J. Inher. Metab. Dis. 15 (1992) 342-346 9 SSIEM and KluwerAcademicPublishers. Printed in the Netherlands
Short Communication
Gene Diagnosis and Carrier Detection in Hunter Syndrome by the Iduronate2-Sulphatase cDNA Probe A. GAL l, M. BECK2, A. C. SEWELL3, C. P. MORRIS4, E. SCHWINGER1 and J. J. HoPWOOD4 llnstitut ffir Humangenetik der Medizinischen Universit~t, Ratzeburger Allee 160, D-2400 Liibeck 1, Germany; 2Kinderklinik der Universitgt, Mainz, Germany; 3Kinderklinik der Universitdt, Frankfurt, Germany," 4L ysosomal Diseases Research Unit, Department of Chemical Pathology, Adelaide Children's Hospital, Adelaide, Australia Hunter disease (McKusick 309900) is an X-chromosomal mucopolysaccharidosis due to deficiency of the lysosomal enzyme iduronate-2-sulphatase (IDS; EC 3.1.6.13). Diagnosis is based on both the typical clinical features of patients and the lack/reduction of IDS activity. Female carriers show no symptoms of the disease. In the past, several different assays were elaborated for measuring enzyme activity in carriers but none of them proved to be suitable for detecting heterozygotes reliably (Zlotogora and Bach 1984). Accordingly, accurate genetic counselling of at-risk female relatives of patients was not possible. The IDS locus has been assigned to Xq28 by both physical and genetic mapping studies. The availability of polymorphic DNA markers, the loci of which showed close linkage with the IDS locus (for summary see Mandel et al 1989), made possible an indirect genotype analysis and, consequently, a more reliable estimation of the carrier risk. Recently, a cDNA clone including the entire coding region of human IDS mRNA has been isolated (Wilson et al 1990). Using this cDNA probe, two intragenic restriction fragment length polymorphisms (RFLP) have been detected (Suthers et al 1991) that now allow a highly accurate genotype diagnosis and carrier detection in informative families. In this paper, we report the results of our analysis using the IDS cDNA probe in two families. In one of them we could follow the segregation of the defective IDS allele over three generations. The data obtained suggest that in that family the mutation arose in male meiosis. PATIENTS A N D METHODS The pedigree of the family of patient 1 is shown in Figure 1A. The patient (IV.2) is the only child of healthy non-consanguineous parents. In the first 2 years of life the child's development was normal. Later he had recurrent middle-ear infections and a herniotomy had to be performed at the age of 15 months. No IDS activity could be 342
343
Gene Diagnosis in Hunter Syndrome
A
((tl[~'
I
(T)L~Jtf [ III
~a) (~aa L~~Tt
f,~AA ~Jtt
L~ ~. Jtt ~
A
_ 3
7-9
IV
Figure 1 (A) Pedigree of family 1 and segregation of the RFLP alleles (a/A and t/T) detected by the IDS cDNA probe after digestion of genomic DNA by StuI and TaqI, respectively. (B) Hybridization pattern seen after StuI digestion in three control females and (C) TaqI digestion in patient 2 (left) and a control female (right). The size of DNA fragments is given in kilobases. I> marks the allelic StuI and TaqI fragments and * the altered-size TaqI fragment
J. Inher. Metab. Dis. 15 (1992)
344
Gal et al.
detected in skin fibroblasts. Presently, at the age of 8 years, typical clinical features of Hunter disease are evident. Mental development is mildly retarded, the boy understands quite well and can speak a few words. A younger brother (111.5) of the patient's mother was repolted to be affected by the same disorder and died several years ago. An enzymatic diagnosis in this latter patient has not been performed. In patient 2 the diagnosis of Hunter disease was ascertained at the age of 3 years by the lack of IDS activity in fibroblasts. At the age of 10 years he shows typical features of Hunter disease and a severe mental retardation. A maternal uncle of the patient was living in an institution for the handicapped and died at the age of 26 years. Both patients 1 and 2 and the 12 others studied here were diagnosed by and are now under the care of one of us (M.B.). DNA studies were performed according to standard protocols and essentially as previously described (Gal et al 1985). For details on the IDS cDNA probe used and the R F L P detected, see Wilson et al (1990) and Suthers et al (1991), respectively.
RESULTS The DNA of a total of 14 patients with Hunter syndrome was studied following digestion by the restriction enzymes StuI, TaqI, or PstI and Southern hybridization with the IDS cDNA probe. In two patients a complete deletion of the IDS coding sequence (Gal et al 1991) and in a third patient an altered TaqI hybridization pattern (see below) were revealed. In the remaining nine patients examined in this study, including the one shown in Figure 1A, the autoradiographic patterns obtained were similar to those found in controls (data not shown). The cousin (IV.l) of the patient (IV.2) shown in Figure 1A asked for genetic counselling and wanted to know her risk of being a carrier. Since the molecular defect of the IDS gene has not yet been identified in this family and, hence, a direct genotype analysis can not be offered, a segregation analysis was performed using the RFLPs detected by the IDS cDNA probe at the Hunter locus. After either StuI or TaqI digestion of genomic DNA, a two-allele R F L P can be revealed (Suthers et al 1991; designated here as A (17.8 kb) and a (15.0 + 2.8) for StuI and T (5.1) and t (3.8) for TaqI, respectively; Figures 1B and 1C). Since the patient's X chromosome - - with the IDS mutation - - carries the StuI IDS allele a, and the maternal great-grandmother (I.2) of the patient shows the StuI alleles A, one can conclude that, most probably, 1.2 is not a carrier. However, based on pedigree information, it is obvious that the maternal grandmother (II.2) of the patient is a carrier for the IDS defect. Taken together these suggest that the mutation of the IDS gene arose during the spermiogenesis of the great-grandfather (I.1). Since the maternal X chromosome of the patient's aunt (III.2), the mother of our counsellee (IV.l), carries the StuI allele a, as is the case in the patient's mother (III.4), these data indicate that III.2 is also a carrier. Under the assumption that the mutation occurred in the paternal meiosis, one can also infer that the carrier risk of the grandmother's sisters is very low. Since the mother (III.2) of our counsellee is homozygote for the StuI RFLP, the segregation of the mutant IDS allele cannot be followed by this RFLP. However, J. Inher. Metab. Dis. 15 (1992)
Gene Diagnosis in Hunter Syndrome
345
since 111.2 is most probably a carrier and since she is heterozygous for the TaqI RFLP at the IDS locus, we can use this latter intragenic RFLP for genotype analysis. Indeed, Ill.2 has passed on her maternal TaqI IDS allele (t) to her daughter (IV.I), i.e. the carrier risk of the counsellee must be very high. In patient 2, Southern hybridization reveals an altered TaqI restriction pattern (Figure 1C); the 1.7-kb fragment is missing and an additional fragment migrating somewhat slower than the alMic 5.1-kb fragment appears. The hybridization patterns obtained after PstI or StuI digestion were identical to those seen in controls (data not shown). Nevertheless, the fact that the altered-size TaqI fragment can also be detected in the mother's DNA, together with the observation that the relative intensity of the 1.7-kb fragment was reduced in her DNA (data not shown), suggests that the mother carries the same structural rearrangement as the patient. DISCUSSION We show that molecular studies can provide valuable information for genetic counselling of at-risk relatives of patients with Hunter syndrome. The distinctive advantage of an indirect genotype analysis is that it can be offered before the molecular defect has been identified in the family seeking advice. However, in case of the IDS eDNA probe, even by scoring for both the StuI and TaqI RFLP, only about half of the women will be heterozygous for at least one of them, which is a prerequisite of applying this method. The exact nature of the alteration of the IDS gene of patient 2 is unknown. Most likely it is a point mutation or a small deletion destroying/removing a TaqI restriction site. Although it remains to be proved that the rearrangement detected is the primary cause of the disease in that family, for diagnostic purposes the novel fragment can be considered as an intragenic RFLP marker and used for tracing the segregation of the defect allele in the family. In about 10-20% of the patients studied so far a complete deletion of the IDS coding sequences has been detected (Gal et al 1991; Wilson et al 1991; Wraith et al 1991). In these families, too, segregation analysis by intragenic RFLPs can be more helpful than a quantitative evaluation of the autoradiogram after Southern hybridization (Beck et al 1992), which is often unreliable. For diagnostic purposes, the superiority of intragenic RFLPs to flanking marker loci is due to the negligible likelihood of a meiotic recombination in the case of the former. However, since the size of the IDS gene is not yet known, and since reciprocal genetic exchanges seem to occur frequently in distal Xq, recombinations within the IDS gene cannot be completely ruled out at present. And finally, if a 'key person' in the family to be studied is homozygous for the intragenic IDS RFLPs, flanking RFLP loci closely linked to the IDS locus in Xq28 should be tested in addition. In family 1 our data suggest that the mutation in the IDS gene responsible for the disease occurred during spermiogenesis of the patient's great-grandfather. Interestingly, in two other families with Hunter syndrome studied so far, it was also suggested that the mutation happened in male meioses (Bakker et al 1991; Beck et al 1992). Whether the mutation rate in Hunter syndrome is indeed higher in males than in females cannot be answered yet and the analysis of additional families is needed J. lnher. Metab. Dis. 15 (1992)
346
Gal et al.
before a solid conclusion can be drawn. Similarly to other X-chromosomal disorders, male gonadal mosaicism can also account for such a phenomenon. In the past great efforts were made to reliably detect carriers by enzyme assays. Presently, molecular analysis of the IDS gene and/or indirect genotype analysis by appropriate D N A probes represents a reliable and easy method for genetic counselling of at-risk females among relatives of Hunter patients and for prenatal diagnosis of this X-linked condition. ACKNOWLEDGEMENTS
We thank the G e r m a n MPS Society for support and the patients' families for cooperation. This study was financially supported by the Deutsche Forschungsgemeinschaft (Ga 210/2-4 and Be 932/2-3). REFERENCES
Bakker E, Kneppers ALJ, Deutz-Terlouw PP et al (1991) Partial IDS gene deletions in 3 out of 12 Dutch Hunter patients: direct and indirect carrier detection by DNA analysis. (Abstract). Eleventh International Workshop on Human Gene Mapping (HGMll). Cytogenet Cell Genet 58: 2055. Beck M, Steglich C, Zabel Bet al (1992) Deletion of the Hunter gene and both DXS466 and DXS304 in a patient with mucopolysaceharidosis type II. Am J Med Genet (in press). Gal A, Miicke J, Theile H, Wieacker PF, Ropers H-H, Wienker TF (1985) X-linked dominant Charcot-Marie-Tooth disease: Suggestion of linkage with a cloned DNA sequence from the proximal Xq. Hum Genet 70: 38-42. Gal A, Steglich C, Beck M, Morris CP, Schwinger E, Hopwood JJ (1991) Deletion of the iduronate-2-sulfatase gene and DXS304 in a patient with Hunter syndrome. (Abstract). Eleventh International Workshop on Human Gene Mapping (HGMll). Cytogenet Cell Genet 58: 2064. Mandel J-L, Willard HF, Nussbaum RL, Romeo G, Puck JM, Davies KE (1989) Report of the committee on the genetic constitution of the X chromosome. Tenth International Workshop on Human Gene Mapping (HGM10). Cytogenet Cell Genet 51: 384-437. Suthers GK, Oberle I, Nancarrow J et al (1991) Genetic mapping of new RFLPs at Xq27q28. Genomics 9: 37-43. Wilson PJ, Morris CP, Anson DS et al (1990) Hunter syndrome: Isolation of an iduronate2-sulfatase eDNA clone and analysis of patient DNA. Proc Natl Acad Sci USA 87: 8531-8535. Wilson PJ, Suthers GK, Callen DF et al (1991) Frequent deletions at Xq28 indicate genetic heterogeneity in Hunter syndrome. Hum Genet 86: 505-508. Wraith JE, Cooper A, Thornley M e t al (1991) The clinical phenotype of two patients with a complete deletion of the iduronate-2-sulfatase gene (mucopolysaccharidosis II - - Hunter syndrome). Hum Genet 87: 205-206. Zlotogora J, Bach G (1984) Heterozygote detection in Hunter syndrome. Am J Med Genet 17: 661-665.
J, Inher. Metab. Dis. 15 (1992)
Short Communication
Gene Diagnosis and Carrier Detection in Hunter Syndrome by the Iduronate2-Sulphatase cDNA Probe A. GAL l, M. BECK2, A. C. SEWELL3, C. P. MORRIS4, E. SCHWINGER1 and J. J. HoPWOOD4 llnstitut ffir Humangenetik der Medizinischen Universit~t, Ratzeburger Allee 160, D-2400 Liibeck 1, Germany; 2Kinderklinik der Universitgt, Mainz, Germany; 3Kinderklinik der Universitdt, Frankfurt, Germany," 4L ysosomal Diseases Research Unit, Department of Chemical Pathology, Adelaide Children's Hospital, Adelaide, Australia Hunter disease (McKusick 309900) is an X-chromosomal mucopolysaccharidosis due to deficiency of the lysosomal enzyme iduronate-2-sulphatase (IDS; EC 3.1.6.13). Diagnosis is based on both the typical clinical features of patients and the lack/reduction of IDS activity. Female carriers show no symptoms of the disease. In the past, several different assays were elaborated for measuring enzyme activity in carriers but none of them proved to be suitable for detecting heterozygotes reliably (Zlotogora and Bach 1984). Accordingly, accurate genetic counselling of at-risk female relatives of patients was not possible. The IDS locus has been assigned to Xq28 by both physical and genetic mapping studies. The availability of polymorphic DNA markers, the loci of which showed close linkage with the IDS locus (for summary see Mandel et al 1989), made possible an indirect genotype analysis and, consequently, a more reliable estimation of the carrier risk. Recently, a cDNA clone including the entire coding region of human IDS mRNA has been isolated (Wilson et al 1990). Using this cDNA probe, two intragenic restriction fragment length polymorphisms (RFLP) have been detected (Suthers et al 1991) that now allow a highly accurate genotype diagnosis and carrier detection in informative families. In this paper, we report the results of our analysis using the IDS cDNA probe in two families. In one of them we could follow the segregation of the defective IDS allele over three generations. The data obtained suggest that in that family the mutation arose in male meiosis. PATIENTS A N D METHODS The pedigree of the family of patient 1 is shown in Figure 1A. The patient (IV.2) is the only child of healthy non-consanguineous parents. In the first 2 years of life the child's development was normal. Later he had recurrent middle-ear infections and a herniotomy had to be performed at the age of 15 months. No IDS activity could be 342
343
Gene Diagnosis in Hunter Syndrome
A
((tl[~'
I
(T)L~Jtf [ III
~a) (~aa L~~Tt
f,~AA ~Jtt
L~ ~. Jtt ~
A
_ 3
7-9
IV
Figure 1 (A) Pedigree of family 1 and segregation of the RFLP alleles (a/A and t/T) detected by the IDS cDNA probe after digestion of genomic DNA by StuI and TaqI, respectively. (B) Hybridization pattern seen after StuI digestion in three control females and (C) TaqI digestion in patient 2 (left) and a control female (right). The size of DNA fragments is given in kilobases. I> marks the allelic StuI and TaqI fragments and * the altered-size TaqI fragment
J. Inher. Metab. Dis. 15 (1992)
344
Gal et al.
detected in skin fibroblasts. Presently, at the age of 8 years, typical clinical features of Hunter disease are evident. Mental development is mildly retarded, the boy understands quite well and can speak a few words. A younger brother (111.5) of the patient's mother was repolted to be affected by the same disorder and died several years ago. An enzymatic diagnosis in this latter patient has not been performed. In patient 2 the diagnosis of Hunter disease was ascertained at the age of 3 years by the lack of IDS activity in fibroblasts. At the age of 10 years he shows typical features of Hunter disease and a severe mental retardation. A maternal uncle of the patient was living in an institution for the handicapped and died at the age of 26 years. Both patients 1 and 2 and the 12 others studied here were diagnosed by and are now under the care of one of us (M.B.). DNA studies were performed according to standard protocols and essentially as previously described (Gal et al 1985). For details on the IDS cDNA probe used and the R F L P detected, see Wilson et al (1990) and Suthers et al (1991), respectively.
RESULTS The DNA of a total of 14 patients with Hunter syndrome was studied following digestion by the restriction enzymes StuI, TaqI, or PstI and Southern hybridization with the IDS cDNA probe. In two patients a complete deletion of the IDS coding sequence (Gal et al 1991) and in a third patient an altered TaqI hybridization pattern (see below) were revealed. In the remaining nine patients examined in this study, including the one shown in Figure 1A, the autoradiographic patterns obtained were similar to those found in controls (data not shown). The cousin (IV.l) of the patient (IV.2) shown in Figure 1A asked for genetic counselling and wanted to know her risk of being a carrier. Since the molecular defect of the IDS gene has not yet been identified in this family and, hence, a direct genotype analysis can not be offered, a segregation analysis was performed using the RFLPs detected by the IDS cDNA probe at the Hunter locus. After either StuI or TaqI digestion of genomic DNA, a two-allele R F L P can be revealed (Suthers et al 1991; designated here as A (17.8 kb) and a (15.0 + 2.8) for StuI and T (5.1) and t (3.8) for TaqI, respectively; Figures 1B and 1C). Since the patient's X chromosome - - with the IDS mutation - - carries the StuI IDS allele a, and the maternal great-grandmother (I.2) of the patient shows the StuI alleles A, one can conclude that, most probably, 1.2 is not a carrier. However, based on pedigree information, it is obvious that the maternal grandmother (II.2) of the patient is a carrier for the IDS defect. Taken together these suggest that the mutation of the IDS gene arose during the spermiogenesis of the great-grandfather (I.1). Since the maternal X chromosome of the patient's aunt (III.2), the mother of our counsellee (IV.l), carries the StuI allele a, as is the case in the patient's mother (III.4), these data indicate that III.2 is also a carrier. Under the assumption that the mutation occurred in the paternal meiosis, one can also infer that the carrier risk of the grandmother's sisters is very low. Since the mother (III.2) of our counsellee is homozygote for the StuI RFLP, the segregation of the mutant IDS allele cannot be followed by this RFLP. However, J. Inher. Metab. Dis. 15 (1992)
Gene Diagnosis in Hunter Syndrome
345
since 111.2 is most probably a carrier and since she is heterozygous for the TaqI RFLP at the IDS locus, we can use this latter intragenic RFLP for genotype analysis. Indeed, Ill.2 has passed on her maternal TaqI IDS allele (t) to her daughter (IV.I), i.e. the carrier risk of the counsellee must be very high. In patient 2, Southern hybridization reveals an altered TaqI restriction pattern (Figure 1C); the 1.7-kb fragment is missing and an additional fragment migrating somewhat slower than the alMic 5.1-kb fragment appears. The hybridization patterns obtained after PstI or StuI digestion were identical to those seen in controls (data not shown). Nevertheless, the fact that the altered-size TaqI fragment can also be detected in the mother's DNA, together with the observation that the relative intensity of the 1.7-kb fragment was reduced in her DNA (data not shown), suggests that the mother carries the same structural rearrangement as the patient. DISCUSSION We show that molecular studies can provide valuable information for genetic counselling of at-risk relatives of patients with Hunter syndrome. The distinctive advantage of an indirect genotype analysis is that it can be offered before the molecular defect has been identified in the family seeking advice. However, in case of the IDS eDNA probe, even by scoring for both the StuI and TaqI RFLP, only about half of the women will be heterozygous for at least one of them, which is a prerequisite of applying this method. The exact nature of the alteration of the IDS gene of patient 2 is unknown. Most likely it is a point mutation or a small deletion destroying/removing a TaqI restriction site. Although it remains to be proved that the rearrangement detected is the primary cause of the disease in that family, for diagnostic purposes the novel fragment can be considered as an intragenic RFLP marker and used for tracing the segregation of the defect allele in the family. In about 10-20% of the patients studied so far a complete deletion of the IDS coding sequences has been detected (Gal et al 1991; Wilson et al 1991; Wraith et al 1991). In these families, too, segregation analysis by intragenic RFLPs can be more helpful than a quantitative evaluation of the autoradiogram after Southern hybridization (Beck et al 1992), which is often unreliable. For diagnostic purposes, the superiority of intragenic RFLPs to flanking marker loci is due to the negligible likelihood of a meiotic recombination in the case of the former. However, since the size of the IDS gene is not yet known, and since reciprocal genetic exchanges seem to occur frequently in distal Xq, recombinations within the IDS gene cannot be completely ruled out at present. And finally, if a 'key person' in the family to be studied is homozygous for the intragenic IDS RFLPs, flanking RFLP loci closely linked to the IDS locus in Xq28 should be tested in addition. In family 1 our data suggest that the mutation in the IDS gene responsible for the disease occurred during spermiogenesis of the patient's great-grandfather. Interestingly, in two other families with Hunter syndrome studied so far, it was also suggested that the mutation happened in male meioses (Bakker et al 1991; Beck et al 1992). Whether the mutation rate in Hunter syndrome is indeed higher in males than in females cannot be answered yet and the analysis of additional families is needed J. lnher. Metab. Dis. 15 (1992)
346
Gal et al.
before a solid conclusion can be drawn. Similarly to other X-chromosomal disorders, male gonadal mosaicism can also account for such a phenomenon. In the past great efforts were made to reliably detect carriers by enzyme assays. Presently, molecular analysis of the IDS gene and/or indirect genotype analysis by appropriate D N A probes represents a reliable and easy method for genetic counselling of at-risk females among relatives of Hunter patients and for prenatal diagnosis of this X-linked condition. ACKNOWLEDGEMENTS
We thank the G e r m a n MPS Society for support and the patients' families for cooperation. This study was financially supported by the Deutsche Forschungsgemeinschaft (Ga 210/2-4 and Be 932/2-3). REFERENCES
Bakker E, Kneppers ALJ, Deutz-Terlouw PP et al (1991) Partial IDS gene deletions in 3 out of 12 Dutch Hunter patients: direct and indirect carrier detection by DNA analysis. (Abstract). Eleventh International Workshop on Human Gene Mapping (HGMll). Cytogenet Cell Genet 58: 2055. Beck M, Steglich C, Zabel Bet al (1992) Deletion of the Hunter gene and both DXS466 and DXS304 in a patient with mucopolysaceharidosis type II. Am J Med Genet (in press). Gal A, Miicke J, Theile H, Wieacker PF, Ropers H-H, Wienker TF (1985) X-linked dominant Charcot-Marie-Tooth disease: Suggestion of linkage with a cloned DNA sequence from the proximal Xq. Hum Genet 70: 38-42. Gal A, Steglich C, Beck M, Morris CP, Schwinger E, Hopwood JJ (1991) Deletion of the iduronate-2-sulfatase gene and DXS304 in a patient with Hunter syndrome. (Abstract). Eleventh International Workshop on Human Gene Mapping (HGMll). Cytogenet Cell Genet 58: 2064. Mandel J-L, Willard HF, Nussbaum RL, Romeo G, Puck JM, Davies KE (1989) Report of the committee on the genetic constitution of the X chromosome. Tenth International Workshop on Human Gene Mapping (HGM10). Cytogenet Cell Genet 51: 384-437. Suthers GK, Oberle I, Nancarrow J et al (1991) Genetic mapping of new RFLPs at Xq27q28. Genomics 9: 37-43. Wilson PJ, Morris CP, Anson DS et al (1990) Hunter syndrome: Isolation of an iduronate2-sulfatase eDNA clone and analysis of patient DNA. Proc Natl Acad Sci USA 87: 8531-8535. Wilson PJ, Suthers GK, Callen DF et al (1991) Frequent deletions at Xq28 indicate genetic heterogeneity in Hunter syndrome. Hum Genet 86: 505-508. Wraith JE, Cooper A, Thornley M e t al (1991) The clinical phenotype of two patients with a complete deletion of the iduronate-2-sulfatase gene (mucopolysaccharidosis II - - Hunter syndrome). Hum Genet 87: 205-206. Zlotogora J, Bach G (1984) Heterozygote detection in Hunter syndrome. Am J Med Genet 17: 661-665.
J, Inher. Metab. Dis. 15 (1992)
