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). the SSCP analyses, the AGU patient (see below). The individual mutations are first PCR product was reamplified as four overlapping frag- summarized in Fig. 2. ments with the following four pairs of primers (numbers Missense Mutations. As according to ref. la described above, all analyzed 5): (nt -39 to -21) and lb (nt 324-304), Scandinavian patients (two Norwegians and one Swede) 2a (nt 257-277) and 2b (nt 613-593), 3a (nt 542-560) and 3b (nt were homozygous for the allele the 849-829), and 4a AGUFin containing Cys'63 (nt 692-711) and 4b (nt 1047-1027). The -- Ser and Arg161 - Gln substitutions. A single missense PCRs were carried out as described (5), except that in the mutation was found in five patients, in two of them an reamplification the products were radiolabeled by adding 10 arginine was introduced: in a Turkish patient a G' -- A ,uCi of [a-32P]dCTP (Amersham; 1 Ci = 37 GBq) to the PCR. substitution changed Gly302 to Arg, and in a white American The temperature profile was 1 min at 95°C, 1 min at 60°C, and patient a T916 -) C change caused a Cys306 -+ Arg change. 1 min at 72°C. The amplification products were diluted 1:5 in A G179 -- A transition in a German patient caused an 1% SDS/10 mM EDTA and mixed with an equal volume of uncharged glycine to be replaced by a negatively charged 95% (vol/vol) formamide/20 mM EDTA containing 0.05% aspartic acid (Gly' -- Asp). The Italian patient had a C302 -+ bromophenol blue and 0.05% xylene cyanol. After denatur- T transition that changed Ala'0' -- Val. The same mutation ation by heating at 80°C for 2 min, 2 ,ul of the sample was was found in the other allele of the English patient (see analyzed by electrophoresis in a neutral 5% single-strand below). separation gel containing 10% (vol/vol) glycerol at 30 W at Deletions and Insertions. Two patients showed a short room temperature for an appropriate time (26). deletion: a Dutch patient had a deletion of a thymidine at Amplification and Sequencing of cDNA and Genomic DNA. position 336 resulting in a shift of the reading frame and The coding region of the AGA cDNA was amplified for premature termination of the polypeptide chain after 126 sequencing using the primers la-4b (see above). The PCR amino acids. The English patient, having the Ala'0' -- Val products were sequenced by the dideoxynucleotide chain- mutation in one allele (see above), had a 7-nt deletion (nt termination method (27) with modifications (28). The primers 102-108) in the other allele, predicting the formation of a used for amplifying and sequencing the mutations from truncated polypeptide chain of only 33 amino acids. genomic DNA were from the sequence of the AGA cDNA. Insertion mutations were also found in two patients: one Enzyme Assay. The AGA activity assay based on colori- Spanish-American patient had an insertion of one thymidine metric measurement of liberated N-acetylglucosamine (29) after nt 800. The thymidine insertion caused a shift in the was performed from fibroblasts as described (30). The en- reading frame and the premature stop codon caused a trun- zyme activity was also assayed after immunoprecipitating cated polypeptide chain with 318 amino acids of which the AGA from fibroblast lysate of one confluent 75-cm2 culture first 267 amino acids represent the normal AGA polypeptide. flask by using the antiserum prepared against purified AGA A Tunisian patient had an insertion of 6 nt (ATGCGG) after (9) and Staphylococcus aureus cells (Immuno-Precipitin, nt 127 causing an in-frame insertion of aspartic acid and BRL) to precipitate the antigen-antibody complexes (31). alanine after the amino acid 42. It is possible that the insertion Downloaded by guest on September 26, 2021 11224 Genetics: Ikonen et al. Proc. Natl. Acad. Sci. USA 88 (1991)

Patient AM

v(.1. .de C. > II 1. AGA eDNA

-39 - 324 Z. ./a- 613 5342

55CC- ana1ys1w 've.E.j4

_s i_ *,* * 4* MC

AM C C AM C JJ CM AM C JJC3 A FIG. 1. SSCP analysis ofthe AGU mutations. The coding region ofthe AGA cDNA is presented as a horizontal bar and the four overlapping fragments used in SSCP analyses are indicated below. The first and last nucleotides ofeach fragment are given. Patient AM was a heterozygote, whose two mutations were located in the most 5' SSCP fragment, and patient JJ was a homozygote with the mutation in the most 3' SSCP fragment. Only the corresponding fragments demonstrate a mobility shift. is a consequence ofa defective splicing event. Genomic DNA quence. The consequences of the splicing mutation are could not be amplified using primers only 54 nt apart in the schematically presented in Fig. 3. AGA cDNA and flanking the insertion, suggesting the exis- Confirmtion of the Mutations in Both Afleles of AGU tence of a long intronic sequence adjacent to it. The insertion Patients. To confirm that the established mutations were the of2 amino acids occurs in the middle ofa computer-predicted only changes in the analyzed AGA cDNAs, we sequenced the helical region in the AGA polypeptide chain. amplified overlapping cDNA fiagments covering all the cod- Splicing Mutation. The black American patient demon- ing regions of the patients. We found three polymorphisms strated a significantly shortened amplification product of the that did not cause amino acid changes: C90 -k T, C186 - A, AGA cDNA with one set ofprimers. However, amplification and T" -* G. These polymorphisms were found in two or of genomic DNA from the same region revealed an amplifi- three AGU individuals, but their population frequency has so cation product of the same size from both the patient and the far not been determined. The mutations found were con- controls. Further sequence analysis of both the AGA cDNA firmed by direct sequencing of the amplified genomic DNA. and genomic DNA confirmed one missing 134-bp exon in the Cosegregation of the mutation and the affection status (af- cDNA and a G -+ T substitution in the adjacent 3' intron. This fected or carrier) confirmed the mutation in the Tunisian mutation at position + 1 of a splice donor site at the exon- family, the only mutation that we were not able to verify by intron boundary causes skipping of the preceding 44-amino amplification and sequencing of genomic DNA (see above). acid exon and results in a shortened transcript. As shown in Since only 1 of the established 10 mutations, C32 -* T Fig. 3, we obtained no amplification product with one set of substitution causing the Ala"'1 -+ Val change, was found in primers, when the other primer was designed according to the two nonrelated AGU patients, we wanted to confirm that it sequence of the deleted exon. The Northern blot analysis of did not represent a common protein polymorphism, espe- the mRNA of the patient confirmed the data obtained with cially since the changes predictable by computer programs at sequencing and the PCR analysis: the mutation results in a the polypeptide level were negligible. We designed primers 134-bp-shorter transcript from both alleles. for specific detection of this mutation using the minisequenc- Due to different codon splicing of the flanking exons, this ing method analogously to the detection ofAGUFin. The C302 mutation also results in the shift of the reading frame and in -+T mutation was not found in 100 chromosomes represent- a premature termination codon in the beginning of the fol- ing a random sample of the Finnish population. lowing exon. The length ofthe predicted polypeptide chain is In one AGU individual, a Mexican-Italian patient, we 273 amino acids, of which the 5 amino acids at the carboxyl- found only neutral polymorphisms (see above). This excep- terminal end differ from the normal AGA polypeptide se- tion confirms that we have sequenced the coding region of AGUFIn +D,A A 1V c 16rS 3oi--* R C306 I' R aa 24 kD_ 17 kD 1001 300 346 -v ;'1 200 600 800 1(VITr 'V IAAI104V-to bp del 102-108 G179 A del T336 G4 C tIpc80-4s 9IA, 127, ins 6 bp C302 T 800, ins T Gg04A FIG. 2. Map of the AGU mutations found. The numbers of the nucleotides corresponding to the coding region of the AGA cDNA are presented below the horizontal line and the numbers ofthe deduced amino acids (aa, single-letter code) are indicated above the line. The region of the putative signal peptide is hatched. The amino-terminal ends of the 24-kDa and 17-kDa subunits are shown as r. Each mutation is represented by a vertical line and an x (point mutation), a triangle pointing upward (ins, insertion), or a triangle pointing downward (del, deletion; splc, splicing mutation). Downloaded by guest on September 26, 2021 Genetics: Ikonen et al. Proc. Natl. Acad. Sci. USA 88 (1991) 11225 1-3 M.. x4_vA C l -2P) C A C A T G C A T G C A

A

-- A _ 3.4 kb- .x 12;. T m_ *:. f n G r 1-2 G t%.A i A r' xn C 2.1 kb -- 0 A A /

-W dew AM, al"Im,

806 807 940 941 CTG CCA AG CAATC AGIC TAC CAG TA GTAT GGT GOT TGC AAT AAA

T 1 2 3 T 4-

806 941 CTG CCA AGIG TGC TGC TTG CAA TAA

FIG. 3. Splicing mutation resulting in AGU (patient BR, fibroblast line GM03560). The exonic nucleotides are boxed and numbered at the intron-exon boundaries. The G -- T point mutation affecting the junctional consensus sequence (indicated with boldface letters) and causing the splicing of an exon (nt 807-940) is shown. The consequent shift of the reading frame and premature termination codon are demonstrated below. The numbered arrows refer to PCR primers used to amplify the products shown in the upper left. By using the primer pair 1-3, a smaller fragment was obtained from the cDNA ofthe AGU patient (lanes A) than from control cDNA (lanes C). No amplification product was obtained from the patient cDNA using the primers 1-2. In the autoradiography of the sequencing gel of genomic DNA of the patient and a control, the site of the mutation is indicated by an arrow and a star. In the Northern blot analysis, 3 I&g ofpoly(A)+ RNA from the fibroblasts of the patient and a control was hybridized with the 32P-labeled AGA cDNA. The indicated sizes of the mRNA species are based on the relative migration of the ribosomal subunits.

both of the patient's alleles and also suggests that the Screening for the unknown gene defects was here carried mutation lies in the noncoding region of the cDNA or in the out using the SSCP method, which detected the AGU mu- regulatory elements of the AGA gene. tations in all analyzed cases. One mutation resided in the Enzyme Activities in the AGU Cell Lines. To study the effect overlapping part of two consecutive amplification fragments ofthe various mutations on the catalytic function ofAGA, the and should actually have been visualized in two SSCP enzyme activity was assayed from cultured fibroblasts of the analyses. However, it remained undetected in one of them patients using two approaches: (i) the standard diagnostic (Fig. 1). Only in one Mexican-Italian patient was no mutation assay system and (ii) measuring the enzyme activity after identified either in the SSCP analysis or by sequencing the concentration of enzyme by immunoprecipitation. Both as- coding region of the AGA cDNA. Since we sequenced the say methods gave equally low activities in all AGU cell lines entire coding region of all the analyzed patients, also the ranging from 1 to 9%o of the activity in control fibroblasts fragments with no migration shifts in the SSCP analysis, we (Table 1). The observed enzyme activities in AGU cells were conclude that preliminary localization of the mutations was on the borderline of the detection limit of the assay. Conse- efficient using the SSCP technique. Although the reproduc- quently, no significant differences in the enzyme activity ibility and interpretation ofthe migration shifts are somewhat between the AGU cell lines containing different mutations problematic, this method proved to be reliable, when applied could be verified. to amplification products of about 300 bp. The 10 mutations found were relatively evenly distributed DISCUSSION in the two subunits of AGA, with possibly one exception: a region of only 46 amino acids in the carboxyl-terminal part of Molecular studies in single gene disorders have as a rule the AGA polypeptide contained two point mutations, one displayed a wide spectrum of different gene defects in phe- splicing mutation and one nucleotide insertion. Interestingly, notypically uniform patients. The mutations described here, similar clustering of the mutations in the carboxyl-terminal all resulting in the lysosomal accumulation disease AGU, also end has been observed in the a subunit of lysosomal ,-hex- demonstrate significant diversity: missense mutations, small osaminidase. Moreover, the mutations affecting the carboxyl insertions and deletions, and splicing mutations are all rep- terminus both in the a and P subunit of (3-hexosaminidase resented, providing a spectrum of 10 gene defects. However (causing Tay-Sachs and Sandhoff disease, respectively) ap- surprisingly, many of the analyzed AGU patients (11/12) pear to result in the improperfolding and transport ofthe gene were homozygotes. This finding would suggest that the products (35). The clustering of the AGU mutations could appearance of the AGU phenotype is almost without excep- analogously indicate that the carboxyl-terminal end of the tion associated with consanguinity, although confirmed here AGA polypeptide is important for the formation ofthe proper only in two families. protein structure and transport of the enzyme. Downloaded by guest on September 26, 2021 11226 Genetics: Ikonen et al. Proc. Natl. Acad. Sci. USA 88 (1991) The mutations resulting in truncated polypeptide chains stein, J. L. & Brown, M. S. (McGraw-Hill, New York), Vol. 5, pp. premature 797-799. most probably result in the defective folding and 4. Aula, P., Renlund, M., Raivio, 0. & Koskela, S.-L. (1986) J. Ment. intracellular degradation of the AGA polypeptide chain. Defic. Res. 30, 365-368. These AGU mutations include, in addition to the splicing 5. Ikonen, E., Baumann, M., Gr6n, K., Syvftnen, A.-C., Enomaa, N., mutation and thymidine insertion discussed above, two de- Halila, R., Aula, P. & Peltonen, L. (1991) EMBO J. 10, 51-58. letions in the amino-terminal subunit. Especially the trans- 6. Mononen, I., Heisterkamp, N., Kaartinen, V., Williams, J. C., Yates, J. R., Griffin, P. R., Hood, L. E. & Groffen, J. (1991) Proc. lation products of only 33 and 126 amino acids will almost Nati. Acad. Sci. USA 88, 2941-2945. inevitably face rapid intracellular degradation. 7. Ikonen, E., Enomaa, N., Ulmanen, I. & Peltonen, L. (1991) The point mutations resulting in substitutions of glycine Genomics 11, 206-211. may affect the folding process, since due to the lack of a 8. Halila, R., Baumann, M., Ikonen, E., Enomaa, N. & Peltonen, L. (1991) Biochem. J. 276, 251-256. f3-carbon this amino acid is involved in positive dihedral 9. Toliersrud, 0. K. & Aronson, N. N. (1989) Biochem. J. 260, angles, energetically unfavorable for other amino acids, and 101-108. leads to a high degree of conservation in structurally related 10. Isenberg, J. N. & Sharp, H. L. (1975) J. Pediatr. 86, 713-717. enzymes (36, 37). 11. Borud, 0. & Torp, K. H. (1976) Lancet 1, 1082-1083. 12. Borud, O., Torp, K. H. & Dahl, T. (1978) in Monographs in Human The folding defect also is the most logical explanation for Genetics, eds. Sperling, O., de Vries, A. & Tiqva, P. (Karger, the loss of AGA activity resulting from the two cysteine Basel), Vol. 10, pp. 23-26. mutations. Both the abolition of a disulfide bridge and the 13. Gehler, J., Sewell, A. C., Becker, C., Hartmann, J. & Spranger, J. appearance of a free cysteine residue have been shown to (1981) Helv. Paediatr. Acta 36, 179-189. 14. Gatti, R., Buttitta, P., Michalski, J. C., Strecker, G., Borrone, C. have a destabilizing effect (38). Cysteine-reacting chemicals & Salemi, D. (1982) Riv. Ital. Pediatr. 8, 87-91. like p-hydroxymercuribenzoate and N-ethylmaleimide do 15. Stevenson, R. E., Taylor, H. A. & Wilkes, G. (1982) Proc. Green- not inactivate the AGA enzyme (39, 40), indicating that free wood Genet. Center 1, 69-72. cysteine residues are not involved in the active site. 16. Hreidarsson, S., Thomas, G. H., Valle, D. L., Stevenson, R. E., Taylor, H., McCarty, J., Coker, S. B. & Green, W. R. (1983) Clin. For the cysteine mutation in AGUFin, we have demon- Genet. 23, 427-435. strated the absence of one disulfide bridge both in vitro and 17. Musumeci, S., Salvati, A., Schiliro, G., Salvo, G., Di Dio, R. & in vivo. In vitro expression of the mutated construct has Caprari, P. (1984) Am. J. Med. Genet. 19, 643-650. further revealed intracellular degradation of newly synthe- 18. Chitayat, D., Nakagawa, S., Marion, R. W., Sachs, G. S., Hahm, S. Y. E. & Goldman, H. S. (1988) Am. J. Med. Genet. 31, 527-532. sized AGA polypeptide chain most probably due to disturbed 19. Ziegler, R., Schmidt, H., Sewell, A. C., Weglage, J., v Lengerke, folding (7). Similar effect could be predicted for the other J. H. & Ullrich, K. (1989) Monatsschr. Kinderheilkd. 137,454-457. found cysteine mutations. These cysteine residues are lo- 20. Gordon, B. A., Rupar, C. A., Rip, J. W., Haust, M. D., Scott, E. cated 10 and 15 amino acids from the neighboring cysteine & Jung, J. H. (1990) in Abstracts ofthe Vth International Congress Inborn Errors of Metabolism, P58. and separated by (-turns. According to Thornton (38) this 21. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular provides favorable conditions for the formation of a disulfide Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold bridge. Spring Harbor, NY), pp. 9.16-9.19. One AGU mutation found resulted in the insertion of two 22. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, the amino-terminal end of the W. J. (1979) Biochemistry 18, 5294-5299. amino acids close to AGA 23. Beaucage, S. L. & Caruthers, M. H. (1982) Tetrahedron Lett. 22, polypeptide. This may have a destabilizing effect on the 1859-1862. enzyme protein, since the insertion occurs in the middle of a 24. Bengtstrom, M., Jungell-Nortano, A. & Syvanen, A.-C. (1990) predicted helical region (41). The mutation changing alanine Nucleosides Nucleotides 9, 123-127. to valine can cause spatial problems and could again result in 25. Syvinen, A.-C., Aalto-Setfi, K., Harju, L., Kontula, K. & S6d- erlund, H. (1990) Genomics 8, 684-692. destabilization of the AGA polypeptide chain. Alanine has a 26. Orita, M., Suzuki, Y., Sekiya, T. & Hayashi, K. (1989) Genomics small hydrophobic side chain, which is typically buried inside 5, 874-879. the folded protein, whereas valine carries a more sizable one. 27. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. This phenomenon has been demonstrated [e.g., in the lambda Sci. USA 74, 5463-5467. repressor, in which an alanine can only be replaced by a 28. Casanova, J.-L., Pannetier, C., Jaulin, C. & Kourilsky, P. (1990) Nucleic Acids Res. 18, 4028. serine with a side chain of the same size (36)]. 29. Makino, M., Kojima, T. & Yamashina, I. (1966) Biochem. Biophys. To conclude, it seems that all AGU mutations described Res. Commun. 24, 961-966. here primarily affect folding and stability and do not directly 30. Aula, P., Mattila, K., Piiroinen, O., Ammila, P. & von Koskull, H. affect the catalytic activity of the AGA enzyme. This hy- (1989) Prenatal Diagn. 9, 617-620. pothesis is reinforced by the lack of mutations in the vicinity 31. Kessler, S. W. (1981) in Methods Enzymol. 73, 442-443. of T06, believed to be located in the proximity of the active 32. Karplus, P. A. & Schulz, G. E. (1985) Naturwissenschaften 72, 212-213. site (42). So far, we have demonstrated the consequences of 33. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132. only one cysteine mutation by using in vitro expression: 34. Gamier, J., Osguthorpe, D. J. & Robson, B. (1978) J. Mol. Biol. increased degradation probably due to improper folding (7). 120, 97-120. It remains to be confirmed whether the hypothesis of de- 35. Neufeld, E. F. (1989) J. Biol. Chem. 264, 10927-10930. creased stability and deficient folding as major consequences 36. Pakula, A. A. & Sauer, R. T. (1989) Annu. Rev. Genet. 23, 289-310. also to the other found AGU mutations. 37. Creighton, T. E. (1983) Proteins (Freeman, New York), p. 112. applies 38. Thornton, J. M. (1981) J. Mol. Biol. 151, 261-287. 39. Dugal, B. & Stromme, J. (1977) Biochem. J. 165, 497-502. We thank Dr. Anu Suomalainen for advice in the SSCP technique 40. Dugal, B. (1978) Biochem. J. 171, 799-802. and Ms. Helena Rantanen for professional secretarial work. This 41. Zebala, J. & Barany, F. 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