Clinical Genetics 1979: 15: 1 13-1 I7
Prenatal monitoring for the Hunter syndrome: The heterozygous female fetus w.J. KLEIJER',
P. D. MOOY', I. LIEBAERS3*, J. J . P. VAN D E KAMP2 AND M. F. NIERMEIJER'
Department of Cell Biology and Genetics, Erasmus University, Rotterdam; Department of Pediatrics, University Hospital, Leiden, The Netherlands; and Section on Human Biochemical Genetics, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland, U. S. A. 2
3
An abnormal level of 35S-sulfate labeled mucopolysaccharides was found in cultured amniotic fluid cells from a pregnancy, at risk for the Hunter syndrome, with a female fetal karyotype. Subsequent prenatal analyses suggested heterozygosity for the X-linked Hunter syndrome, and this was confirmed by clonal analysis of fibroblasts of the child after birth. The possible implications of abnormal biochemical results in association with a female karyotype in the prenatal diagnosis of the Hunter syndrome are discussed. Received 3 August, accepted f o r publication 15 September 1978 Key words: Heterozygote detection; Hunter's syndrome; mucopolysaccharidosis, type 11;
prenatal diagnosis; X-linked disorders.
In the Hunter syndrome (mucopolysaccharidosis type 11, MPS II), the defective inucopolysaccharide degradation is caused by a deficiency of iduronate sulfatase (Bach et al. 1973, Coppa et al. 1973, Sjoberg et al. 1973). Clinically mild and severe types of the disease, both with X-linked inheritance, are known (McKusick 1975). Recently, two female patients have been described who had MPS I1 a n d iduronate-sulfatase deficiency, which may have been an autosoma1 recessive type of the disease or the result of selection in a heterozygote (Epstein e t al. 1976, Neufeld et al. 1977). Prenatal monitoring for MPS I1 is car-
*
ried o u t by the determination of fetal sex and biochemical analysis. Iduronate sulfatase deficiency in the amniotic fluid as well as in the cultured cells (Liebaers e t al. 1977) and increased 3%-sulfate incorporation in intracellular mucopolysaccharides in cultured amniotic fluid cells (Fratantoni et al. 1969) have both been used t o establish a prenatal diagnosis of MPS 11. This report describes the prenatal monitoring of a pregnancy a t risk f o r MPS 11, in which a female fetus was found, and in which there was abnormal mucopolysaccharide metabolism in the cultured amniotic fluid cells.
Present address: Department of Pediatrics, University Hospital St. Pieter, Brussels, Belgium 0009-9163/79/020113-05 $02.5010 0 1979 Munksgaard, Copenhagen
114
K L E IJ E R ,
M 0 0 Y , L I E B A E R S , V A N D E K A M P A N D N I E R M E IJ E R
Case Report
The Caucasian parents are healthy and of Dutch origin; consanguinity is present in the ninth generation. Their first son (D.B.) shows clinical symptoms of MPS I1 with increased levels of urinary mucopolysaccharides (Dr. M. Giesberts, Leiden) and an iduronate sulfatase deficiency in leucocytes and cultured fibroblasts (Dr. H. Kresse and Dr. K. von Figura, Miinster). No other relatives are affected with MPS 11. During the second pregnancy, amniocentesis was carried out in the 15th week (Dr. M. Jahoda, Rotterdam).
sent on dry ice from Rotterdam to the N.I.H., Bethesda and analysed for iduronate sulfatase activity as previously described (Liebaers et al. 1977, Hall et al. 1978). Cloning of fibroblasts was performed in wells of Terasaki tissue typing plates (1 mm diameter, Falcon plastics) after inoculation with 25 ~1 of a cell suspension containing one cell on average, obtained from the early outgrowths of a skin biopsy. The wells were screened under the microscope 1 day after plating, and cell growth was allowed only in wells containing one cell. After about 3 weeks confluent clones could be subcultured.
Materials and Methods
Cells from 20 ml amniotic fluid were cultured in Falcon dishes (35 mm diameter) on cover slips (Niermeijer et al. 1976). After 10 days, cell clones in two separate primary cultures were processed for chromosome analysis (Q-banding). The incorporation of 36-sulfate in the intracellular mucopolysaccharide fraction in confluent cultures of amniotic fluid cells and skin fibroblasts was measured according to a modified procedure of Fratantoni et al. (1969) (Fortuin & Kleijer 1978). Cell-free amniotic fluids and sera were
Results
Chromosome analysis in two separate primary amniotic fluid cultures from the pregnancy at risk showed a normal female karyotype, 46,XX. The other primary cultures were subcultured and used at confluency for 35Ssulfate incorporation assays in the first subculture (assay I; Table 1), the second subculture (assay II), and the fourth subculture (assay 111). The cells used in assays I and I1 showed epithelial morphology, whereas those used in assay I11 were pre-
Table 1 JSS04-incorporation in amniotic fluid cells and skin fibroblasts Assay I cprnimg' ratio'*
Assay I I ratio CPrnh3
x 10-3
Assay 111 cprnimg ratio x 10-3
Amnlotlc fluid cells At risk (Mrs. B.) Control
31.O 6.8
4.6
46.2 8.5
5.4
12.4 10.5
1.2
Fibroblasts Index patient (D.B.) Control
21.4 10.4
2.1
27.4 9.2
3.0
34.1
2.5
13.8
' cpm incorporated per mg cell protein of triplicate confluent cultures after 5 days in medium with 5 mCi/ nmol Na? J$SO+ * * ratio of 3sS-incorporation in patient amniotic fluid cells or fibroblasts and matched controls.
P R E N A T A L D I A G N O S I S OF T H E H U N T E R S Y N D R O M E
115
Table 2
lduronate sulfatase activity in cell free amniotic fluid and serum Amniotic fluid
Activity (unitslmg protein)
Mrs. B Controls 1 ; 2; 3 ; 4 Control range' (n=20) MPS II range' (n=4)
10 27: 30; 18; 25 1133 1- 7
Activity (unitslmg protein)
Serum
MPSll (D.B.) control control range' (n = 10) MPS II range' (n=lO)
0.0
3.0 1.6-4.8
0.&0.02
Ranges from previous investigations.
dominantly fibroblast-like. The results (Table 1) of assays I and I1 showed an increased level of 35S-labeled mucopolysaccharides in the cultured amniotic fluid cells from the pregnancy at risk (Mrs. B); the values were even higher than in fibroblasts of the index patient (D.B.). The amniotic fluid cells in assay 111, however, showed a normal level of 3%-sulfate incorporation. Cell-free amniotic fluids from the pregnancy a t risk and four controls were assayed for iduronate sulfatase activity; the results (Table 2) showed a relatively low activity in the sample from Mrs. B., which
Table 3
Demonstration of heterozygosity by 35SOdincorporation analysis of fibroblast clones No. of clones
cpmlmg Protein relative to control.
A. Mother (Mrs. B) Clones - normal type - MPS Il-type - indeterminate Uncloned - MPS Il-patient
13 3 1
0.41.4 2.e-6.0 2.0 3.4
8. Daughter (S.B.) Clones -normal type - MPS Il-type - indeterminate Uncloned - MPS Il-patient
5 14 2
O.el.3 2.23.0 1.7-1.7 4.9
Confluent cultures of about 105 cells were grown for 5 days in medium with 20 rnCi1mmol Na2 %O,. Control fibroblasts incorporated 40.000 and 45,000 cpmlmg protein in assays A and B, respectively.
was compatible with a heterozygous state of the fetus. Fibroblast clones were grown from skin biopsies of Mrs. B. and subsequently of her daughter (S.B.) after birth. The 35s-sulfate incorporation of these clones showed increased levels typical for MPS I1 in 3 of 17 clones cultured from the mother and in 14 of 21 clones grown from her daughter (Table 3). Discussion
In the present prenatal analysis, female fetal sex was determined, which seemed to exclude X-linked MPS I1 in the fetus. However, 3%-sulfate incorporation analyses in two separate amniotic fluid cell cultures showed an accumulation level characteristic for MPS 11. The following explanations for these results should be considered: (1) In vitro or in vivo selection of the mutant type of cells in a case of a heterozygote for X-linked MPS 11. In vitro selection may not be a rare event since the amniotic fluid cultures often originate from only a few cells; Liebaers et al. (1977) reported abnormal 8%-sulfate incorporation in one of the three amniotic fluid cultures tested from a pregnancy with a fetus heterozygous for MPS 11. Non-random Xchromosome inactivation or (in vivo) selection of mutant cells co'uld result in female patients with X-linked MPS 11, but so far
116
KLElJER,
M O O Y , L I E B A E R S , VAN D E K A M P A N D N I E R M E IJ ER
no such patients have been reported (Neufeld et al. 1977).
(2) Homozygous X-linked MPS TI seems unlikely, since in addition to heterozygosity of the mother this would require either a new mutation in the X-chromosome donated by the father or the father being affected with a mild type of MPS I1 (McKusick 1975, Hobolth & Pedersen 1978).
(3) An autosomal recessive type of MPS 11; two female patients with MPS II and iduronate sulfatase deficiency were recently described and evidence was presented suggesting autosomal inheritance (Neufeld et al. 1977). Further investigations in the present case showed normal V ~ s u l f a t eincorporation in a later subculture grown from the same amniotic fluid and a relatively low iduronate sulfatase activity in cell-free amniotic fluid. Conclusive evidence for heterozygosity for X-linked MPS 11 was obtained by the demonstration of both normal and mutant cells in clonal analysis of skin fibroblasts of the girl after birth. A majority (7470) of the clones were of the mutant phenotype. In the mother 18 70 of the clones showed abnormal 3%-sulfate incorporation. As demonstrated here and previously by Danes & Bearn (1967), Capobianchi & Romeo (1976), Migeon et al. (1977), heterozygosity for X-linked MPS 11 can be established by clonal analysis of cultured fibroblasts or by the recently reported method of hair-root analysis (Fluharty et al. 1977, Nwokoro et al. 1977). However both methods are time-consuming and probably not sufficiently reliable to exclude heterozygcsity. Prenatal diagnosis should therefore be offered not only to obligate carriers of MPS I1 but also to possible carriers. The present observations suggest that if
abnormal biochemical results are found in a female fetus in a pregnancy of a possible carrier for MPS 11, clonal analysis of the amniotic fluid cells should be carried out to exclude the presumed autosomal form of MPS II. Acknowledgments
The authors are grateful to Mrs. G . Hensing-Wolffers, Mrs. A. M. Zandvoort-Waayers and Miss Th. Zwaanenburg for performing cell cultures and analyses. This study was supported in part by “Het Praeventiefonds”, The Hague, The Netherlands. References
Bach, G., F. Eisenberg, M. Cantz & E. F. Neufeld (1973). The defect in the Hunter syndrome: Deficiency of suIfoiduronate sulfatase. Proc. nut. Acad. Sci. (Wash) 70, 2134-2138. Capobianchi, M. R. & G. Romeo (1976). Mosaicism for sulfoiduronate sulfatase deficiency in carriers of Hunter’s syndrome. Experientb 32, 459460. Coppa, G. B., J. Singh, B. L. Nichols & N. DiFerrante (1973). Urinary excretion of disulfated disaccharides in Hunter syndrome: Correction by infusion of a serum fraction. Anal. Lett. 6, 225-233. Danes, B. S. & A. G . Bearn (1967). Hunter’s syndrome: A genetic study of clones in cell culture with particular reference to the Lyon hypothesis. J . exp. Med. 126, 509-522. Epstein, C. J., S. Yatziv, E. F. Neufeld & I. Liebaers (1976). Genetic counseling for Hunter syndrome. Lancet ii, 737-738. Fluharty, A. L., T. Yutaka, R. L. Stevens & H. Kihara (1977). Hair-root analysis of sulfoiduronate sulfatase in a Hunter syndrome heterozygote. Amer. J . hum. Genet. 29, 43A. Fortuin, J. J. H. & W. J. Kleijer (1978). Pericellular glycosaminiglycans in cultured human cells. A possible source of error in prenatal diagnosis of mucopolysaccharidoses. Clin. Chim. Acta 82, 79-83. Fratantoni, J. C. E. F. Neufeld, W. B. Uhlendorf & C. B. Jacobson (1969). Intrauterine
P R E N A T A L D I A G N O S I S OF T H E H U N T E R S Y N D R O M E
diagnosis of the Hurler and Hunter syndromes. N e w Engl. J. Med. 280, 686-688. Hall, C. W., I. Liebaers, P. DiNatale & E. F. Neufeld (1978). Enzymic diagnosis of the genetic mucopolysaccharide storage disorders. Methods in Enzymology 50, 439-456. Hobolth, N. & C . Pedersen (1978). Six cases of a mild form of the Hunter syndrome in five generations. Three affected males with progeny. Clin. Genet. 13, 121. Liehaers, I., P. DiNatale & E. F. Neufeld (1977). Iduronate sulfatase in amniotic fluid: An aid in the prenatal diagnosis of the Hunter syndrome. J . Pediat. 90, 423-425. McKusick, V. A. (1975). Mendelian Inheritance in Man, 4th Ed. John Hopkins University Press, p. 643. Migeon, B. R., J. A. Sprenkle, I. Liebaers, J. F. Scott & E. F. Neufeld (1977). X-linked Hunter syndrome: The heterozygous phenotype in cell culture. Amer. J . hum. Genet. 29, 448-454. Neufeld, E. F., I. Liebars, C. J. Epstein, S. Yatziv, A. Milunsky & B. R. Migeon (1977). The Hunter syndrome in females: Is there
117
an autosornal recessive form of iduronate sulfatase deficiency? Amer. J . hum. Genet. 29, 455-461. Niermeijer, M. F., E. S. Sachs, M. Jahodova, C. Tichelaar-Klepper, W. J. Kleijer & H. Galjaard (1976). Prenatal diagnosis of genetic disorders. J . med. Genet. 13, 182-194. Nwokoro, N., I. Liebaers & E. F. Neufeld (1977). Iduronate sulfatase activity in hair roots of Hunter heterozygotes. Amer. J . hum. Genet. 29, 82A. Sjoberg, I., L. A. Fransson, R. Matalon & A. Dorfrnan (1973). Hunter’s syndrome: A deficiency of L-iduronosulfatase. Biochem. Biophys. Res. Commun. 54, 1125-1132.
Address: Dr. W . J . Kleijer Department of Cell Biology & Genetics Erasmus University P. 0. Box 1738 Rotterdam The Netherlands
Prenatal monitoring for the Hunter syndrome: The heterozygous female fetus w.J. KLEIJER',
P. D. MOOY', I. LIEBAERS3*, J. J . P. VAN D E KAMP2 AND M. F. NIERMEIJER'
Department of Cell Biology and Genetics, Erasmus University, Rotterdam; Department of Pediatrics, University Hospital, Leiden, The Netherlands; and Section on Human Biochemical Genetics, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland, U. S. A. 2
3
An abnormal level of 35S-sulfate labeled mucopolysaccharides was found in cultured amniotic fluid cells from a pregnancy, at risk for the Hunter syndrome, with a female fetal karyotype. Subsequent prenatal analyses suggested heterozygosity for the X-linked Hunter syndrome, and this was confirmed by clonal analysis of fibroblasts of the child after birth. The possible implications of abnormal biochemical results in association with a female karyotype in the prenatal diagnosis of the Hunter syndrome are discussed. Received 3 August, accepted f o r publication 15 September 1978 Key words: Heterozygote detection; Hunter's syndrome; mucopolysaccharidosis, type 11;
prenatal diagnosis; X-linked disorders.
In the Hunter syndrome (mucopolysaccharidosis type 11, MPS II), the defective inucopolysaccharide degradation is caused by a deficiency of iduronate sulfatase (Bach et al. 1973, Coppa et al. 1973, Sjoberg et al. 1973). Clinically mild and severe types of the disease, both with X-linked inheritance, are known (McKusick 1975). Recently, two female patients have been described who had MPS I1 a n d iduronate-sulfatase deficiency, which may have been an autosoma1 recessive type of the disease or the result of selection in a heterozygote (Epstein e t al. 1976, Neufeld et al. 1977). Prenatal monitoring for MPS I1 is car-
*
ried o u t by the determination of fetal sex and biochemical analysis. Iduronate sulfatase deficiency in the amniotic fluid as well as in the cultured cells (Liebaers e t al. 1977) and increased 3%-sulfate incorporation in intracellular mucopolysaccharides in cultured amniotic fluid cells (Fratantoni et al. 1969) have both been used t o establish a prenatal diagnosis of MPS 11. This report describes the prenatal monitoring of a pregnancy a t risk f o r MPS 11, in which a female fetus was found, and in which there was abnormal mucopolysaccharide metabolism in the cultured amniotic fluid cells.
Present address: Department of Pediatrics, University Hospital St. Pieter, Brussels, Belgium 0009-9163/79/020113-05 $02.5010 0 1979 Munksgaard, Copenhagen
114
K L E IJ E R ,
M 0 0 Y , L I E B A E R S , V A N D E K A M P A N D N I E R M E IJ E R
Case Report
The Caucasian parents are healthy and of Dutch origin; consanguinity is present in the ninth generation. Their first son (D.B.) shows clinical symptoms of MPS I1 with increased levels of urinary mucopolysaccharides (Dr. M. Giesberts, Leiden) and an iduronate sulfatase deficiency in leucocytes and cultured fibroblasts (Dr. H. Kresse and Dr. K. von Figura, Miinster). No other relatives are affected with MPS 11. During the second pregnancy, amniocentesis was carried out in the 15th week (Dr. M. Jahoda, Rotterdam).
sent on dry ice from Rotterdam to the N.I.H., Bethesda and analysed for iduronate sulfatase activity as previously described (Liebaers et al. 1977, Hall et al. 1978). Cloning of fibroblasts was performed in wells of Terasaki tissue typing plates (1 mm diameter, Falcon plastics) after inoculation with 25 ~1 of a cell suspension containing one cell on average, obtained from the early outgrowths of a skin biopsy. The wells were screened under the microscope 1 day after plating, and cell growth was allowed only in wells containing one cell. After about 3 weeks confluent clones could be subcultured.
Materials and Methods
Cells from 20 ml amniotic fluid were cultured in Falcon dishes (35 mm diameter) on cover slips (Niermeijer et al. 1976). After 10 days, cell clones in two separate primary cultures were processed for chromosome analysis (Q-banding). The incorporation of 36-sulfate in the intracellular mucopolysaccharide fraction in confluent cultures of amniotic fluid cells and skin fibroblasts was measured according to a modified procedure of Fratantoni et al. (1969) (Fortuin & Kleijer 1978). Cell-free amniotic fluids and sera were
Results
Chromosome analysis in two separate primary amniotic fluid cultures from the pregnancy at risk showed a normal female karyotype, 46,XX. The other primary cultures were subcultured and used at confluency for 35Ssulfate incorporation assays in the first subculture (assay I; Table 1), the second subculture (assay II), and the fourth subculture (assay 111). The cells used in assays I and I1 showed epithelial morphology, whereas those used in assay I11 were pre-
Table 1 JSS04-incorporation in amniotic fluid cells and skin fibroblasts Assay I cprnimg' ratio'*
Assay I I ratio CPrnh3
x 10-3
Assay 111 cprnimg ratio x 10-3
Amnlotlc fluid cells At risk (Mrs. B.) Control
31.O 6.8
4.6
46.2 8.5
5.4
12.4 10.5
1.2
Fibroblasts Index patient (D.B.) Control
21.4 10.4
2.1
27.4 9.2
3.0
34.1
2.5
13.8
' cpm incorporated per mg cell protein of triplicate confluent cultures after 5 days in medium with 5 mCi/ nmol Na? J$SO+ * * ratio of 3sS-incorporation in patient amniotic fluid cells or fibroblasts and matched controls.
P R E N A T A L D I A G N O S I S OF T H E H U N T E R S Y N D R O M E
115
Table 2
lduronate sulfatase activity in cell free amniotic fluid and serum Amniotic fluid
Activity (unitslmg protein)
Mrs. B Controls 1 ; 2; 3 ; 4 Control range' (n=20) MPS II range' (n=4)
10 27: 30; 18; 25 1133 1- 7
Activity (unitslmg protein)
Serum
MPSll (D.B.) control control range' (n = 10) MPS II range' (n=lO)
0.0
3.0 1.6-4.8
0.&0.02
Ranges from previous investigations.
dominantly fibroblast-like. The results (Table 1) of assays I and I1 showed an increased level of 35S-labeled mucopolysaccharides in the cultured amniotic fluid cells from the pregnancy at risk (Mrs. B); the values were even higher than in fibroblasts of the index patient (D.B.). The amniotic fluid cells in assay 111, however, showed a normal level of 3%-sulfate incorporation. Cell-free amniotic fluids from the pregnancy a t risk and four controls were assayed for iduronate sulfatase activity; the results (Table 2) showed a relatively low activity in the sample from Mrs. B., which
Table 3
Demonstration of heterozygosity by 35SOdincorporation analysis of fibroblast clones No. of clones
cpmlmg Protein relative to control.
A. Mother (Mrs. B) Clones - normal type - MPS Il-type - indeterminate Uncloned - MPS Il-patient
13 3 1
0.41.4 2.e-6.0 2.0 3.4
8. Daughter (S.B.) Clones -normal type - MPS Il-type - indeterminate Uncloned - MPS Il-patient
5 14 2
O.el.3 2.23.0 1.7-1.7 4.9
Confluent cultures of about 105 cells were grown for 5 days in medium with 20 rnCi1mmol Na2 %O,. Control fibroblasts incorporated 40.000 and 45,000 cpmlmg protein in assays A and B, respectively.
was compatible with a heterozygous state of the fetus. Fibroblast clones were grown from skin biopsies of Mrs. B. and subsequently of her daughter (S.B.) after birth. The 35s-sulfate incorporation of these clones showed increased levels typical for MPS I1 in 3 of 17 clones cultured from the mother and in 14 of 21 clones grown from her daughter (Table 3). Discussion
In the present prenatal analysis, female fetal sex was determined, which seemed to exclude X-linked MPS I1 in the fetus. However, 3%-sulfate incorporation analyses in two separate amniotic fluid cell cultures showed an accumulation level characteristic for MPS 11. The following explanations for these results should be considered: (1) In vitro or in vivo selection of the mutant type of cells in a case of a heterozygote for X-linked MPS 11. In vitro selection may not be a rare event since the amniotic fluid cultures often originate from only a few cells; Liebaers et al. (1977) reported abnormal 8%-sulfate incorporation in one of the three amniotic fluid cultures tested from a pregnancy with a fetus heterozygous for MPS 11. Non-random Xchromosome inactivation or (in vivo) selection of mutant cells co'uld result in female patients with X-linked MPS 11, but so far
116
KLElJER,
M O O Y , L I E B A E R S , VAN D E K A M P A N D N I E R M E IJ ER
no such patients have been reported (Neufeld et al. 1977).
(2) Homozygous X-linked MPS TI seems unlikely, since in addition to heterozygosity of the mother this would require either a new mutation in the X-chromosome donated by the father or the father being affected with a mild type of MPS I1 (McKusick 1975, Hobolth & Pedersen 1978).
(3) An autosomal recessive type of MPS 11; two female patients with MPS II and iduronate sulfatase deficiency were recently described and evidence was presented suggesting autosomal inheritance (Neufeld et al. 1977). Further investigations in the present case showed normal V ~ s u l f a t eincorporation in a later subculture grown from the same amniotic fluid and a relatively low iduronate sulfatase activity in cell-free amniotic fluid. Conclusive evidence for heterozygosity for X-linked MPS 11 was obtained by the demonstration of both normal and mutant cells in clonal analysis of skin fibroblasts of the girl after birth. A majority (7470) of the clones were of the mutant phenotype. In the mother 18 70 of the clones showed abnormal 3%-sulfate incorporation. As demonstrated here and previously by Danes & Bearn (1967), Capobianchi & Romeo (1976), Migeon et al. (1977), heterozygosity for X-linked MPS 11 can be established by clonal analysis of cultured fibroblasts or by the recently reported method of hair-root analysis (Fluharty et al. 1977, Nwokoro et al. 1977). However both methods are time-consuming and probably not sufficiently reliable to exclude heterozygcsity. Prenatal diagnosis should therefore be offered not only to obligate carriers of MPS I1 but also to possible carriers. The present observations suggest that if
abnormal biochemical results are found in a female fetus in a pregnancy of a possible carrier for MPS 11, clonal analysis of the amniotic fluid cells should be carried out to exclude the presumed autosomal form of MPS II. Acknowledgments
The authors are grateful to Mrs. G . Hensing-Wolffers, Mrs. A. M. Zandvoort-Waayers and Miss Th. Zwaanenburg for performing cell cultures and analyses. This study was supported in part by “Het Praeventiefonds”, The Hague, The Netherlands. References
Bach, G., F. Eisenberg, M. Cantz & E. F. Neufeld (1973). The defect in the Hunter syndrome: Deficiency of suIfoiduronate sulfatase. Proc. nut. Acad. Sci. (Wash) 70, 2134-2138. Capobianchi, M. R. & G. Romeo (1976). Mosaicism for sulfoiduronate sulfatase deficiency in carriers of Hunter’s syndrome. Experientb 32, 459460. Coppa, G. B., J. Singh, B. L. Nichols & N. DiFerrante (1973). Urinary excretion of disulfated disaccharides in Hunter syndrome: Correction by infusion of a serum fraction. Anal. Lett. 6, 225-233. Danes, B. S. & A. G . Bearn (1967). Hunter’s syndrome: A genetic study of clones in cell culture with particular reference to the Lyon hypothesis. J . exp. Med. 126, 509-522. Epstein, C. J., S. Yatziv, E. F. Neufeld & I. Liebaers (1976). Genetic counseling for Hunter syndrome. Lancet ii, 737-738. Fluharty, A. L., T. Yutaka, R. L. Stevens & H. Kihara (1977). Hair-root analysis of sulfoiduronate sulfatase in a Hunter syndrome heterozygote. Amer. J . hum. Genet. 29, 43A. Fortuin, J. J. H. & W. J. Kleijer (1978). Pericellular glycosaminiglycans in cultured human cells. A possible source of error in prenatal diagnosis of mucopolysaccharidoses. Clin. Chim. Acta 82, 79-83. Fratantoni, J. C. E. F. Neufeld, W. B. Uhlendorf & C. B. Jacobson (1969). Intrauterine
P R E N A T A L D I A G N O S I S OF T H E H U N T E R S Y N D R O M E
diagnosis of the Hurler and Hunter syndromes. N e w Engl. J. Med. 280, 686-688. Hall, C. W., I. Liebaers, P. DiNatale & E. F. Neufeld (1978). Enzymic diagnosis of the genetic mucopolysaccharide storage disorders. Methods in Enzymology 50, 439-456. Hobolth, N. & C . Pedersen (1978). Six cases of a mild form of the Hunter syndrome in five generations. Three affected males with progeny. Clin. Genet. 13, 121. Liehaers, I., P. DiNatale & E. F. Neufeld (1977). Iduronate sulfatase in amniotic fluid: An aid in the prenatal diagnosis of the Hunter syndrome. J . Pediat. 90, 423-425. McKusick, V. A. (1975). Mendelian Inheritance in Man, 4th Ed. John Hopkins University Press, p. 643. Migeon, B. R., J. A. Sprenkle, I. Liebaers, J. F. Scott & E. F. Neufeld (1977). X-linked Hunter syndrome: The heterozygous phenotype in cell culture. Amer. J . hum. Genet. 29, 448-454. Neufeld, E. F., I. Liebars, C. J. Epstein, S. Yatziv, A. Milunsky & B. R. Migeon (1977). The Hunter syndrome in females: Is there
117
an autosornal recessive form of iduronate sulfatase deficiency? Amer. J . hum. Genet. 29, 455-461. Niermeijer, M. F., E. S. Sachs, M. Jahodova, C. Tichelaar-Klepper, W. J. Kleijer & H. Galjaard (1976). Prenatal diagnosis of genetic disorders. J . med. Genet. 13, 182-194. Nwokoro, N., I. Liebaers & E. F. Neufeld (1977). Iduronate sulfatase activity in hair roots of Hunter heterozygotes. Amer. J . hum. Genet. 29, 82A. Sjoberg, I., L. A. Fransson, R. Matalon & A. Dorfrnan (1973). Hunter’s syndrome: A deficiency of L-iduronosulfatase. Biochem. Biophys. Res. Commun. 54, 1125-1132.
Address: Dr. W . J . Kleijer Department of Cell Biology & Genetics Erasmus University P. 0. Box 1738 Rotterdam The Netherlands
