Am J Hum Genet 29:455-461, 1977

The Hunter Syndrome in Females: Is There an Autosomal Recessive Form of Iduronate Sulfatase Deficiency? ELIZABETH F. NEUFELD,1 INGEBORG LIEBAERS,' CHARLES J. EPSTEIN,2 SHAUL YATZIV,2 3 AUBREY MILUNSKY,4 AND BARBARA R. MIGEON5

The Hunter syndrome (mucopolysaccharidosis II) is a disorder of mucopolysaccharide metabolism which is transmitted as an X-linked recessive trait [1]. The lysosomal storage of mucopolysaccharide which underlies the numerous clinical problems is the result of a specific enzyme defect-a nearly total absence of iduronate sulfatase activity [2]. Biochemical tests for diagnosing the Hunter syndrome have evolved from determination of urinary mucopolysaccharides, through studies of [35S]-mucopolysaccharide accumulation and correction in cultured fibroblasts, to the direct assay of enzyme activity in cells, body fluids, and tissues [3]. The Hunter syndrome is thought to occur only in hemizygous males. Heterozygous females, in accord with the Lyon hypothesis, have two populations of cells, one of which is as deficient in iduronate sulfatase activity as that of affected patients [4]. However, the heterozygotes are probably protected from the disease by a transfer of enzyme from the normal cell population to the abnormal, analogous to the correction which occurs in cultured fibroblasts [4, 5]. We wish to report our studies of two karyotypically normal girls with clinical manifestations of mucopolysaccharidosis II and with profound iduronate sulfatase deficiency in all tissues examined. (A preliminary report about one of these patients has been published [6].) Although it is not possible to rule out with complete certainty that these girls are manifesting heterozygotes of the X-linked disorder, the data suggest that Received April 22, 1977. This work was supported in part by National Institutes of Health Genetic Center grant GM 19527, Maternal and Child Health Project no. 445, and a National Foundation-March of Dimes Birth Defects Center grant awarded to C. J. Epstein; grants HD05515, HD09281, and GM07015 from the National Institutes of Health awarded to A. Milunsky; and grant HD05465 to B. Migeon. C. J. Epstein is an investigator of the Howard Hughes Medical Institute. S. Yatziv was a recipient of the U.S. Public Health Service International Fellowship. I. Liebaers was a recipient of the U.S. Public Health Service International Fellowship and a Belgian Nationaal Fonds voor Wetenshappelijk Onderzoek Fellowship. 1 Section on Human Biochemical Genetics, National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland 20014. 2 Departments of Pediatrics and Biochemistry and Biophysics, University of California, San Francisco, California 94143. 3Present address: Department of Pediatrics, Hadassah Hebrew University Medical Center, Jerusalem, Israel. 4Eunice Kennedy Shriver Center for Mental Retardation, Waltham, Massachusetts 02154. 5 Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. © 1977 by the American Society of Human Genetics. All rights reserved.

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they are homozygous for a previously unsuspected autosomal recessive form of iduronate sulfatase deficiency disease. CASE REPORTS

The first patient, T. L., had an affected brother, A. L., and two normal siblings. Although all four Ashkenazi Jewish grandparents came from the same small town in Lithuania, there is no known consanguinity. T. L.'s mother has adult Gaucher disease, but both T. L. and A. L. were found to have normal 0-glucosidase activity in their cultured skin fibroblasts. At 22 months of age, T. L. had coarse facies, blond curly hair, speckled blue irises, but no corneal clouding. She appeared deaf, and her psychomotor retardation, associated with moderate spasticity, was severe. Liver enlargement (2 cm) and splenomegaly (1-2 cm) were observed. T. L. died at 71/2 years of age. Analysis of a urine sample taken when she was 2-years-old showed elevated mucopolysaccharide with heparan sulfate as the major component. The karyotype of T. L.'s cultured fibroblasts was 46,XX, with no apparent morphological abnormalities. Banding studies were not performed. The second patient, M. C., whose parents are second cousins, has no family history of mucopolysaccharidosis. She has two siblings, one of whom (a male) has bilateral sensory neural hearing loss of unknown etiology. She had numerous upper respiratory infections as an infant, and delayed motor development was noted at 1 year. At 2 years of age, physical manifestations including hepatosplenomegaly, coarse facial features with a broad nasal bridge, deafness, and a tense thick skin suggested mucopolysaccharidosis. The urinary excretion of both dermatan sulfate and heparan sulfate was markedly elevated. During the next 3 years, there was a progressive development of the typical Hunter syndrome phenotype. Cardiac evaluation revealed mitral valve incompetence, but the corneas were clear on slit lamp examination. Although she was deaf and did not speak, mental development was considered normal. Because of continued upper respiratory infections and chronic otitis media, she underwent an uneventful tonsillectomy and adenoidectomy at age 5. Analysis of cultured lymphocytes by the Giemsa banding technique revealed a 46,XX karyotype, and careful study of the X chromosomes in 20 high quality metaphases evidenced no abnormality. EXPERIMENTAL

[35S]-Mucopolysaccharide Accumulation Accumulation of [35S]-mucopolysaccharide was carried out as described [7], in the presence of Hunter corrective factor [8], with fibroblasts cultured from skin biopsies of T. L., her brother (A. L.), and M. C. After 2 days in radioactive medium, the three lines had accumulated an abnormal amount of [35S]-mucopolysaccharide, which was reduced in the presence of the factor (table 1). Correctability by the factor, which has iduronate sufatase activity [9], indicates that the elevated [35S]-mucopolysaccharide is specifically the result of a deficiency of iduronate sulfatase and that the other enzymes required for mucopolysaccharide catabolism function adequately. This interpretation is supported by normal values obtained for the activity of other enzymes of mucopolysaccharide degradation (a-L-iduronidase, heparan N-sulfatase, N-acetyl-aglucosaminidase, f3-glucuronidase, aryl sulfatase B and 83-galactosidase) in the fibroblasts of M. C. Iduronate Sulfatase Activity The activity of the enzyme in lymphocytes, plasma, and serum was determined

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TABLE 1

MUCOPOLYSACCHARIDE ACCUMULATION

AND

CORRECTIBILITY

IN

CULTURED FIBROBLASTS MUCOPOLYSACCHARIDE ACCUMULATION (cpm/mg protein)

CELL LINE

-Factor

T. L ............................ A. L. ........................... M .C Mother L ......... ............... Father L ......................... Mother C ........................ Father C ............ ............. Normal control ........ ............ Adult Gaucher patientt ...... ....... ...........................

+Factor

47,200

19,900

41,900

16,400

Ratio* (-Factor/ +Factor)

53,300

14,700

10,900 11,000 10,500

10,300 10,500 10,800

2.4 2.6 3.6 1.1 1.0 1.0

11,700

9,700 11,700 10,100

1.2 1.1 1.0

12,700 10,400

* A ratio of I is characteristic of normal cells or cells in which iduronate sulfatase is not limiting; a ratio of 2 or more is characteristic of Hunter cells. t Cell strain GM 852 was obtained from the Human Mutant Cell Repository, Camden, N. J.

under conditions previously described and expressed in nmol per 24 hr/mg protein; the results in table 2 can therefore be compared to published values [3]. For fibroblasts and solid tissues (table 3), a reduced substrate concentration (0. 1 mM) was used; the activity is expressed in units, 1 U being defined as that level of enzyme activity which catalyses the hydrolysis of 1% of the substrate per hour [10]. In all the assays, products and residual substrate were separated from each other on minicolumns of anion exchange resin [10]. Tables 2 and 3 reveal that all specimens from M. C. and T. L. were almost totally devoid of iduronate sulfatase activity. However, serum, plasma, and lymphocyte homogenates from parents did not show reduced activity. In fact, the lymphocytes of the parents of family L and concurrent controls were unusually high, perhaps because TABLE 2 IDURONATE SULFATASE ACTIVITY IN BLOOD IDURONATE SULFATASE ACTIVITY

(nmol/24 hr/mg protein) SUBJECTS

Family L: Mother ........................ Father ......................... Control ........................ Family C: M. C. ......................... Mother ........................ Father ......................... Control ........................ *

Three individuals.

t Two individuals.

Lymphocytes

198 74 127 3.4 47 41 58

Plasma

7 6 8 (7-9)* ... ... . . ...

Serum

...

...

...

0.08 10 8

10; 7t

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TABLE 3 IDURONATE SULFATASE ACTIVITY IN HOMOGENATES IDURONATE SULFATASE ACTIVITY

T. L.

M. C.

TISSUE

0.3 0.1 0.02

......... Cultured fibroblasts* ...... Tonsilst ............ ............. ............. Adenoidst .......... Livert 1............................ Spleent .............i............. Braint

Control

31 36 58

.

131 1.3 0.4 .1. 1.8

..

111 44

..

.

...........................

* Activity is expressed in units per mg protein. Fibroblast homogenates were prepared and assayed as described for amniotic fluid cells [10]. t Activity is expressed in units per gram wet weight of tissue. Tissues were ground in 1-3 vol of 0.15 M NaCl. The homogenate was centrifuged at 12,000 g for 20 min, the pellet washed with 0. 15 M NaCl, and the combined supernatant solutions were made 80% saturated with respect to (NH4)2S04. The precipitated protein, collected by centrifugation, was dissolved in saline and thoroughly dialyzed against 0.15 M NaCI (four changes, 61 each, over 20 hr). All the preceding steps were carried out at 0-4cC. The homogenates were assayed under the conditions described for amniotic fluid cells [10].

of some difference in the method of preparation; in no case was the activity in the specimens from the mother lower than in those from the father.

Study of Cloned Fibroblasts Fibroblasts from the two mothers as well as from M. C. were tested for the mosaicism expected of heterozygotes for an X-linked trait. Over 30 clonal cultures were obtained from each individual and tested for [35S]-mucopolysaccharide accumulation and correctability as described [4]. Not a single abnormal clone was obtained from the mothers of T. L. and M. C. nor a single normal clone from the culture of M. C. (table 4). DISCUSSION

We have presented evidence for a profound deficiency of iduronate sulfatase in the serum, cells, and tissues of two karyotypically normal girls with normal fathers. We consider several explanations for this occurrence, which is unexpected in an X-linked

disease. 1. The patients are homozygous for the X-linked mutation which causes iduronate sulfatase deficiency, as suggested by Punnett [11]. This could occur either by TABLE 4 PHENOTYPE OF FIBROBLAST CLONES No. OF CLONES INDIVIDUAL

M. C.

............................... ...

Mother C ......................... Mother L .........................

...

34 34

Normal

Hunter

0

33 0 0

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simultaneous mutations affecting both parental X chromosomes or by transmission of a mutant X chromosome from a carrier mother and a newly mutant X from the father. The likelihood of the former is g2 (where u is the mutation rate, assumed to be the same for egg and sperm); the likelihood of the latter is 4,u (a priori probability that a woman is carrier) x 0.5 (probability of transmitting the mutant X) x u (probability of mutant X in sperm) = 2 g2. The incidence of the Hunter syndrome has been estimated at approximately 0.6 x 10-5 [12]; the mutation rate, g, for a lethal X-linked disorder is one-third the incidence or about 0.2 x 10-5. Therefore, the prior probabilities of homozygosity are 4 x 10-12 and 8 x 10-12, respectively. The ratio of homozygous females to hemizygous males would be (4 + 8) x 10-12/0.6 x 10-5, or of the order of 10-6. But, we have detected two families with female patients among the approximately 100 Hunter families diagnosed biochemically in Bethesda. Such a large discrepancy between the expected and the observed ratios forces us to reject the hypothesis of homozygosity at the X locus for iduronate sulfatase. 2. The patients are heterozygous for the X-linked mutation, but the chromosome bearing the normal gene for iduronate sulfatase is always i1active. This would occur either because of nonrandom X-chromosome inactivation, which is not known to occur in humans [13], or because of some subsequent selection of the cells in which the mutant X chromosome is active. Evidence for this possibility would have been the demonstration of Hunter clones among the fibroblasts obtained from the mothers. If the mutation is X-linked, Mrs. C, with only one affected child and one normal son, has a 1/2 posterior probability of being a carrier [14], whereas Mrs. L, with two affected children, is an obligate heterozygote. Yet we could find no deficient clones among over 30 clones for either mother. We have had no difficulty previously in demonstrating the presence of Hunter clones in three cultures of Hunter carriers [4] and occasionally observed, as have others [15, 16], increased accumulation of [35S]-mucopolysaccharide even in uncloned cultures. To account for the disease in heterozygotes, we would have to assume a new mutation followed by selection of the mutant cells in the case of M. C.; in the case of T. L., we would have to postulate selection against the cells bearing the normal X chromosome in the daughter and selection in the opposite direction in the mother. It is not possible to assign a statistical probability to such an unprecedented situation, since it would presumably be caused not by random events but by some unknown interactions with other parts of the genome. For example, Mrs. L has Gaucher disease; although there is no evidence that ,8-glucosidase deficiency affects mucopolysaccharide metabolism, we cannot exclude the possibility that the deficiency of a second lysosomal enzyme might have been selectively detrimental to the cells deficient in iduronate sulfatase. 3. The patients are homozygous for an autosomal gene which controls iduronate sulfatase activity. The strongest support for this possibility comes from the fact that M. C.'s parents are second cousins and that the grandparents of the children in family L are from the same ethnic group and the same small town. Assuming that the two affected girls represent such autosomal recessive cases, then the frequency, q2, of this genetic form would be 0.02 x 0.6 x 10-5 = 1.2 x 10-7. This rough approximation, based on the Bethesda experience, would lead to estimated values for the gene frequency, q, of 3.5 x 10-4, and for the heterozygote frequency, 2pq, of 7 x 10-4.

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Such a rare gene is unlikely to find homozygous expression except in consanguinous or endogamous marriages. The mutations in the two families are probably different (but perhaps allelic) since the children in family L are among the most severely affected Hunter patients ever described, whereas the clinical manifestations of M. C. are typical of the milder form of the Hunter syndrome. The presumed autosomal recessive locus could encode a subunit of iduronate sulfatase or regulate its production. Discrimination between these alternatives will require examination of the enzyme at a molecular level and studies of complementation in somatic cell hybrids. SUMMARY

Profound iduronate sulfatase deficiency, characteristic of the Hunter syndrome, has been found in cultured fibroblasts, serum, lymphocytes, and tissues of two clinically affected girls. The patients are karyotypically normal and have normal fathers; cloning of the mothers' fibroblasts did not reveal the mosaicism expected of carriers of an X-linked disease. Homozygosity for a previously unsuspected autosomal recessive gene for iduronate sulfatase is considered the most likely explanation, although heterozygosity for the X-linked gene and subsequent selection cannot be completely excluded. ACKNOWLEDGMENTS We thank Dr. Julian Kanfer of the University of Manitoba for ,3-glucosidase determinations on the fibroblasts of family L, Dr. Trefor Jenkins of the South African Institute for Medical Research for preparation of lymphocytes from family L, Dr. William van Robertson of the Children's Hospital at Stanford for analysis of M. C.'s urinary mucopolysaccharide, and Mrs. Joyce Sprenkle for her assistence in obtaining clonal cultures.

1.

2. 3. 4. 5.

6. 7.

8. 9. 10.

REFERENCES McKusIcK VA: Heritable Disorders ofConnective Tissue, 4th ed. St. Louis, Mosby, 1972, pp 521-687 NEUFELD EF, LIM TW, SHAPIRO LJ: Inherited disorders of lysosomal metabolism. Ann Rev Biochem 44:357-376, 1975 LIEBAERS I, NEUFELD EF: Iduronate sulfatase in serum, lymphocytes and fibroblasts. Simplified diagnosis of the Hunter syndrome. Pediatr Res 10:733-736, 1976 MIGEON BR, SPRENKLE JA, LIEBAERS I, SCOTT JF, NEUFELD EF: X-linked Hunter syndrome: the heterozygous phenotype in cell culture. Am JHum Genet 29:448-454, 1977 FRATANTONI JC, HALL CW, NEUFELD EF: Hurler and Hunter syndromes: mutual correction of the defect in cultured fibroblasts. Science 162:570-572, 1968 MILUNSKY A, NEUFELD EF: The Hunter syndrome in a 46,XX girl. N Engl J Med 288:106-107, 1973 CANTZ M, KRESSE H, BARTON RW, NEUFELD EF: Corrective factors for inborn errors of mucopolysaccharide metabolism, in Methods in Enzymology, vol 28, edited by GINSBURG V, New York, Academic Press, 1972, pp 884-897 CANTZ M, CHRAMBACH A, BACH G, NEUFELD EF: The Hunter corrective factor. Purification and preliminary characterization. J Biol Chem 247:5456-5462, 1972 BACH G, EISENBERG F JR, CANTZ M, NEUFELD EF: The defect in the Hunter syndrome: deficiency of sulfoiduronate sulfatase. Proc Natl Acad Sci USA 70:2134-2 138, 1973 LIEBAERS I, DI NATALE P, NEUFELD EF: Iduronate sulfatase in amniotic fluid: an aid in the prenatal diagnosis of the Hunter syndrome. J Pediatr 90:423-425, 1977

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11. PUNNETT HH: Hunter syndrome in girl. N Engl J Med 288:856, 1973 12. LOWRY RB, RENWICK DHG: Relative frequency of the Hurler and Hunter syndromes. N Engl J Med 284:221, 1971 13. MIGEON BR: X-chromosome inactivation as determinant of the female phenotype, in Genetic Mechanism of Sexual Development, edited by VALLET L, PORTER I, New York, Academic Press, 1977 14. CHASE GA, MURPHY EA: Risk of recurrence and carrier frequency for X-linked lethal recessives. Hum Hered 23:19- 26, 1973 15. BOOTH CW, NADLER HL: Demonstration of the heterozygous state in Hunter's syndrome. Pediatr 53:396-399, 1974 16. DONNELLY PW, DIFERRANTE N: Reliability of the Booth-Nadler technique for the detection of Hunter heterozygotes. Pediatr 56:429-433, 1975

Joint Congress of International Society of Hematology and International Society of Blood Transfusion A joint congress of the International Society of Hematology and International Society of Blood Transfusion will be held in Paris, July 23-29, 1978. The principal topics to be covered are: basic and clinical hematology, hemostasia, immunohematology, and blood transfusion; special symposia will cover: thrombopathies, hemoglobin, cryobiology and blood preservation, viral hepatitis, oncornavirus, and leukemia. The deadline for registration for papers and submission of abstracts is February 1, 1978. Only the papers of registered participants will be accepted and published. For scientific information, contact Professor C. Salmon, Scientific Secretary, B.P. 8, 75560 Paris, Cedex 12, France. For practical and registration information, contact CongresServices, 1 rue Jules Lefeibvre, 75009 Paris, France.

The Hunter syndrome in females: is there an autosomal recessive form of iduronate sulfatase deficiency?

Am J Hum Genet 29:455-461, 1977 The Hunter Syndrome in Females: Is There an Autosomal Recessive Form of Iduronate Sulfatase Deficiency? ELIZABETH F...
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