity in the low normal or heterozygous r a n g e s h o u l d be further investigated by determination of the fucosidase activity in isolated mononuclear cells. We acknowledge the highly skilled assistance of Miss Roberta Weiss. We also wish to thank Mrs. Minnie Woodson for her help in the prepai:ation of this manuscript. REFERENCES
1. Dawson G, and Spranger JW: Fucosidosis: A glycosphingolipidosis, N Engl J Med 285:122, 1971. 2. Matsuda I, Arashima S, Anakura M, Ege A, and Hayata I: Fucosidosis, Tohoku J Exp Meal 109:41, 1973. 3. Van Hoof F, and Hers HG: Mucopolysaccharidosis by absence of a-fucosidase, Lancet 1:1198, 1968. 4. Wiederschain GY, and Rosenfeld EL: Two forms of a-Lfucosidase from pig kidney and their action on natural olyg0saccharides, Biochem Biophys Res Comm 44:1008, 1971. 5: Wiederschain GY, Kolibata LG, and Rosenfeld EL: Human a-L-fucosidases, Clin Chim Acta 46:305, 1973. 6. KousseffBG, Beratis NG, Danesino C, and Hirschhorn K: Genetic heterogeneity in fucosid0sis, Lancet 2: J387, 1973. 7. Kousseff BG, Beratis NG, Strauss L, Brill PW, Rosenfeld RE, Kaplan B, and Hirschhorn K: Fucosidosis type 2, Pediatrics (in press).
The Journal of Pediatrics December 1975
8. Van Hoof F, and Hers HG: The abnormalities oflysosomaI enzymes in mucopolysaccharidoses, Eur0p J Biochem 7i34, 1968. 9. Loeb H,. Tondeur M, Jonr/iaux G, Mockel-Pohl S, and Vamos-Hurwitz E: Biochemical and ultrastructural studies in a case of mucopolysaccharidosis "F" (Fucosidosis), Helv Paediatr Acta 36:519, 1969. 10. Zielke K , Okada SI and O'Brien J: Fucosidosis: Diagnosis by serum assay of a-L-fucosidase, J Lab Clin Med 79:164, 1972. 11. Zielke K, Veath ML, and O'Brien JS: Fucosidosis: Deficiency o f alfa-L-fucosidase in cultured skin fibroblasts, J Exp Med 136:197, 1972. 12. Borrone C, Gatti R,Trias X, and Durand P: Fucosidosis: Clinical, biochemical, immunologic, and genetic studies in two new cases, J P~DIATR84:727, 1974. 13. Matsuda I, Ai'ashima S, Anakura M, and Oka Y: a-LFucosidase and a-D~mannosidase activity in the white blood cells in the disease and carrier state of fucosidosis, Clin Chim Acta 48;9, 1973. 14. Boyum A i Separation of leukocytes from blood and bone marrow, Scand J Clin Lab Invest 21(Suppl):97, 1968. 15. Lowry OH, Rosebrough NJ, Farr AL, and Randall R J: Protein measurement with the Folin phenol reagent, J Biol Chem 193:265, 1951. 16. Robinson D, and Thorpe R: Fluorescent assay of a-Lfucosidase, Clin Chim Acta 55:65, !974. 17. Turner BM, Turner vS, Beratis NG, and Hirschhorn K: Polymorphism of huma n a-fucosidase, Am J Hum Genet Am J Hum Genet 27:651, 1975. 18. Turner BM, Beratis NG, Turner VS, and Hirschhorn K: Isozymes & human a-L-fucosidase detectable by starch gel electrophoresis, Clin Chim Acta 57:29, 1974. 19. Wiederschain GY, Kolibaba LG, and Rosenfeld EL: Human a-L-fucosidase, Clin Chim Acta 46:305, 1973. 20. Robinson D, and Thorpe R: Human liver a-L-fUcosidases, Clin Chim Acta 47:403, 1973.
The Journal o f P E D I A T R I C S
1193
Fucosidosis: Detection of the carrier state in peripheral blood leukocytes We have utilized the fluorogenic substrate 4-methylumbelliferyl-c~-L-fucoside to measure the activity of aL-fucosidase in white blood cells and serum, l,Ve have compared the findings with those using the pnitrophenyl derivative, p H activity curves showed two major peaks of activity in leukocyte lysates, with different specificities to these substrates. C~-L-Fucosidase activity was determined in peripheral leukocytes. isolated mononuclear cells (mainly lymphocytes), and granulocytes in 21 members of a family in which fucosidosis had occurred and in normal control subjects. The activity in the leukocytes, lymphocytes, and granulocytes of the normal subjects was 300. 7 +_ 79.8, 190.1 +_ 43.9, and 281.9 +_ 73.1 nmoles 4methylumbelliferone/mg protein~hour with the fluorogenic substrate, and 150.0 +_ 31.8, 154.8 + 21.0, and 148.3 +_ 48.3 nmoles p-nitrophenol/mg protein~hour with the colorigenic substrate, respectively. No activity was detected in the patients" cells with the colorigenic substrate, whereas with the fluorogenic substrate the apparent activity varied from 0.5 to 1.1. In the lymphocytes of both of the patients' parents, two grandparents, and six other potential carriers, the activity fell between the normal and patients" values. Great variation in a-t-fucosidase activity, with broad overlap between normal subjects and heterozygotes, was observed in serum and plasma. Our findings" indicate that detection of carriers for fucosidosis is possible by direct fucosidase determinations in isolated mononuclear cells.
Nicholas G. Beratis, M.D., Bryan M. Turner, Ph.D., and Knrt Hirschhorn, M.D.,* New York, N. Y.
FUCOSIDOSIS is a lysosomal storage disease characterized by an autosomal recessive mode of inheritance and accumulation in the tissues of fucose containing sphingolipids, glycoproteins, and smaller amounts of mucopolysaccharides. ' z The basic metabolic defect has been found to be severe deficiency of ~-L-fucosidase,:' an enzyme that catalyzes the cleavage of c~-L-fucoside bonds. ~ We have recently identified genetic heterogeneity in fucosidosis~ and we have designated the two distinct forms of the disease as types t and 2. 7 Patients with type 1 develop psychomotor retardation, severe neurologic signs, and bony deformities during the second year of life. They From the Division of Medical Genetics, Department of Pediatrics, Mount Sinai School o f Medicine of the City University of New York. Supported by United States Public Health Service Grant HD02552 and Genetics Center Grant GM19443. *Career Scientist of the Health Research Council of the City of New York (1-513).
also demonstrate increased concentration of sodium and chloride in the sweat; none has survived beyond the age of six years. In type 2 the psychomotor retardation and neurologic signs are less severe; patients develop skin lesions of angiokeratoma corporis diffusum, and the concentration of electrolytes in the sweat is normal, This form is compatible with survival into early adulthood. Detection of the enzymatic defect in fucosidosis has been reported in brain, lung, pancreas, and kidney, ~ liver,~. 8. ~ serum,tO cultured skin fibroblasts,2- ~1 and peripheral leukocytes} t_~.x3 However, data pertinent to the identification of the heterozygotes for fucosidosis are very limited. Matsuda and associatesz failed to reliably identify the carrier state of fucosidosis in peripheral leukocytes and cultured skin fibroblasts. More recently the same group 18 reported detection of heterozygosity for fucosidosis by measuring the ratio of a-L-fuc0sidase to a-Dmannosidase activity in the peripheral leukocytes of two obligate heterozygotes for this disorder. In this paper we report the activity of C~-L-fucosidase in
Vol. 87, No. 6, part 2, pp. 1193-1198
1 19 4
Beratis, Turner, and Hirschhorn
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The Journal of Pediatrics December 1975
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Fig. 1. Pedigree of the kindred. peripheral leukocytes, isolated mononuclear cells (mainly lymphocytes), polymorphonuclear leukocytes, and plasma or serum obtained from 21 members of a family in which two cases of fucosidosis type 2 had Occurred. We have developed a technique that utilizes the fluorogenic substrate 4-methylumbelliferyl-c~-L-fucoside and the results are compared with those obtained from the hydrolysis of the p-nitrophenyl derivative. MATERIALS
AND METHODS
Heparinized blood was obtained from two patients with fucosidosis type 2, their parents and grandparents, and 11 other family members who were at risk for carrying the fucosidosis gene (Fig. 1). Blood was also obtained from two individuals who had married into the family and were, therefore, not at risk and from 12 other normal controls. Ten milliliters of blood were mixed with 2 ml of 6% dextran, and the erythrocytes were allowed to sediment for 30 minutes. The supernatant leukocyte-rich plasma was centrifuged at 150 x g .for 15 minutes. For lysis of contaminating erythrocytes the leukocyte pellet was resuspended in 5 ml of ice cold solution of 0.83% NH4C1 containing 0.1% KHCO~ and 0.0037% sodium ethylenediamine tetra-acetic acid. The cell suspension was kept at 4~ and was agitated every minute. After six minutes it was centrifuged and leukocytes were washed twice with cold saline. Cells were stored at --80~ until enzyme determinations were performed. Separation of the mononuclear cells from the granulocytes was accomplished by differential flotation using a modification of the Ficoll-Hypaque system5 ~ Twenty milliliters of heparinized blood were carefully layered on top of an equal volume of Ficoll-Hypaque mixture, and centrifuged at 600 x g for 40 minutes. The mononuclear cells which accumulated at the interface were collected with a Pasteur pipet; Since the vast majority of the cells in the mononuclear-cell layer were lymphocytes, cells obtained in this layer will be referred to as lymphocytes
For recovery of the granulocytes the erythrocytic-granulocyte layer derived from 20 ml of blood was suspended in the NH4C1 solution described above to a final volume of 50 ml. The cell suspension was kept at 4~ and was agitated every minute for 10 minutes. Subsequently it was centrifuged and the pellet was resuspended in 10 ml of cold NH~CI solution. After ten minutes the cell suspension was centrifuged and the granulocyte pellet was washed twice with saline. Cells were lysed by eight cycles of rapid freezing and thawing. The lysates were centrifuged at 1,800 x g for five minutes and the supernatant fractions were used for enzymatic determinations. a-L-Fucosidase activity in the cell lysates was determined using 4-methylumbelliferyl-a-L-fucoside as substrate. One hundred microliters of 0.78 mM substrate in 0.1M citrate-phosphate buffer, pH 5.7, were incubated with 20/B lysate (5 to 7/~g of protein) for 30 minutes at 37~ and the reaction was stopped with 5.0 ml 0.25M glycine-carbonate buffer, pH 10.0. The amount of 4methylumbelliferone released was measured against a blank in a Beckman Model 772 ratio fluorometer at 450 nm after excitation at 360 nm. A linear relationship between fluorescence and amount of protein used per assay was observed between 4 and 8/xg of protein. Extrapolation of the line passing through these points also passed through zero. Eight micrograms of protein per assay was the highest amount used, whereas less than 4/~g resulted in a decrease of specific activity. Maximum velocity was obtained at substrate concentration of 0.5 raM. The reaction was linear for at least one hour. When the p-nitrophenyl glycoside was used as substrate, optimum activity was reached at a substrate concentration of 3.0 raM. a-L-Fucosidase activity was also measured using the pnitrophenyl substrate by the method of Zielke and associates" except that the concentration of the substrate (pnitrophenyl-a-L-fucoside) in the reaction mixture was increased to 3.0 raM, and the incubation time was reduced to 2 hours, a-o-Mannosdase activity was estimated by incubating 100 ttl of 7.2 mM 4-methylumbelliferyl-a-Dmannoside in 0.1M acetate buffer, pH 4.4, with 20 td of cell lysate (6 to 8/zg of protein). After 30 minutes at 37~ the reaction Was stopped with 4.0 ml 0.25M glycinecarbonate buffer, pH 10.0. Protein was determined according to Lowry and associates. 15 All determinations were carried out in duplicate. Activity is expressed in nmoles of 4-methylumbelliferone or nmoles of p-nitrophenol per milligram of protein per hour, when the fluorogenic or col0rigenic substrate was used. C~-L-Fucosidase activity was also measured in serum and/or plasma of the 21 family members and 50 control
Volume 87 Number 6, part 2
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360
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Carrier detection in fucosidosis
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PERIPHERALLEUKOCYTES MONONUCLEARCELLS 4-MUI~-L-FU p-NP-oL-UFU 4-MUi-o~-L-FUp-NP-oeL-FU c FM C FM C FM C FM
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subjects. Serum was diluted tenfold with water and assayed by the method described above. Activity is expressed in nmoles of 4-methylumbelliferone per milliliter of serum per hour. RESULTS The pH-activity curve for a-L-fucosidase, when the 4methylumbelliferyl glycoside was used as substrate, had a maximum between pH 5.0 and 5.8 (Fig. 2). Within this pH range all samples showed two separate peaks, one at pH 5.0 and one at pH 5.7. A major shoulder of activity was at pH 6.0 to 7.0 and a smaller one at pH 4.5 to 4.75. There was some variation in the pattern of activity between samples, but the general shape of the profile remained the same a n d the two peaks at pH 5.0 and 5.7 were always present. It was observed that in some samples the activity of the first peak was lower than the second, whereas in other samples the two peaks were approximately equal. Granulocytes and lymphocytes obtained from the same donor gave strikingly similar pH profiles. With the pnitrophenyl glycoside as substrate, a slightly different pH profile was obtained with. optimum activity at pH 5.8 (Fig. 2). Assays performed under optimum conditions for both substrates showed a faster hydrolysis of the 4-methylumbelliferyl glycoside than the p-nitrophenyl derivative. The mean hydrolysis of the 4-methylumbelliferyl and p-nitrophenyl glycosides by the leukocyte lysates of 14 normal subjects was 300.7 and 150.0, respectively, giving a ratio of 2:1. In granulocytes from these individuals a very simitar
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Fig. 2. PH-activity profiles of polymorphonuclear (PMN) cells
and lymphocytes (L Y) using 4-methylumebellifeJ/yl-a-L-fucoside (4-MU) and p-nitrophenyl-a-L-fucoside (p-nitrophenol) substrates.
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Fig. 3. a-L-Fucosidase activity in 14 control subjects, two obligate heterozygotes for fucosidosis, two patients with fucosidosis, and 15 family members at risk for carrying the fucosidosis gene. The family members I-4 and 1-9 who are not at risk are listed as controls. 4MU-a-L-FU = 4-methylumbelliferyl-a-L-fucoside; pNP-a-FU = p-nitrophenyl-a-L-fucoside; C = Controls; FM = family members ratio (1.9:1) was obtained, but in lymphocytes the ratio was only 1.2:1. Table I lists the a-L-fucosidase and a-D-mannosidase activities in peripheral leukocytes, lymphocytes, and granulocytes of 14 normal subjects and 21 members of the family in which fucosidosis had occurred (Fig: 1). By using the fluorogenic substrate a-L-fucosidase activity in the mixed leukocytes of two control subjects overlapped with the activity in the leukocytes of an obligate heterozygote. Although there was no overlap between normal subjects and carriers in the leukocytes, when the pnitrophyenyl glycoside was used as substrate, the activity between these two groups was very close (Fig. 3). Overlap between two normal subjects and an obligate heterozygote was also present in the granulocytes. The smallest variation in enzyme activity among normal individuals was observed in the lymphocytes. With both the 4-methylumbelliferyl and the p-nitrophenyl substrates there was no overlap between normal subjects and carriers (Fig. 3). In both of the patients' parents, their
1 19 6
Beratis, Turner, and Hirschhorn
The Journal of Pediatrics December 1975
Table I. a-L-Fucosidase a n d a - D - m a n n o s i d a s e activity in p e r i p h e r a l leukocytes, lymphocytes, a n d granulocytes
Leukocytes Fucosidase Subject Normal (14)~ Range I- 1 I-2 I-3 I-4 1-5 I-6 I-7 1-8 I-9 II-1 II-2 II-3 II-4 II-5 1I-6 II-7 II-8 II-9 III- 1 Ill-2 1II-3
4-MU
p-NP
Lymphocytes* Mannosidase 4-MU
4-MU
M annosidase
Mannosidase
4-MU
p-NP
4-MU
154.8_+21.0
378.9_+76.0
281.9_+73.1
t48.3_+48.3
999.4_+221.2
94.4-217.8 556.2- 1248,4 160.l -288.4 117.0-202.2 115.5 824.0 181.2 150.8 33.l 660.4 80.0 57.9 60.1 714.6 91.6 68.1 162.3 888.0 288.4 123.6 148.0 604.4 257.8 160.0 124.6 616.0 213.0 140.5 80.5 899.0 93.6 43.1 53.4 684.8 69.2 44.4 159.4 895.6 216.2 163.7 89.0 562.4 80.4 81.7 153.3 49 t .6 214.8 132.5 96.8 841.9 150.3 124.9 69.7 666.4 103.8 61.5 72.7 1016.6 127.2 87.6 74.6 947.0 125.2 68.2 47.6 913.0 95.2 59.1 96.7 816.0 197.9 111.5 124.4 649.4 167.2 151.6 0.0 1548.8 0.6 0.0 0.0 1357.0 0.8 0.0 53.4 1005.2 90.8 44.4
296.6-544.5 735,9 497.2 631.2 325.2 515.0 370.4 624.0 459.9 330.4 558.6 292.4 314.4 367.4 526.5 543.9 345:9 869.0 432.6 1194.8 849.4 470.6
144.9-446.8 359.4 106.8 153.4 304.8 318.8 ---314.2 108.0 404.2 185.6 191.4 120.6 218.2 -242.0 0.5 0:7 --
55.0-208.1 638.2- 1549.0 121.2 1578.9 58.3 1064.5 53.2 534.3 146.5 1038.2 135.1 952.2 -----154.1 868.4 70.7 628.4 164.7 1396.6 68.3 607.0 46.2 1038.5 82.2 1512.8 57.7 907.0 --168.6 1285.0 0.0 1318.4 0.0 713.8 -
190.1_+43.9
p-NP
Fucosidase
4-M U
300.7_+ 79.8 150.0_+31.8 861.2_+179.6 169.6-422.6 230.8 110.0 | 16.5 351.0 322.6 210.3 132.7 122.2 307.1 104.6 219.5 184.2 124.4 173.9 163.4 139.7 264.2 251.9 0.8 1.1 128.5
Fucosidase
Granulocytes
Activity is expressed in nmoles/mg protein/hour. 4-MN = 4-methylumbellferone; F-NP = p-nitrophenol. *Mainly lymphocytes; also monocytes and platelets. tMean = SD.
grandmothers, a n d six other potential carriers for the fucosidosis gene, the a-L-fucosidase activity fell b e t w e e n that of the patients a n d control subjects. All p o t e n t i a l carriers who were identified as heterozygotes for fucosidosis in lymphocyte p r e p a r a t i o n s also h a d e n z y m e activities in the leukocytes that were b e t w e e n those o f the patients a n d the control subjects, a l t h o u g h the activity in some of t h e m was close to the lowest region o f the n o r m a l range. T h e fucosidase to m a n n o s i d a s e ratios o b t a i n e d in the peripheral leukocytes of some heterozygotes a n d n o r m a l donors were very close or o v e r l a p p i n g and, therefore, reliable distinction between the two groups could not b e m a d e by using this enzyme ratio. No a-L-fucosidase activity was detected in the leukocyte, lymphocyte, a n d granulocyte lysates o f b o t h p a t i e n t s by using the colorigenic substrate. W h e n the 4 - m e t h y l u m belliferyl glycoside was used some a p p a r e n t residual activity was present in the cell lysates. Hydrolysis o f the substrate failed to increase f u r t h e r by increasing the a m o u n t of cellular protein per assay.
T a b l e . l I summarizes the a-g-fucosidase activity f o u n d in the serum a n d plasma o f the 21 family m e m b e r s a n d 50 control subjects. Since n o significant differences were f o u n d between the enzymatic activity of serum a n d plasma, results from b o t h types o f s a m p l e h a v e b e e n combined. In the control g r o u p activity o f the e n z y m e varied over a wide range; two individuals were f o u n d with extremely low levels of activity. In these two samples activity was only slightly greater t h a n t h a t in s e r u m a n d plasma from the two affected children. I n m e m b e r s o f the family serum a-g-fucosidase varied over a similar r a n g e to that f o u n d in the general p o p u l a t i o n . A l t h o u g h family m e m b e r s who h a d b e e n diagnosed as carriers by assay o f the white cell enzyme had, o n average, r a t h e r lower activity t h a n noncarriers, the difference is not significant (p > 0.2). a-L-Fucosidase activity was assayed in leukocytes, lymphocytes, a n d granulocytes o f one o f the two n o r m a l individuals with low serum activity. W i t h the fluorogenic substrate values were, respectively, 218.1, 166.6, a n d 288.5. These values are within the n o r m a l range. Activity
Volume 87 Number 6, part 2
was also within the normal range when the p-nitrophenyl substrate was used.
Carrier detection in fucosidosis
1 19 7
Table II. a-L-Fucosidase activity in serum or plasma from 50 normal subjects and from 20 members of the family shown in Fig. 1
DISCUSSION Our findings demonstrate that a-L-fucosidase activity in the leukocytes of heterozygotes for fucosidosis is generally lower than in normal subjects. In a small number of normal donors, however, the enzyme activity in the peripheral leukocytes fell within the carrier range, when the fluorogenic substrate was used. Also in the leukocytes, by using the colorigenic substrate, the upper level of activity of the carriers was too close to the lower level of activity of the normal controls to allow reliable identification of the heterozygotes. There was no overlap between normal subjects and heterozygotes, when the enzyme activity was determined in the mononuclear cells (mainly lymphocytes) of the peripheral blood. It appears, however, that the colorigenic substrate may be more suitable for the identification of the carriers. Homozyg0tes, heterozygotes, and normal subjects were clearly delineated in the lymphocytes by using the p-nitrophenyl glycoside as substrate (Fig. 3). The granulocytes demonstrated the greatest variability of all types of white blood cells in a-L-fucosidase activity. In some preparations the activity was lower and in others higher than in the leukocytes and lymphocytes. This may be due to the fact that some of the enzyme was released from the granulocytes during the isolation of the cells. The apparent small amount of residual a-L-fucosidase activity present in the lysates of the two patients with fuc0sidosis, when the 4-methylumbelliferyl glycoside was utilized, appears to be due to nonspecific hydrolysis of the substrate. This is because increasing the amount of cellular protein per assay did not increase further the fluorescence of the final reaction mixture. Matsuda and associates 13 found elevated a-D-mannosidase activity in the leukocytes of a patient with fucosidosis and our findings confirm this. Contrary to this previous report, however, the ratio of fucosidase to mannosidase activity was not always diagnostic of the carrier state. The fluorometric assay is both simpler and more sensitive than the spectrophotometric assay. These properties make the fluorogenic substrate the substrate of choice for kinetic studies of the enzyme. a-L-Fucosidase has been shown to exist in a number of distinct isozymic forms. We recently described a common genetic polymorphism of the enzyme in the North American population." Each of the three different genetically determined phenotypes consists of six or more isozymes which differ in the number of sialic acid residues bound to them. TM In addition, two forms of the enzyme with differing molecular weights have been described. 1~ 2o The
Enzyme activity*
Subject (No.) Family members Normal in lymphocytes (9) Heterozygotes in lymphocytes (8) Obligate heterozygotes (2) Homozygores (2) Control subjects (50)
Pedigree No.
Mean _+SD
(21) I-1,4,5,6,9, 340.0_+ 153.3 II2,3,8,9 I-2,7,8, 276.6_+174.5 II-1,4,7, III-3 II-5,6 219.7 IlI-1,2
Range
134.5-618,5
37.0-435.0 201.0-238.5
10.7
10.2- 11.3
326.9 _+182.4~
13.2-660.0
*Activity is expressed in nmoles 4-methylumbelliferone released per ml of serum per hour. tin two control subjects a very low enzyme activity of 13.2 and 19.2 was found.
complex pH activity profile of a-L-fucosidase in lymphocytes and granulocytes (Fig. 2) is probably due to the underlying heterogeneity of the enzyme. The two different molecular weight forms have been shown to have different pH optima. 19, 20 Possible differences in the genetically determined and sialic acid containing isozymes remain to be studied. The existence of these various different isozymes is probably also responsible for the variation from one tissue to another in the ratio of activity toward the two different substrates. In leukocytes the activity toward the fluorogenic substrate was two times greater than that toward the colofigenic. In peripheral lymphocytes the ratio was only 1.2 to 1. The observed ratio of 1.9 to 1 in granulocytes, rather than the expected higher one, could possibly be attributed to changes that occurred during the isolation of the granulocytes. Ratios' of 4 to 1 and 1.3 to 1 have previously been reported for the liver and serum enzymes, respectively?~ The great variability of the a-L-fucosidase activity in sera from normal subjects indicates that this system cannot be used for assessment of carrier status or even for reliable diagnosis of fucosidosis. Two normal donors with very low serum activities were found. The a-L-fucosidase activity in the leukocytes and lymphocytes Of one of these individuals was measured and was within the normal range. This finding suggests that individuals with low serum activity can be distinguished from homozygotes
1 19 8
Beratis, Turner, and Hirschhorn
and heterozygotes for fucosidosis by studying the enzyme levels in leukocytes. Since mixed peripheral leukocyte preparations o f normal subjects only occasionally g a v e borderline or overlapping values with the heterozygotes, use o f leukQcytes for the identification of the carrier state of fucosidosis is feaSible. For more reliable identification o f the heterozygotes, howeVer, subjects with enzymatic ~ictivity in the low normal or heterozygous r a n g e s h o u l d be further investigated by determination of the fucosidase activity in isolated mononuclear cells. We acknowledge the highly skilled assistance of Miss Roberta Weiss. We also wish to thank Mrs. Minnie Woodson for her help in the prepai:ation of this manuscript. REFERENCES
1. Dawson G, and Spranger JW: Fucosidosis: A glycosphingolipidosis, N Engl J Med 285:122, 1971. 2. Matsuda I, Arashima S, Anakura M, Ege A, and Hayata I: Fucosidosis, Tohoku J Exp Meal 109:41, 1973. 3. Van Hoof F, and Hers HG: Mucopolysaccharidosis by absence of a-fucosidase, Lancet 1:1198, 1968. 4. Wiederschain GY, and Rosenfeld EL: Two forms of a-Lfucosidase from pig kidney and their action on natural olyg0saccharides, Biochem Biophys Res Comm 44:1008, 1971. 5: Wiederschain GY, Kolibata LG, and Rosenfeld EL: Human a-L-fucosidases, Clin Chim Acta 46:305, 1973. 6. KousseffBG, Beratis NG, Danesino C, and Hirschhorn K: Genetic heterogeneity in fucosid0sis, Lancet 2: J387, 1973. 7. Kousseff BG, Beratis NG, Strauss L, Brill PW, Rosenfeld RE, Kaplan B, and Hirschhorn K: Fucosidosis type 2, Pediatrics (in press).
The Journal of Pediatrics December 1975
8. Van Hoof F, and Hers HG: The abnormalities oflysosomaI enzymes in mucopolysaccharidoses, Eur0p J Biochem 7i34, 1968. 9. Loeb H,. Tondeur M, Jonr/iaux G, Mockel-Pohl S, and Vamos-Hurwitz E: Biochemical and ultrastructural studies in a case of mucopolysaccharidosis "F" (Fucosidosis), Helv Paediatr Acta 36:519, 1969. 10. Zielke K , Okada SI and O'Brien J: Fucosidosis: Diagnosis by serum assay of a-L-fucosidase, J Lab Clin Med 79:164, 1972. 11. Zielke K, Veath ML, and O'Brien JS: Fucosidosis: Deficiency o f alfa-L-fucosidase in cultured skin fibroblasts, J Exp Med 136:197, 1972. 12. Borrone C, Gatti R,Trias X, and Durand P: Fucosidosis: Clinical, biochemical, immunologic, and genetic studies in two new cases, J P~DIATR84:727, 1974. 13. Matsuda I, Ai'ashima S, Anakura M, and Oka Y: a-LFucosidase and a-D~mannosidase activity in the white blood cells in the disease and carrier state of fucosidosis, Clin Chim Acta 48;9, 1973. 14. Boyum A i Separation of leukocytes from blood and bone marrow, Scand J Clin Lab Invest 21(Suppl):97, 1968. 15. Lowry OH, Rosebrough NJ, Farr AL, and Randall R J: Protein measurement with the Folin phenol reagent, J Biol Chem 193:265, 1951. 16. Robinson D, and Thorpe R: Fluorescent assay of a-Lfucosidase, Clin Chim Acta 55:65, !974. 17. Turner BM, Turner vS, Beratis NG, and Hirschhorn K: Polymorphism of huma n a-fucosidase, Am J Hum Genet Am J Hum Genet 27:651, 1975. 18. Turner BM, Beratis NG, Turner VS, and Hirschhorn K: Isozymes & human a-L-fucosidase detectable by starch gel electrophoresis, Clin Chim Acta 57:29, 1974. 19. Wiederschain GY, Kolibaba LG, and Rosenfeld EL: Human a-L-fucosidase, Clin Chim Acta 46:305, 1973. 20. Robinson D, and Thorpe R: Human liver a-L-fUcosidases, Clin Chim Acta 47:403, 1973.
