1473
band. Activity of this enzyme was increased in both patients (189 and 102 IU/1, normal 10-50) and similar to values in patients with CDG syndrome (40-112 IU/1, n = 10). A repeat analysis after 1 week of a galactose-free diet showed a normal isoenzyme pattern (data of patient 2 in figure) and normal activity. a-fucosidase had a normal activity but a similarly disturbed isoenzyme pattern with abnormal cathodal bands that normalised during treatment. These data are similar to those found in CDG syndromes (figure).4 Taken together with our findings of decreased serum TBG and abnormal sialotransferrin pattern, these results are a further argument for grouping classic galactosaemia as a CDG syndrome, albeit a secondary and treatable one. In serum glycoproteins of patients with CDG syndrome there is a partial deficiency of the terminal trisaccharide sialic acid, galactose, and Nacetylglucosamine.5 Sialic acid bears a negative electric charge and the deficiency of this carbohydrate residue explains the cathodal shift of sialotransferrins and other glycoproteins. We postulate that in classic galactosaemia the accumulated galactose-1-phosphate interferes with the carbohydrate processing at the Golgi galactosyltransferase step and leads to a partial deficiency of the terminal disaccharide (sialic acid and galactose) of glycoproteins such as transferrin, TBG, and the lysosomal enzymes. Defective galactosylation of proteins in cultured skin fibroblasts and deficiency of glycolipids containing galactose or Nacetylgalactosamine in brain and lymphocytes with classical galactosaemia patients has been reported.6,7 Classical galactosaemia and CDG syndromes are both multisystemic disorders affecting the nervous system, liver, gastrointestinal tract, kidneys, and gonads. We suggest that these glycoprotein abnormalities play a major part in the pathogenesis of classic galactosaemia that thus may be regarded as a secondary "Golgi disorder". Department of Paediatrics, University of Leuven, 3000 Leuven, Belgium
JAAK JAEKEN
Department of Paediatrics, University of Ghent,
Jos KINT
Department of Molecular Cell Biology and Genetics, University of Limburg, Maastricht, Netherlands
LEO SPAAPEN
Vulsma T, Theunissen PMVM, van der Meer SB, Jaeken J. Galactosaemia, a carbohydrate-deficient glycoprotein syndrome. Society for the Study of Inborn Errors of Metabolism, 30th Annual Symposium, Leuven, Sept 8-11, 1992 (abstr). 2. Jaeken J, Vanderschueren-Lodeweyckx M, Casaer P, et al. Familial psychomotor retardation with markedly fluctuating serum prolactin, FSH and GH levels, partial TBG deficiency, increased serum arylsulphatase A and increased CSF protein: a new syndrome? Pediatr Res 1980; 14: 179. 3. Jaeken J, Stibler H, Hagberg B. The carbohydrate-deficient glycoprotein syndrome. Acta Paediatr Scand 1991; suppl: 375. 4. Jaeken J, Kint J. Abnormal serum lysosomal isoenzymes in leukodystrophy with sialic add deficient proteins. Society for the Study of Inborn Errors of Metabolism, 25th Annual Symposium, Sheffield, Sept 22-25, 1987 (abstr). 5. Stibler H, Jaeken J. Carbohydrate deficient serum transferrin in a new systemic hereditary syndrome. Arch Dis Child 1990; 65: 107-11. 6. Omstein KS, McGuire EJ, Berry GT, Roth S, Segal S. Abnormal galactosylation of complex carbohydrates in cultured fibroblasts from patients with galactose-1phosphate uridyltransferase deficiency. Pediatr Res 1992; 31: 508-11. 7. Petry K, Greinix HT, Nudelman E, et al. Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes. Biochem Med Metab Biol 1991; 46: 93-104.
1. Spaapen LJM,
Nested PCR of PAH cDNA and sequence analysis of V388M.
Upper= PCR lane 1 =size marker, 2=first PCR with lymphoblast cDNA, 3=second PCR with lymphoblast cDNA, 4=first PCR with liver cDNA, and 5=second PCR with lymphoblast poly(A+) RNA without reverse transcription. PCR product was not detected after first PCR with lymphoblast cDNA. Negative control with lymphoblast poly (A+) RNA without reverse transcription proves no contamination of cDNA. Primer sequences for first PCR: 5’dAGGGAAACCTGCCTGTACGT, 3’dCTCCATCAACAGATTCACAGC, and for second PCR: 5’dAAAAGCCAGAGACCTCACTC, 3’dACAGACCACATTCTGTCCATG. can be detected by nested polymerase chain reaction (PCR).2-4 We used this method to analyse PAH mRNA from lymphoblasts of a Japanese phenylketonuria patient and identified a novel mutation. Poly (A + ) RNA was isolated from a patient-derived EpsteinBarr-virus-immortalised lymphoblastoid cell line. Single-stranded cDNA was synthesised by reverse transcription with oligo(dT) primer and subjected to nested PCR with two sets of primers to amplify the entire coding region of PAH cDNA. A DNA fragment of the predicted size (1416 bp) was obtained (figure). Sequencing analysis of the amplified DNA revealed two point mutations: a single base substitution of G to A at base 1384 that alters valine at codon 388 in exon 11 to methionine (figure), and another substitution of G to C at base 1460 that alters arginine at codon 413 in exon 12 to proline. The valine-to-arginine mutation (V388M) has not been reported, whereas the arginine-to-proline mutation (R413P) has been described.6 Analysis of genomic DNA of the patient as well as the parents showed that this patient is indeed a compound heterozygote of V388M and R413P, whereas the father is a heterozygote of V338M and the mother is a heterozygote of R413P. Our study demonstrates that PAH mRNA in a phenylketonuria patient can be analysed with lymphoblasts and there is no need for risking liver biopsy. The method is not only rapid and reliable but also advantageous for the analysis of compound heterozygotes, because each allele can be examined separately. Various types of mutations responsible for the disease have been reported by genomic analysis of the PAH gene. Despite extensive studies for nearly a decade, less than 50% of the mutations have been identified in most populations.’ The analysis of ectopically transcribed PAH mRNA would facilitate the discovery of unknown mutations.
Department of Biochemical Genetics, Tohoku University School of Medicine, Aoba-ku, Sendai 980, Japan
KAZUTOSHI TAKAHASHI SHIGEO KURE YOICHI MATSUBARA KUNIAKI NARISAWA
Novel phenylketonuria mutation detected by
analysis of ectopically transcribed phenylalanine hydroxylase mRNA from lymphoblast is one of the most common inherited of aminoacid metabolism and is caused by altered activity of phenylalanine hydroxylase (PAH). To date molecular analysis of phenylketonuria has focused on extensive sequencing of 13 exons in the PAH gene’ because it is believed that PAH mRNA is expressed only in liver, and biopsy or necropsy liver specimens are required for the analysis of mRNA. Sequencing of each exon is laborious. Extremely low concentrations of mRNA synthesised by "ectopic" or "illegitimate" transcription in non-expressing tissues
SIR,-Phenylketonuria
inborn
errors
RC, Woo SLC. Phenylketonuria and the phenylalanine hydroxylase gene. Mol Biol Med 1991; 8: 3-18. 2. Sarkar G, Sommer SS. Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 1989; 244: 331-34. 3. Chelly J, Concordet JP, Kaplan JC, Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Proc Natl Acad Sci USA 1989; 86: 2617-21. 4. Roberts RG, Bently DR, Barby TFM, Manners E, Bobrow M. Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA. Lancet 1990; 336: 1523-26. 5. Chomczynski P, Sacchi N. Single-step method of RNA isolation by add guanidium thiocyanate-phenol-chloroform extraction Anal Biochem 1987; 162: 156-59. 6. Wang T, Okano Y, Eisensmith R, et al. Molecular genetics of PKU in onentals. Am J Hum Genet 1989; 45: A228. 7. Konecki DS, Lichter-Konecki U. The phenylketonuria locus: current knowledge about alleles and mutations of the phenylalanine hydroxylase gene in various populations. Hum Genet 1991; 87: 377-88. 1. Eisensmith
band. Activity of this enzyme was increased in both patients (189 and 102 IU/1, normal 10-50) and similar to values in patients with CDG syndrome (40-112 IU/1, n = 10). A repeat analysis after 1 week of a galactose-free diet showed a normal isoenzyme pattern (data of patient 2 in figure) and normal activity. a-fucosidase had a normal activity but a similarly disturbed isoenzyme pattern with abnormal cathodal bands that normalised during treatment. These data are similar to those found in CDG syndromes (figure).4 Taken together with our findings of decreased serum TBG and abnormal sialotransferrin pattern, these results are a further argument for grouping classic galactosaemia as a CDG syndrome, albeit a secondary and treatable one. In serum glycoproteins of patients with CDG syndrome there is a partial deficiency of the terminal trisaccharide sialic acid, galactose, and Nacetylglucosamine.5 Sialic acid bears a negative electric charge and the deficiency of this carbohydrate residue explains the cathodal shift of sialotransferrins and other glycoproteins. We postulate that in classic galactosaemia the accumulated galactose-1-phosphate interferes with the carbohydrate processing at the Golgi galactosyltransferase step and leads to a partial deficiency of the terminal disaccharide (sialic acid and galactose) of glycoproteins such as transferrin, TBG, and the lysosomal enzymes. Defective galactosylation of proteins in cultured skin fibroblasts and deficiency of glycolipids containing galactose or Nacetylgalactosamine in brain and lymphocytes with classical galactosaemia patients has been reported.6,7 Classical galactosaemia and CDG syndromes are both multisystemic disorders affecting the nervous system, liver, gastrointestinal tract, kidneys, and gonads. We suggest that these glycoprotein abnormalities play a major part in the pathogenesis of classic galactosaemia that thus may be regarded as a secondary "Golgi disorder". Department of Paediatrics, University of Leuven, 3000 Leuven, Belgium
JAAK JAEKEN
Department of Paediatrics, University of Ghent,
Jos KINT
Department of Molecular Cell Biology and Genetics, University of Limburg, Maastricht, Netherlands
LEO SPAAPEN
Vulsma T, Theunissen PMVM, van der Meer SB, Jaeken J. Galactosaemia, a carbohydrate-deficient glycoprotein syndrome. Society for the Study of Inborn Errors of Metabolism, 30th Annual Symposium, Leuven, Sept 8-11, 1992 (abstr). 2. Jaeken J, Vanderschueren-Lodeweyckx M, Casaer P, et al. Familial psychomotor retardation with markedly fluctuating serum prolactin, FSH and GH levels, partial TBG deficiency, increased serum arylsulphatase A and increased CSF protein: a new syndrome? Pediatr Res 1980; 14: 179. 3. Jaeken J, Stibler H, Hagberg B. The carbohydrate-deficient glycoprotein syndrome. Acta Paediatr Scand 1991; suppl: 375. 4. Jaeken J, Kint J. Abnormal serum lysosomal isoenzymes in leukodystrophy with sialic add deficient proteins. Society for the Study of Inborn Errors of Metabolism, 25th Annual Symposium, Sheffield, Sept 22-25, 1987 (abstr). 5. Stibler H, Jaeken J. Carbohydrate deficient serum transferrin in a new systemic hereditary syndrome. Arch Dis Child 1990; 65: 107-11. 6. Omstein KS, McGuire EJ, Berry GT, Roth S, Segal S. Abnormal galactosylation of complex carbohydrates in cultured fibroblasts from patients with galactose-1phosphate uridyltransferase deficiency. Pediatr Res 1992; 31: 508-11. 7. Petry K, Greinix HT, Nudelman E, et al. Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes. Biochem Med Metab Biol 1991; 46: 93-104.
1. Spaapen LJM,
Nested PCR of PAH cDNA and sequence analysis of V388M.
Upper= PCR lane 1 =size marker, 2=first PCR with lymphoblast cDNA, 3=second PCR with lymphoblast cDNA, 4=first PCR with liver cDNA, and 5=second PCR with lymphoblast poly(A+) RNA without reverse transcription. PCR product was not detected after first PCR with lymphoblast cDNA. Negative control with lymphoblast poly (A+) RNA without reverse transcription proves no contamination of cDNA. Primer sequences for first PCR: 5’dAGGGAAACCTGCCTGTACGT, 3’dCTCCATCAACAGATTCACAGC, and for second PCR: 5’dAAAAGCCAGAGACCTCACTC, 3’dACAGACCACATTCTGTCCATG. can be detected by nested polymerase chain reaction (PCR).2-4 We used this method to analyse PAH mRNA from lymphoblasts of a Japanese phenylketonuria patient and identified a novel mutation. Poly (A + ) RNA was isolated from a patient-derived EpsteinBarr-virus-immortalised lymphoblastoid cell line. Single-stranded cDNA was synthesised by reverse transcription with oligo(dT) primer and subjected to nested PCR with two sets of primers to amplify the entire coding region of PAH cDNA. A DNA fragment of the predicted size (1416 bp) was obtained (figure). Sequencing analysis of the amplified DNA revealed two point mutations: a single base substitution of G to A at base 1384 that alters valine at codon 388 in exon 11 to methionine (figure), and another substitution of G to C at base 1460 that alters arginine at codon 413 in exon 12 to proline. The valine-to-arginine mutation (V388M) has not been reported, whereas the arginine-to-proline mutation (R413P) has been described.6 Analysis of genomic DNA of the patient as well as the parents showed that this patient is indeed a compound heterozygote of V388M and R413P, whereas the father is a heterozygote of V338M and the mother is a heterozygote of R413P. Our study demonstrates that PAH mRNA in a phenylketonuria patient can be analysed with lymphoblasts and there is no need for risking liver biopsy. The method is not only rapid and reliable but also advantageous for the analysis of compound heterozygotes, because each allele can be examined separately. Various types of mutations responsible for the disease have been reported by genomic analysis of the PAH gene. Despite extensive studies for nearly a decade, less than 50% of the mutations have been identified in most populations.’ The analysis of ectopically transcribed PAH mRNA would facilitate the discovery of unknown mutations.
Department of Biochemical Genetics, Tohoku University School of Medicine, Aoba-ku, Sendai 980, Japan
KAZUTOSHI TAKAHASHI SHIGEO KURE YOICHI MATSUBARA KUNIAKI NARISAWA
Novel phenylketonuria mutation detected by
analysis of ectopically transcribed phenylalanine hydroxylase mRNA from lymphoblast is one of the most common inherited of aminoacid metabolism and is caused by altered activity of phenylalanine hydroxylase (PAH). To date molecular analysis of phenylketonuria has focused on extensive sequencing of 13 exons in the PAH gene’ because it is believed that PAH mRNA is expressed only in liver, and biopsy or necropsy liver specimens are required for the analysis of mRNA. Sequencing of each exon is laborious. Extremely low concentrations of mRNA synthesised by "ectopic" or "illegitimate" transcription in non-expressing tissues
SIR,-Phenylketonuria
inborn
errors
RC, Woo SLC. Phenylketonuria and the phenylalanine hydroxylase gene. Mol Biol Med 1991; 8: 3-18. 2. Sarkar G, Sommer SS. Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 1989; 244: 331-34. 3. Chelly J, Concordet JP, Kaplan JC, Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Proc Natl Acad Sci USA 1989; 86: 2617-21. 4. Roberts RG, Bently DR, Barby TFM, Manners E, Bobrow M. Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA. Lancet 1990; 336: 1523-26. 5. Chomczynski P, Sacchi N. Single-step method of RNA isolation by add guanidium thiocyanate-phenol-chloroform extraction Anal Biochem 1987; 162: 156-59. 6. Wang T, Okano Y, Eisensmith R, et al. Molecular genetics of PKU in onentals. Am J Hum Genet 1989; 45: A228. 7. Konecki DS, Lichter-Konecki U. The phenylketonuria locus: current knowledge about alleles and mutations of the phenylalanine hydroxylase gene in various populations. Hum Genet 1991; 87: 377-88. 1. Eisensmith
