© 1992 S. Karger AG, Basel 0301-0171 /92/0604-0208S2.75/0
Cytogenet Cell Genet 60:208-209 ( 1992)
Assignment of the aspartylglucosaminidase gene (AGA) to 4q33—>q35 based on decreased activity in a girl with a 46,XX,del(4)(q33) karyotype J. Engelen,1A. Hamers,1C. Schrander-Stumpel,1 H. Mulder,2 and B. Poorthuis3 1Department of Molecular Cell Biology and Genetics, University of Limburg. M aastricht.2Department of Pediatrics, Jans Gasthuis. Weert. and 3 Department of Pediatrics, University of Leiden. Leiden (The Netherlands)
Abstract. Aspartylglucosaminuria (AGU) is a recessive autosomally inherited lysosomal storage disorder due to deficiency of the enzyme aspartylglucosaminidase (AGA). The structural gene for this human enzyme (AGA) has been assigned to the region 4q21->qter. We determined the AGA activity in
The lysosomal enzyme aspartylglucosaminidase (1-aspartamido-P-N-acetylglucosamine amidohydrolase. E.C.3.5.1.26: AGA) cleaves the N-acetylglucosamine-asparagine linkage of oligosaccharide chains in a variety of glycoproteins and glycopeptides (Makino et al„ 1966. 1968: Komfeld and Kornfeld. 1976). A deficiency of aspartylglucosaminidase causes aspartylglu cosaminuria (AGU). a rare disorder of glycoprotein metabo lism. AGU is inherited as an autosomal recessive trait and occurs in relatively high frequency in individuals of Finnish ori gin (Aula et al„ 1982). The structural gene for the human lysosomal enzyme AGA has been assigned to the chromosome region 4q21 qter. using somatic cell hybridization techniques (Aula et a!., 1984). We report on a girl with a 46,XX.del(4)(q33) karyotype and an AGA enzyme activity in fibroblasts in the range of obligate heterozy gotes.
Materials and methods The patient was referred for diagnostic examination at the age of 4 months because of developmental retardation and generalised hypotonia, splenomegalia and slight hepatomegalia. epicanthal folds, hypoplasticalac nasi, macroglossia. and high small palate. Chromosome analysis in cultured lymphocytes revealed a deletion of the terminal portion of the long arm of chromosome 4 (Fig. 1). The karyotype could be delineated as 46.XX.del(4)(q33). Both parents showed normal karyotypes.
cultured fibroblasts of a girl with a 46,XX.del(4)(q33) karyo type. The results indicate that the girl is a hemizygote for AGA. permitting the assignment of human AGA to the region 4q33-»qter.
4
del ( 4 ) (q 3 3 )
Fig. I. Chromosome pair No. 4 in a girl with a 46.XX.del(4)(q33) chromo some composition. Normal (left) and abnormal (right) G-banded chromo somes and their idiograms.
For enzyme assays, a skin biopsy was obtained from the patient and her parents. Fibroblasts were cultured by routine techniques using Medium-FlO enriched with 10% fetal bovine scrum. Cells were passed at least five times after primary growth. Aspartylglucosaminidase activity was measured against 2-acclamido-1 -()-(L.-aspartamido)-1,2-dideoxy-p-D-glucose according to the method of Aula et al. (1973, 1974). AGA activity is expressed as nmol of N-acetylglucosamine liberated per min per g of protein.
Received 30 October 1991: accepted 23 January 1992. Request reprints from J.J.M. Engelen. Department of Molecular Cell Biology and Genetics, University of Limburg. P.O. Box 616.6200 MD Maastricht (The Nether lands).
The activity of aspartylglucosaminidase in extracts of skin Fibroblasts of our patient has a mean of 32.5 ± 15 and her par ents 145.6 ± 28.5 nmol/min/g protein, respectively (Table 1). The AGA activities of cell lines of AGU siblings and normal
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/10/2018 9:36:50 AM
Results and discussion
209
Assignment of the human aspartylglucosaminidase gene (AGA)
controls are also given in Table I. In the interpretation of their results, the discrimination line between normals and heterozy gotes was set by Aula et al. (1974) at 58 nmol/min/g protein (mean + 2 SD of obligate hetcrozygotes). For an individual with an activity level of this value or higher, the probability of a false negative heterozygote diagnosis is 2.5% or less. According to this interpretation our patient’s cell line indicates heterozygos ity for AGA. The gene locus of the AGA enzyme has been physically map ped to the region 4q21-» qter using somatic cell hybrids (Aula et al., 1984). Linkage data analysis in AGU families tentatively placed the locus of AGA to the distal end of chromosome 4. the most probable gene order being: cen-ADH-EGF-FG-MNSAGA (Grdn et al., 1990). Recently, Halal et al. (1991) suggested a possible assignment of the AGA gene to 4q23-»q27 based on an increased AGA activity (161 nmol/min/g protein) in a patient with an intersti tial duplication of 4q. However, our data, the data of Aula et al. (1974), and results of a recent unpublished series of Aula (per-
T able I. A spartylglucosam inidase (AGA) activity in fibroblasts o f the p atien t, her parents, an d controls Subject
AGA activity (nmol/min/g protein)
Patient Mother Father Normals (our laboratory) Heterozygotes (our laboratory) Normals (Aula ct al.. 1974) Heterozygotes (Aula et al.. 1974) Patients (Aula ct al., 1974)
32.5± 15 125.7 165.5 130 ±35 48 + 35 152 + 62 26+ 16 3.3 ±3.3
sonal communication) indicate that an AGA activity of 161 nmol/min/g protein is within the normal range. In conclusion, the chromosomal deletion of 4q33—»qter and the heterozygous value of AGA in our patient justify the assign ment of the gene locus to the region 4q 33-»qter.
References Aula P, Nanto V. Laipio M. Autio S: Aspartylglucosam inuria; deficiency of aspartylglucosaminidase in cultured fibroblasts of patients and their heterozy gous parents. Clin Genet 4:297-300 ( 1973). Halal F, Vekcmans M. Chitayat D: Interstitial tandem direct duplication of the long arm of chromosome 4 (q23-q27) and possible assignment of the structural gene encoding human aspartylglucosaminidase to this segment. Am J med Genet 39:418-421 (1991). Gron K, Aula P. Peltoonen L: Linkage of aspartylglucos aminuria (AGU) to marker loci on the long arm of chromosome 4. Hum Genet 85:233-236 (1990).
Kornfeld R. Kornfeld D: Comparative aspects of glyco protein structure. A Rev Biochem 45:217-237 (1976). Makino M. Kojima T. Ohgushi T. Yamashina I: Studies on enzvmes acting on glvcopeptides. J Biochem (Tokyo) 63:186-192 (1968). Makino M. Kojima T. Yamashina I: Enzymatic cleav age of glycopeptides. Biochem biophys Res Commun 24:961-966 (1966).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/10/2018 9:36:50 AM
Aula P. Astrin K. Francke U. Desnick R: Assignment of the structural gene encoding human aspartylglucos aminidase to the long arm of chromosome 4 (4q214qter). Am J hum Genet 36:1215-1224(1984). Aula P. Autio S. Raivio K. Nanto V: Detection of het erozygotes for aspartylglucosaminuria (AGU) in cultured fibroblasts. Humangenetik 25:307-314 (1974). Aula P. Autio S. Raivio K. Rapola P: Aspartylglucosam inuria. in Durand P. O'Brien J (eds): Genetic Errors in Glycoprotein Metabolism, pp 123-152. (Springer Verlag. Berlin 1982).
Cytogenet Cell Genet 60:208-209 ( 1992)
Assignment of the aspartylglucosaminidase gene (AGA) to 4q33—>q35 based on decreased activity in a girl with a 46,XX,del(4)(q33) karyotype J. Engelen,1A. Hamers,1C. Schrander-Stumpel,1 H. Mulder,2 and B. Poorthuis3 1Department of Molecular Cell Biology and Genetics, University of Limburg. M aastricht.2Department of Pediatrics, Jans Gasthuis. Weert. and 3 Department of Pediatrics, University of Leiden. Leiden (The Netherlands)
Abstract. Aspartylglucosaminuria (AGU) is a recessive autosomally inherited lysosomal storage disorder due to deficiency of the enzyme aspartylglucosaminidase (AGA). The structural gene for this human enzyme (AGA) has been assigned to the region 4q21->qter. We determined the AGA activity in
The lysosomal enzyme aspartylglucosaminidase (1-aspartamido-P-N-acetylglucosamine amidohydrolase. E.C.3.5.1.26: AGA) cleaves the N-acetylglucosamine-asparagine linkage of oligosaccharide chains in a variety of glycoproteins and glycopeptides (Makino et al„ 1966. 1968: Komfeld and Kornfeld. 1976). A deficiency of aspartylglucosaminidase causes aspartylglu cosaminuria (AGU). a rare disorder of glycoprotein metabo lism. AGU is inherited as an autosomal recessive trait and occurs in relatively high frequency in individuals of Finnish ori gin (Aula et al„ 1982). The structural gene for the human lysosomal enzyme AGA has been assigned to the chromosome region 4q21 qter. using somatic cell hybridization techniques (Aula et a!., 1984). We report on a girl with a 46,XX.del(4)(q33) karyotype and an AGA enzyme activity in fibroblasts in the range of obligate heterozy gotes.
Materials and methods The patient was referred for diagnostic examination at the age of 4 months because of developmental retardation and generalised hypotonia, splenomegalia and slight hepatomegalia. epicanthal folds, hypoplasticalac nasi, macroglossia. and high small palate. Chromosome analysis in cultured lymphocytes revealed a deletion of the terminal portion of the long arm of chromosome 4 (Fig. 1). The karyotype could be delineated as 46.XX.del(4)(q33). Both parents showed normal karyotypes.
cultured fibroblasts of a girl with a 46,XX.del(4)(q33) karyo type. The results indicate that the girl is a hemizygote for AGA. permitting the assignment of human AGA to the region 4q33-»qter.
4
del ( 4 ) (q 3 3 )
Fig. I. Chromosome pair No. 4 in a girl with a 46.XX.del(4)(q33) chromo some composition. Normal (left) and abnormal (right) G-banded chromo somes and their idiograms.
For enzyme assays, a skin biopsy was obtained from the patient and her parents. Fibroblasts were cultured by routine techniques using Medium-FlO enriched with 10% fetal bovine scrum. Cells were passed at least five times after primary growth. Aspartylglucosaminidase activity was measured against 2-acclamido-1 -()-(L.-aspartamido)-1,2-dideoxy-p-D-glucose according to the method of Aula et al. (1973, 1974). AGA activity is expressed as nmol of N-acetylglucosamine liberated per min per g of protein.
Received 30 October 1991: accepted 23 January 1992. Request reprints from J.J.M. Engelen. Department of Molecular Cell Biology and Genetics, University of Limburg. P.O. Box 616.6200 MD Maastricht (The Nether lands).
The activity of aspartylglucosaminidase in extracts of skin Fibroblasts of our patient has a mean of 32.5 ± 15 and her par ents 145.6 ± 28.5 nmol/min/g protein, respectively (Table 1). The AGA activities of cell lines of AGU siblings and normal
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/10/2018 9:36:50 AM
Results and discussion
209
Assignment of the human aspartylglucosaminidase gene (AGA)
controls are also given in Table I. In the interpretation of their results, the discrimination line between normals and heterozy gotes was set by Aula et al. (1974) at 58 nmol/min/g protein (mean + 2 SD of obligate hetcrozygotes). For an individual with an activity level of this value or higher, the probability of a false negative heterozygote diagnosis is 2.5% or less. According to this interpretation our patient’s cell line indicates heterozygos ity for AGA. The gene locus of the AGA enzyme has been physically map ped to the region 4q21-» qter using somatic cell hybrids (Aula et al., 1984). Linkage data analysis in AGU families tentatively placed the locus of AGA to the distal end of chromosome 4. the most probable gene order being: cen-ADH-EGF-FG-MNSAGA (Grdn et al., 1990). Recently, Halal et al. (1991) suggested a possible assignment of the AGA gene to 4q23-»q27 based on an increased AGA activity (161 nmol/min/g protein) in a patient with an intersti tial duplication of 4q. However, our data, the data of Aula et al. (1974), and results of a recent unpublished series of Aula (per-
T able I. A spartylglucosam inidase (AGA) activity in fibroblasts o f the p atien t, her parents, an d controls Subject
AGA activity (nmol/min/g protein)
Patient Mother Father Normals (our laboratory) Heterozygotes (our laboratory) Normals (Aula ct al.. 1974) Heterozygotes (Aula et al.. 1974) Patients (Aula ct al., 1974)
32.5± 15 125.7 165.5 130 ±35 48 + 35 152 + 62 26+ 16 3.3 ±3.3
sonal communication) indicate that an AGA activity of 161 nmol/min/g protein is within the normal range. In conclusion, the chromosomal deletion of 4q33—»qter and the heterozygous value of AGA in our patient justify the assign ment of the gene locus to the region 4q 33-»qter.
References Aula P, Nanto V. Laipio M. Autio S: Aspartylglucosam inuria; deficiency of aspartylglucosaminidase in cultured fibroblasts of patients and their heterozy gous parents. Clin Genet 4:297-300 ( 1973). Halal F, Vekcmans M. Chitayat D: Interstitial tandem direct duplication of the long arm of chromosome 4 (q23-q27) and possible assignment of the structural gene encoding human aspartylglucosaminidase to this segment. Am J med Genet 39:418-421 (1991). Gron K, Aula P. Peltoonen L: Linkage of aspartylglucos aminuria (AGU) to marker loci on the long arm of chromosome 4. Hum Genet 85:233-236 (1990).
Kornfeld R. Kornfeld D: Comparative aspects of glyco protein structure. A Rev Biochem 45:217-237 (1976). Makino M. Kojima T. Ohgushi T. Yamashina I: Studies on enzvmes acting on glvcopeptides. J Biochem (Tokyo) 63:186-192 (1968). Makino M. Kojima T. Yamashina I: Enzymatic cleav age of glycopeptides. Biochem biophys Res Commun 24:961-966 (1966).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 9/10/2018 9:36:50 AM
Aula P. Astrin K. Francke U. Desnick R: Assignment of the structural gene encoding human aspartylglucos aminidase to the long arm of chromosome 4 (4q214qter). Am J hum Genet 36:1215-1224(1984). Aula P. Autio S. Raivio K. Nanto V: Detection of het erozygotes for aspartylglucosaminuria (AGU) in cultured fibroblasts. Humangenetik 25:307-314 (1974). Aula P. Autio S. Raivio K. Rapola P: Aspartylglucosam inuria. in Durand P. O'Brien J (eds): Genetic Errors in Glycoprotein Metabolism, pp 123-152. (Springer Verlag. Berlin 1982).
