Spectrum of Mutations in Aspartylglucosaminuria E
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Proc. Natd. Acad. Sci. USA Vol. 88, pp. 11222-11226, December 1991 Genetics Spectrum of mutations in aspartylglucosaminuria E. IKONEN*, P. AULAt, K. GR6Nt, 0. TOLLERSRUD*, R. HALILA*, T. MANNINEN*, A.-C. SYVANEN*, AND L. PELTONEN* *National Public Health Institute, Laboratory of Molecular Genetics, Mannerheimintie 166, SF-00300 Helsinki, Finland; and tUniversity of Turku, Department of Medical Genetics, Kiinamyllynkatu 10, SF-20520 Turku, Finland Communicated by Leon E. Rosenberg, September 5, 1991 ABSTRACT Aspartyucaminuria (AGU) Is an inher- AGU mutations consisting ofmissense mutations, insertions, ited lysosomal storage disoder caused by the deficiency of deletions, and a splicing defect. Four ofthem were clustered aspartylguminidase. We have earlier reported a single in the 17-kDa subunit, suggesting that this molecular region missense mutation (Cys"*3 -~Ser) to be responsible for 98% of is important for the three-dimensional structure of the en- the AGU alleles in the isolated Finnish population, which zyme molecule. contains about 90% of the reported AGU patients. Here we describe the spectrum of 10 AGU mutations found in unrelated patients of non-Finnish origin. Since 11 out of 12 AGU patients MATERIALS AND METHODS were homozygotes, con g ity has to be a common denom- Patients. The diagnosis ofAGU in affected individuals from inator in most AGU families. The mutations were distributed 13 patients outside Finland was ascertained by the referring over the entire coding region of the aspartylgi center on the basis of clinical findings, demonstration of cDNA, except in the carboxyl-terminal 17-kDa subunit in urinary glycoasparagines, and assay of AGA activity in which they were clustered within a 46-amino acid region. Based cultured fibroblasts or peripheral blood leukocytes. A sum- on the character of the mutations, most of them are prone to mary of the patient data including the ethnic origin of the affect the folding and stability and not to directly affect the families is given in Table 1. A fibroblast cell line was available active site of the aspartylglucosaminidase enzyme. for the present study from 11 AGU patients. From the Swedish patient and the other Norwegian patient, the anal- Aspartylglucosaminuria (AGU, McKusick 208400) is a re- yses were performed using peripheral blood leukocytes. cessively inherited lysosomal storage disorder resulting from Extraction of DNA and RNA and Northern Blot Analysis. inadequate function of aspartylglucosaminidase (AGA, EC Genomic DNA was isolated from fibroblast cell lines or 3.5.1.26) (1). Phenotypically the disease is relatively homo- peripheral blood leukocytes of the AGU patients using stan- geneous: patients from various populations have demon- dard procedures (21). Total RNA was purified from the strated similar clinical fibroblast cell lines (22) and poly(A)+ RNA was isolated using findings including progressive psych- oligo(dT)-cellulose chromatography. Northern blot analysis omotor retardation, coarse facial features, and mild osseous was performed as described (5). abnormalities (2). Clinically or biochemically distinct sub- Primers. The oligonucleotides were synthesized on an types based on the course of the disease cannot be identified Applied Biosystems model 381A DNA synthesizer (23). The (3). primer PCR5a was biotinylated as described (24). The given The highest prevalence of AGU is reported from Finland nucleotide numbers refer to the numbering used in the AGA with a carrier frequency of 1:30 to 1:40 (4, 43). We have cDNA (5), in which the first nucleotide (+1) corresponds to cloned the cDNA coding for human AGA and shown that one the adenine in the predicted translation initiation ATG codon. mutation is responsible for 98% of the AGU alleles in the Solid-Phase Minisequencing. Known point mutations were Finnish population (refs. 5 and 43). The same mutation was identified by a primer-guided nucleotide incorporation assay recently confired in eight Finnish AGU patients also by (25). Primers and optimized reaction conditions for detecting others (6, 44). The mutation, designated as AGUFil, causes ofthe cysteine mutation in AGUFm, allele were used (43). The a Cys163 -- Ser change and results in the absence ofa disulfide PCR and detection step primers for the identification of the bridge in the AGA enzyme (5, 7). In all the AGUFin alleles, Cm - T mutation were designed based on the intronic and the cysteine mutation is accompanied by an Arg161 -* Gln exonic sequences flanking the mutation. The 5' PCR primer substitution, which alone does not decrease the enzyme (PCR5a, 5'-TCTATGAACCTCTGAAAACTCC) was lo- activity (7). However, the latter does not represent a common cated in the noncoding region of the gene, 53-32 nucleotides polymorphism in this population since so far it has not been (nt) upstream ofthe exon-intron junction at position 281 and found in 200 analyzed control chromosomes but appears only the 3' PCRprimer was designed complementary to nt 351-372 to be coupled to the Cys163 -- Ser mutation. The AGA protein of the coding sequence (PCR5b, 5'-GTGTGTTGTATGTTC- is synthesized as a single polypeptide chain of 346 amino CAGTACT). The amplification product obtained using these acids. After removal of the signal peptide, AGA is most primers is a 145-base-pair (bp) DNA fragment. The detection probably subjected to posttranslational proteolysis resulting step primer was designed to hybridize immediately 3' of the in 24-kDa and 17-kDa subunits (8). Both of the subunits are mutation at nt 302 (PCR6, 5'-TTCGTCTGAGATC- required for the enzyme activity, but it is not known if the TCCTACT, nt 322-303). precursor form is active before the proteolytic cleavage (8, 9). Single-Strand Conformation Polymorphism (SSCP) Analy- About 20 sporadic AGU patients have been reported in the ses. First-strand cDNA was synthesized by reverse transcrip- literature (1, 10-20) in families showing a diversity of ethnic tion from 1 ,ug of total RNA using a primer specific for AGA backgrounds. Here we describe AGU mutations in 12 unre- (primer 4b, see below). Half of the synthesized cDNA was lated affected individuals of non-Finnish origin. We found 10 used for amplifying the coding region ofthe AGA cDNA with The publication costs of this article were defrayed in part by page charge Abbreviations: AGU, aspartylglucosaminuria; AGA, aspartylglu- payment. This article must therefore be hereby marked "advertisement" cosaminidase; SSCP, single-strand conformation polymorphism; nt, in accordance with 18 U.S.C. §1734 solely to indicate this fact. nucleotide(s). 11222 Downloaded by guest on September 26, 2021 Genetics: Ikonen et al. Proc. Natl. Acad. Sci. USA 88 (1991) 11223 Table 1. Summary of the mutations in AGU patients analyzed The precipitated immunocomplexes were dissolved in 10 mM Fibroblast Tris HCl, pH 8.0/150 mM NaCl/0.05% Tween 20 and the Pa- Age, Ethnic AGA activity, enzyme assay was carried out at 37TC for 20 hr. tient year(s) background (ref.) Gene defect % of normal Computer-Assisted Analyses. The flexibility ofthe polypep- tide chain was HV 7 Norwegian (11) predicted by the method ofKarplus and Schulz AGUFin 6 the was the of and ML 19 Norwegian (12) AGUFin ND (32), hydropathy profile by algorithm Kyte LC 8 Swedish* Doolittle (33), and the secondary structures were predicted AGUFin 1 the method of Garnier et al. KF 10 Turkish (FCP)* G904 A 7 by (34). JJ 16 American white T916 - C 6 (NC)* RESULTS WA 3 German (NC) (19) A G179 7 of Mutations. Since we MS 1 Italian (NC)* CO T 4 Preliminary Screening had previ- AM 5 English (NC)* CO - ously found AGUFmi to be the major AGU-causing mutation T/ 9 in the Finnish population, we initially analyzed the genomic del 102-1 DNA of all 13 examined the RV 8 Dutch (NC)* del T336 7 patients using solid-phase DM 17 Spanish-American ins T 5 minisequencing technique (25), which detects specifically both the Cys'63 -_ Ser and the Arg'6' -) Gin mutations found (NC)* after TI°° in the allele. Three Scandinavian two MA 3 Tunisian (FCP)* ins 6 bp 3 AGUFm. patients, from northern Norway and one from northern Sweden, were after G127 shown to be homozygous for the AGUFiN allele. The remain- BR 12 American black del 807-940 4 ing 10 patients gave results corresponding the normal allele in (NC) (16) (Camden no. the minisequencing analysis, and they were further studied GM03560) with the SSCP for SL 5 Mexican-Italian (10) ? 5 technique preliminary identification of the AGA cDNA regions containing a mutation. Total RNA was AGA activity in control fibroblasts is expressed as nmol per min reverse-transcribed and the coding region ofthe AGA cDNA per mg of protein (XT = 134; range, 70-226; n = 10). ND, not was amplified as four overlapping fragments, from 308 to 363 determined; FCP, first-cousin parents; NC, no consanguinity. nt in size (Fig. 1). In the SSCP analyses, we identified *Refemng centers of unpublished AGU patients: MA, M. Rossiter, aberrant Enfield District Hospital, Enfield, UK; SM, L. Pavone, Department migration ofthe labeled DNA strands in the samples of Pediatrics, University of Catania, Italy; FK, B. Poorthuis, from nine patients. In each case only one ofthe fouramplified University Hospital, Leiden, The Netherlands; JJ, B. R. Powell, fragments revealed a mobility shift (Fig. 1). When compared University Hospital, Portland, Oregon, USA; VR, 0. v. Diggelen, to identically amplified controls, there was only one DNA Erasmus Universiteit Rotterdam, Department of Genetics, Rotter- sample, from one Mexican-Italian patient, that showed no dam, The Netherlands; AM, I. Maire, Hospital Debrousse, Lyon, mobility shift. France; MD, S. I. Goodman, Department of Pediatrics, University Nine mutations were identified by sequencing the ampli- of Colorado, Denver, Colorado, USA; CL, L. Skogberg, Central- fied fragments showing a mobility shift in the SSCP analysis. hospital, Boden, Sweden. Only one English AGU individual was a compound heterozy- and primers la and 4b (see For gote shared the other mutation with a homozygous Italian below).