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The Molecular Lesion in the Alpha-N-Acetylgalactosaminidase

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The Molecular Lesion in the Alpha-N-Acetylgalactosaminidase Rapid Publication The Molecular Lesion in the a-N-Acetylgalactosaminidase Gene That Causes Angiokeratoma Corporis Diffusum with Glycopeptiduria Anne M. Wang,* Tamotsu Kanzaki,* and Robert J. Desnick* *Department of Human Genetics, Mount Sinai School of Medicine, New York 10029; and 'Department of Dermatology, Kagoshima University Faculty of Medicine, Kagoshima-shi 890, Japan Abstract roaxonal dystrophy * lysosomal storage disease * a-N-acetyl- galactosaminidase - mutation analysis * transient expression Angiokeratoma corporis diffusum with glycopeptiduria is a recently recognized inborn error of glycoprotein catabolism Introduction resulting from the deficient activity of human a-N-acetylga- lactosaminidase (E.C.3.2.1.49; a-GalNAc). The first patient Angiokeratoma corporis diffusum with glycopeptiduria is a re- with this autosomal recessive disorder, a 46-yr-old consan- cently recognized inborn error of glycoprotein catabolism re- guineous Japanese woman, presented with diffuse angioker- sulting from the deficient activity of the lysosomal glycohydro- atoma, mild intellectual impairment, and peripheral neu- lase, a-N-acetylgalactosaminidase (E.C.3.2.1.49; a-Ga1NAc)1 roaxonal degeneration. Deficient a-GaINAc activity also has (1-3). The enzymatic defect, inherited as an autosomal reces- been reported in consanguineous brothers with an infantile- sive trait, leads to the tissue accumulation and increased urinary onset form of neuroaxonal dystrophy resulting from a mis- excretion of glycopeptides and oligosaccharides containing a- sense mutation (designated E325K) in the a-GaINAc gene. N-acetylgalactosaminyl moieties. The disorder was described To identify the mutation causing the phenotypically distinct in 1989 by Kanzaki et al. (1) in a 46-yr-old consanguineous adult-onset disorder, Southern and Northern hybridization Japanese female who had diffuse angiokeratoma, mild intellec- analyses of DNA and RNA from the affected homozygote tual impairment, and peripheral neuroaxonal degeneration. The were performed which revealed a grossly normal a-GalNAc angiokeratoma, a disseminated petechiae-like eruption, first ap- gene structure and normal transcript size and abundancy. peared on her lower torso at - 28 years of age and then spread Reverse transcription, amplification, and sequencing of the slowly over her body in a distribution typical to that seen in a-GalNAc transcript identified a single C to T transition at hemizygotes with Fabry disease (4). Dilated, tortuous blood nucleotide (nt) 985 that predicted an arginine to tryptophan vessels were observed in her ocular conjunctiva and fundi. substitution in residue 329 (designated R329W) of the a- There were no other manifestations commonly found in other GaINAc polypeptide. This base substitution was confirmed lysosomal storage diseases, including organomegaly, lymphade- by hybridization of PCR-amplified genomic DNA from fam- nopathy and skeletal abnormalities. The proband had a low ily members with allele-specific oligonucleotides. Transient intelligence (IQ = 70) and magnetic resonance imaging of the expression of an a-GalNAc construct containing the R329W brain revealed a few small lacunar infarctions, but no gross mutation resulted in the expression of an immunoreactive parenchymal atrophy. Motor nerve conduction studies were nor- polypeptide which had no detectable a-GalNAc activity. mal, but the sensory nerves had a marked decrease in amplitude Comparison of the biosynthesis and stabilities of the tran- with normal velocity, indicating a peripheral neuroaxonal de- siently expressed and radiolabeled normal, E325K (infan- generation (3). tile-onset) and R329W (adult-onset) a-GalNAc polypeptides The affected homozygote had levels of a-GalNAc activity in COS-1 cells indicated that both the mutant precursors that were < 1% of normal in various sources as assayed with were processed to the mature form; however, the E325K the recently synthesized substrate, 4-methylumbelliferyl-a-N- mutant polypeptide was more rapidly degraded than the acetylgalactosaminide (4MU-a-GalNAc) (2). Her children had R329W subunit, thereby providing a basis for the distinctly intermediate levels of activity consistent with being obligate different infantile- and adult-onset phenotypes. (J. Clin. In- heterozygotes for this autosomal recessive disorder. Using vest. 1994. 94:839-845.) Key words: angiokeratoma - neu- monospecific rabbit anti-human a-GalNAc antibodies, immu- noblots of fibroblast extracts from the affected proband revealed no detectable immunoreactive enzyme protein, while extracts from her heterozygous son had two immunoreactive peptide Address all correspondence to R. J. Desnick, Ph.D., M.D., Mount Sinai species, the - 48-kD monomeric and 117-kD dimeric forms School of Medicine, Fifth Avenue at 100th Street, Department of Human of a-GalNAc observed in normal cultured fibroblasts and in Genetics, New York, NY 10029. preparations of the enzyme. sug- Received for publication IO February 1994 and in revised form 7 purified human These findings April 1994. J. Clin. Invest. 1. Abbreviations used in this paper: a-GalNAc, a-N-acetylgalactosami- © The American Society for Clinical Investigation, Inc. nidase; CRIM, cross-reacting immunological material; Endo H, endogly- 0021-9738/94/08/0839/07 $2.00 cosidase H; nt, nucleotide; PNGase F, peptide N-glycosidase F; 4-MU, Volume 94, August 1994, 839-845 4-methylumbelliferyl. Molecular Lesion in Adult-Onset a-N-Acetylgalactosaminidase Deficiency 839 gested that the a-GalNAc mutation in this family markedly unit, suggesting a molecular basis for these phenotypically dis- altered the enzyme's activity and stability. tinct disorders. Previously, an infantile form of neuroaxonal dystrophy due to the deficient activity of lysosomal a-GalNAc was described Methods in two consanguineous German boys (4-7). The course of this disorder was remarkably distinct from that of the adult-onset Cell lines. Primary cultures of fibroblasts and lymphoblasts were estab- form, and was characterized by normal development until 8- lished from skin biopsies and peripheral blood samples obtained with informed consent from 12 mo of life, followed by a rapidly regressive course resulting members of the infantile- and adult-onset fami- lies and from normal controls. The COS-l cells lines were purchased in profound psychomotor retardation by three years of age. They from the American Type Tissue Collection (Rockville, MD). The fibro- had no apparent or histologic evidence of cutaneous involve- blasts, lymphoblasts and COS-1 cell lines were grown in DME media ment at 8 and 9 years of age. Morphologic analysis of cortical supplemented with 10% fetal bovine serum, 1% penicillin, and 1 mg/ tissue revealed the characteristic "spheroids," which are the ml streptomycin by standard procedures (16). pathologic hallmark of the neuroaxonal dystrophies. The dem- Assays ofa-GalNAc activity and protein. The a-GalNAc activity in onstration of accumulated 0-linked sialoglycopeptides and the cultured cell lines was determined with the synthetic fluorogenic sub- blood group A oligosaccharide in their urines led to the identifi- strate 4MU-a-GalNAc (17) as previously described (7). Protein concen- cation of deficient a-GalNAc activity as the primary enzymatic tration was determined by the fluorescamine assay (18). Immunoblot analyses of a-GaINAc enzyme protein in transfected COS-1 cells was defect causing this form of infantile neuroaxonal dystrophy (5, performed using monospecific rabbit anti-human a-GaINAc antibodies 7, 8). The finding that the German brothers with infantile neu- as described (7, 11). roaxonal dystrophy and the Japanese woman with diffuse angi- Southern and Northern hybridization analyses. Genomic DNA was okeratoma excreted the same sialoglycoproteins (9, 10), led to isolated from at least 106 cultured lymphoblasts (19). DNA digested the discovery that the adult-onset proband also had deficient a- with various restriction endonucleases (e.g., BamHI, PstI, EcoRI, TaqI) GalNAc activity (3). Thus, phenotypically distinct infantile- and (New England Biolabs, Beverly, MA), was electrophoresed in 1% agar- adult-onset forms of a-GaINAc deficiency were delineated. ose, transferred to BioTrace RP charge-modified nylon 66 binding ma- The isolation and expression of the full-length cDNA and trix (Gelman Sciences, Inc., Ann Arbor, MI) (20) and hybridized with nick-translated a-GalNAc cDNA as the probe (21). To determine the genomic sequence encoding human a-GalNAc (11, 12) permit- relative sizes and amounts of the 2.2- and 3.6-kb a-GaINAc transcripts ted investigation of the molecular lesions that cause the pheno- (11), total RNA was isolated from at least 108 cultured lymphoblasts typically distinct forms of a-GalNAc deficiency. The 12-kb a- from the adult-onset proband and normal individuals (22). RNA samples GalNAc gene encodes a polypeptide of 411 amino acids includ- were electrophoresed in 1% agarose/formaldehyde denaturing gels (23), ing a signal peptide of 17 residues. Studies of human a-GalNAc transferred to the BioTrace membranes described above, and analyzed biosynthesis indicated that the precursor polypeptide was co- with the radiolabeled a-GaINAc riboprobe synthesized with SP6 poly- translationally glycosylated in the endoplasmic reticulum, and merase from the a-GaINAc 2.2-kb cDNA cloned into the pGEM 4Z after carbohydrate modifications and phosphorylation of its high vector (Promega, Madison, WI). DNA amplification and sequencing of the mutant allele. Sense and mannose-type oligosaccharide
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