4585 JMed Genet 1996;33:458-464 Human xc-N-acetylgalactosaminidase Qx-NAGA)

deficiency: new mutations and the paradox J Med Genet: first published as 10.1136/jmg.33.6.458 on 1 June 1996. Downloaded from between genotype and phenotype

J L M Keulemans, A J J Reuser, M A Kroos, R Willemsen, M M P Hermans, A M W van den Ouweland, J G N de Jong, R A Wevers, W 0 Renier, D Schindler, M J Coll, A Chabas, H Sakuraba, Y Suzuki, 0 P van Diggelen

Abstract reported the second independent case of a- Up to now eight patients with a-NAGA NAGA deficiency with an entirely different deficiency have been described. This in- clinical phenotype. This patient had a late onset cludes the newly identified patient re- disease with slight facial coarseness, dis- ported here who died unexpectedly aged seminated , and mild intellectual II years of hypoxia during convulsions; impairment (IQ= 70), but without neuro- necropsy was not performed. logical symptoms. Unlike the infantile cases, Three patients have been genotyped pre- this patient had prominent vacuolisation in all viously and here we report the mutations dermal cells, most prominently in vascular and in the other five patients, including two lymphatic endothelial cells and eccrine sweat new mutations (S160C and E193X). The gland cells, but also in dermal neural cells and newly identified patient is consanguineous fibroblasts.' The glomerular endothelial cells with the first patients reported with a- but not the epithelial kidney cells are involved NAGA deficiency and neuroaxonal dys- and also blood lymphocytes are vacuolised.6 trophy and they all had the a-NAGA geno- These three patients shared, however, the type E325KIE325K. abnormal urinary excretion of specific oli- Clinical heterogeneity among patients gosaccharides. The major compounds are si- with x-NAGA deficiency is extreme. Two alylglycopeptides of the 0-glycosidic type with Department of affected sibs, homozygotes for E325K, are serine or threonine linked to the a-N-acet- Clinical Genetics, severely affected and have the signs and ylgalactosamine (aGalNAc) moieties.78 Using Erasmus University, PO Box 1738, 3000 DR symptoms of infantile neuroaxonal dys- an axGalNAc specific lectin, intralysosomal Rotterdam, The trophy, but prominent vacuolisation is storage products were shown in lysosomes of Netherlands lacking. The mildly affected patients (two cultured fibroblasts from the infantile patients.9 http://jmg.bmj.com/ J L M Keulemans families, three patients) at the opposite The a-NAGA codes for 411 amino A J J Reuser M A Kroos end of the clinical spectrum have clear acids'1 and consists of nine exons." Mutation R Willemsen vacuolisation and angiokeratoma but no analysis of the ox-NAGA gene showed the M M P Hermans overt manifestations. Two of missense mutation E325K in the infantile A M W van den neurological Ouweland them are homozygous for the stop muta- cases13; both patients were homozygous for O P van Diggelen tion E193X, leading to complete loss of this mutation. The adult patient was found a-NAGA protein. These observations are to be homozygous for the missense mutation Institute of 14 on September 30, 2021 by guest. Protected copyright. and Department of difficult to reconcile with a simple geno- R329W. Child Neurology, type-phenotype correlation and we sug- Recently, four additional patients with a- of University gest that factors or other than a- NAGA deficiency were reported in two in- Nijmegen, The Netherlands NAGA contribute to the clinical hetero- dependent families. The index case in the J G N de Jong geneity of the eight patients with a-NAGA Dutch family, reported by de Jong et al,'5 had R A Wevers deficiency. psychomotor retardation at the age of 4 years. W 0 Renier At the metabolic level, the patients with Screening her sibs showed that a healthy sib with Institute for Human a-NAGA deficiency are similar. The major a-NAGA deficiency has no overt clinical symp- Genetics, University of abnormal urinary oligosaccharides are si- toms at the age of3 years. Chabas etal'6 reported Germany Wurzburg, 0 with a mild D Schindler alylglycopeptides ofthe linked type. Our adult patients of Spanish origin enzymatic studies indicated that these phenotype consisting of angiokeratoma, slight Institute of Clinical compounds are not the primary lysosomal dysmorphism, lymphoedema, and prominent Biochemistry, storage products. vacuolisation in endothelial cells, resembling Barcelona, Spain Kanzaki et al.5 In this M J Coll ( Med Genet 1996;33:458-464) the case reported by paper A Chabas we report the mutations of these four patients and describe a new German case with infantile a- Department of Key words: ac-N-acetylgalactosaminidase; a-NAGA de- Clinical Genetics, The ficiency; processing. NAGAdeficiency. The genotype andphenotype Tokyo Metropolitan of the presently known eight cases of a-NAGA Institute of Medical deficiency are discussed. Science, Tokyo, Japan In 1987 two German infants were reported by H Sakuraba Van Diggelen et ail with a profound deficiency Y Suzuki of the lysosomal a-N-acetylgalactos- Patients and methods Correspondence to aminidase (ot-NAGA). Schindler et al2 reported CASE REPORTS Dr van Diggelen. that the patients had infantile neuroaxonal dys- The two German brothers, first reported by van Received 18 October 1995 visceral involvement or Diggelen et all and Schindler et aP with a severe Revised version accepted for trophy without dys- publication 9 January 1996 morphism. Two years later Kanzaki et al-5 infantile form of at-NAGA deficiency, are Human a-N-acetylgalactosaminidase (a-NAGA) deficiency: new mutations and the paradox between genotype and phenotype 459

Table 1 Oligonucleotide primers used to sequence and PCR amplify the coding regions of on visual examination, but neurological tests the a-NAGA gene have not been performed. Length offragment sequenced The Spanish patients reported by Chabas et Exon* Primer sequencest and (location in gene*) and (relative position to exont) al'6 are designated El.1 and E1.2. J Med Genet: first published as 10.1136/jmg.33.6.458 on 1 June 1996. Downloaded from 1 s: GCCTAAGGTTGAGGGCGG (1646-1663) 228 bp (-60§ to +31) as: GAAGGGCCAAAGCACTCCTT (1854-1873) 2 s: AGCTGTGGGGGCAACTGATA (3479-3498) 223 bp (-11 to + 2) CELL CULTURE AND ENZYME ASSAYS as: GGCAAGGCTCATCAGGTGA (3683-3701) 3 s: GAGGCAGGGTGTGGGTGA (4101-4118) 308 bp (-29 to +32) Skin fibroblasts were cultured according to as: GCCCTAAGCGAGGTAGGGTG (4389-4408) routine procedures in Ham's F10 medium sup- 4 s: GTCCTGCTTGAGCCCACTGT (4777-4796) 260 bp (-17 to + 23) as: CCCTCCCCGT' TGCCTAC (5019-5036) plemented with 10% fetal bovine serum and 5 s: CCAGACTGCCTCCTGCACTT (5244-5263) 204 bp (-22 to +29) antibiotics. The cells were harvested with tryp- as: AGGTAAGGAGGCCTGGGTGT (5428-5447) 6 s: CTGGTGCTGGACTCTGCCTT (6157-6176) 248 bp (-7 to + 18) sin seven days after the last subculture and as: GCAGGGTCGTCACAGATGG (6386-6404) were stored at - 70°C until use. 7 s: CCTCCTTGAGGAGATGAGAGC (8991-9011) 282 bp (-4 to + 13) as: CTCAGCCCTCCCCTTCCTT (9254-9272) a-N-acetylgalactosaminidase (xNAGA) ac- 8 s: AGATAAGCTGGGGTGCTGGA (10832-10851) 351 bp (-64 to +5) tivity was assayed in homogenates prepared as: CCCACAGAAATCTGAAGCCC (11163-11182) 9 s: CTGTGCCCCACTCTCATGG (11587-11605) 226 bp (-12 to + 9¶) by sonication of cultured fibroblasts in water. as: AGGCTCAGTGGTGCCACC (11795-11812) Reaction mixtures consisted of 10,ul homo- * Exon number and position of nucleotide in a-NAGA gene according to accession No M59199. genate (30 jug protein for patient materials or t s = sense strand; as = antisense. All primers shown in 5' to 3' direction. 5 jug for controls) and 20 p1 1 mmol/l MU- t Relative position to exon; - = upstream and + = downstream of exon. § Position upstream of start codon in exon 1. xGalNAc, Moscerdam Substrates, Rotterdam) ¶ Position downstream of stop codon in exon 9. in McIlvain's phosphate/citrate buffer, pH 4.7. After incubation for one hour at 37°C, the reactions were terminated by the addition of Table 2 The mutations in all patients with a-NAGA deficiency 200,ul 0.5 mol/I Na2CO3/NaHCO3, pH 10.7, Patient Nucleotide change* (exonlexon) Protein change* Reference and the fluorescence of 4-methylumbelliferone (MU) was measured with a Fluoroskan (Titer- D1.1 G11005A (8/8) E325K/E325K 13 D1.2 G11005A (8/8) E325K/E325K 13 tek) fluorimeter. ,B-galactosidase activity, using D2.1 G11005A (8/8) E325K/E325K Present paper an MU substrate, and protein content of cell NLl.I C4969G/G11005A (4/8) S160C/E325K Present paper NL1.2 C4969G/G11005A (4/8) S160C/E325K Present paper homogenates was determined as described pre- Jl.1 C11017T (8/8) R329W/R329W 14 viously.'7 ,B-galactosidase was also determined El.I G5371T (5/5) E193X/E193X Present paper - E1.2 G5371T (5/5) E193X/E193X Present paper with the disaccharide Gal/Il 3GalNAc (Oxford GlycoSystems) in a galactose de- * Nucleotide and amino acid numbering is according to Yamauchi et al'0 and accession No M59199. hydrogenase based assay.'8 Reaction mixtures The exons in which the mutation was found are shown in brackets. (30 pl) contained 20 pg protein from sonicated fibroblast homogenates, 2-7 mmol/l of the dis- accharide, 67 mmol/l NaCl in 67 mmol/l so- designated Dl. 1 and D1.2. The patients have dium acetate buffer, pH 4 3, and were remained in a vegetative state for the last few incubated for three hours at 37°C, after which http://jmg.bmj.com/ years and are at present 12 and 1 1 years of age. 200 pl 0 1 mol/I Tris/HCl, pH 8-6 containing The newly diagnosed German patient D2. 1 0-6 mmol/1 NAD+ and 0-13 U galactose de- lived in the same village as the Dl family and hydrogenase (Boehringer) was added. Free ga- is consanguineous with the German patients in lactose was then determined by measuring the family Dl. The pregnancy and development absorbance at 340 nm, after incubation for 25 up to the age of 6 months were normal. From minutes at room temperature. activity was determined by 7 months, the patient had several convulsions, on September 30, 2021 by guest. Protected copyright. most of them during fever caused by upper the standard method using the MU substrate'9 respiratory tract infections. He died at the age as well as with a disialyltetraose substrate (DST, of 18 months of hypoxia during a prolonged NeuAca2 - 3Gal 1-3 [NeuAcca2 -6] GalNAc, convulsion causing apnoea. During the last 3 Oxford GlycoSystems). Reaction mixtures months of his life, his development seemed to (30 p1) contained 50 pg protein from fibroblast have stopped. He had never been admitted to homogenates (Potter homogenised), 1-3 mmol/l a hospital and necropsy was not performed. DST, 67 mmolll NaCl in 67 mmol/l sodium The two Dutch patients reported by de Jong acetate buffer, pH 4-3, and were incubated for et al 5 are designated NL1. 1 and NL1 .2. Patient three hours at 37°C. Then free N-acetyl- NL . I was last seen at the age of 3 8 years and neuraminic acid (NANA) was purified as pre- was retarded. After the age of 2-4 years she viously described20 and determined with the had four convulsions, mostly associated with thiobarbituric acid method as described by fever, despite treatment with antiepilectic Denny et al.2' drugs. In retrospect, the retardation began after a severe episode around the age of 1 year. IMMUNOBLOTTING The patient had a high fever, uncontrollable The polyclonal antibody against human x- convulsions, and attacks of apnoea and brady- NAGA, raised in rabbits, was used at a 1000 cardia which required admission to the intens- fold dilution and immunoblotting was per- ive care unit. The fever and convulsions formed as previously described.'2 responded to medication, but a few days after detubation she developed pneumonia with high fever and the clinical signs of sepsis with mul- LABELLING OF cx-GALNAc MOIETIES BY tiple organ failure, from which she recovered LECTIN HISTOCHEMISTRY slowly without convulsions. Her brother The lectin from Helix pomatia (Sigma), which (NL1.2) appeared healthy at the age of 3 years is specific for axGalNAc, was used to stain 460 Keulemans et al

sequencing was performed according to Her- mans et al."3 Restriction enzyme digestions (TaqI, Boehringer; BsgI and MseI, BioLabs)

were performed according to the man- J Med Genet: first published as 10.1136/jmg.33.6.458 on 1 June 1996. Downloaded from ufacturers' instructions.

Results THE GENOTYPE OF O-NAGA DEFICIENCY Using the intron primer sets shown in table 1, DNA fragments encompassing the entire open reading frame ofthe nine exons ofthe oc-NAGA gene were amplified and sequenced (except for patient D2.1); this included all flanking intron regions (table 1). Since patient D2.1 is consanguineous with the D 1 patients, only exon 8 was analysed, and the presence of the Gl1005A mutation (E325K) was confirmed (table 2). This muta- tion destroys a TaqI restriction site and its presence could be confirmed by TaqI digestion of the 351 bp long PCR products of exon 8. The digestion of the normal PCR product into fragments of 174 and 177 bp was not observed in the PCR products of the mutant alleles (fig 1). Sequence analysis ofthe sense and antisense strand as well as restriction enzyme analysis Figure 1 Restriction enzyme analysis of PCR amplified a-NAGA exons 8 and 4. Tc showed that patient D2.1 is homozygous for panel: BsgI digests of exon 4 of NLI patients, their parents, and a healthy sib. Bottoi panel: TaqI digests of exon 8 of the NLI patients, their parents, and patient D2. 1. the E325K mutation (fig 1). We obtained the same results for the D 1 patients (data not shown). Sequence analysis of all ac-NAGA exons of the Dutch sibs (NL1) showed two different mutations. The C1 01 7G substitution in exon 4 (S160C) could be confirmed with the re- striction enzyme BsgI (the mutation creates a new restriction site). Fig 1 shows that the affec-

ted sibs are heterozygous for this mutation http://jmg.bmj.com/ which is ofmaternal origin. The paternal muta- tion in these sibs is the previously described E325K mutation of the German patients. The father of the Dutch patients is not of German ancestry. The healthy sib of the NL1 patients showed the normal pattern in both exons 4 and that she is not a carrier of either 8, indicating on September 30, 2021 by guest. Protected copyright. mutation (fig 1). The S160C mutation was not detected in 80 Dutch white control alleles. Sequence analysis showed that both Spanish (El) patients were homozygous for the non- sense mutation E193X. Homozygosity was confirmed using the restriction enzyme MseI (the mutation creates a new restriction site). In agreement with this observation, both par- ents of the El sibs showed a heterozygous pattern after MseI digestion oftheir PCR prod- Figure 2 Restriction enzyme analysis of PCR amplified cs-NAGA exon 5. MseI dige uct of exon 5 (fig 2). of exon 5 of El patients and their parents. The entire sequenced parts of the a-NAGA gene (table 1) were identical in all patients and two controls (with the exception of the axGalNAc containing material in cultured mutations). Differences, however, were found fibroblasts as described.9 with the published sequence of the oc-NAGA gene (Wang and Desnick, accession No METABOLIC LABELLING M59199). In intron 1, starting at position 3516, Cultured fibroblasts were labelled with 35S- we detected CCCTTGCCCCC (discrepancies methionine following the procedure described underlined, three Gs in sequence M59199). In earlier. " exon 3 we detected three polymorphisms (no amino acid changes) in all samples analysed, DNA AMPLIFICATION AND SEQUENCING which had previously been published by Ya- DNA was isolated from cultured fibroblasts by mauchi et al.10 In all the DNA samples we standard procedures." PCR amplification and tested, a C was found at position 4239 instead Human a-N-acetylgalactosaminidase (a-NAGA) deficiency: new mutations and the paradox between genotype and phenotype 461

Table 3 a-NAGA activity in fibroblasts from patients C>tror eli 9! with infantile and adult x-NAGA deficiency x-NAGA activity

(nmollh/mg) J Med Genet: first published as 10.1136/jmg.33.6.458 on 1 June 1996. Downloaded from Infantile cases D1.l 0-5 D1.2 1-0 D2.1 1-4 NL1.1 3-2 Adult patients E1.1 0-2 E1.2 0-2 Control range Range n=34 40-130 (mean) (81)

of an A and at position 4263 there was a T instead ofa C. In both alleles ofthe two controls and in one allele of the NL1 patients, an A-+G substitution at position 4293 was found. In intron 3, starting at position 4348 we found four Cs instead of three Cs and starting at position 4364 we found GGCC instead of Figure 4 Pulse chase labeUling of a-NAGA in fibroblasts GCGC. In intron 5, at position 5404 we found from ,atzents with a-NAGA deficiency. Cells were labelled a C-+G substitution. with 5S-methionine for four hour (pulse), or they were labelledforfour hours and subsequently maintainedfor 17 hours in labelfree medium. a-NAGA was then immunoprecipitated and analysed by SDS-PAGE. THE BIOCHEMICAL PHENOTYPE OF Molecular weight markers are shown on the right hand I-NAGA DEFICIENCY side in kDa. We studied the effect of these point mutations on the steady state level of ax-NAGA activity and observed differences among the various These low residual activities and their differ- patients. The highest activity was measured in ences are genuine since the activities are linear fibroblasts of patient NLl.1. About half this with the amount ofprotein and incubation time activity was present in cells from the D2.1 and and reproducible (data not shown). Using a Dl patients, whereas x-NAGA activity in the polyclonal antibody against a-NAGA, we El patients was barely detectable (table 3). showed that in all cases the deficiency of a- NAGA enzyme activity was associated with undetectable levels of x-NAGA protein (fig 3). kDa Metabolic labelling of cultured fibroblasts for http://jmg.bmj.com/ four hours, followed by immunoprecipitation -200 of native ax-NAGA protein showed that the synthesis of precursor ax-NAGA (50kDa) is normal in the NL1 patients and patient D1.2 (fig 4). The El patients, on the other hand, do - 97 not synthesise any ot-NAGA protein. Mat-

uration of newly synthesised a-NAGA was in- on September 30, 2021 by guest. Protected copyright. vestigated after a 17hour chase period and mature enzyme (45 kDa) was not detectable in the German patient D1.2, whereas a small amount was present in the NL1 patients (fig 4). p, At the cellular level, a-NAGA deficiency was 43 reflected in the lysosomal storage of oaGalNAc containing compounds (fig 5), which were identified with the otGalNAc specific lectin from Hpomatia. On the other hand all patients with aL-NAGA deficiency excrete sialylglyco- -- 26 peptides, the new patient D2.1 included (data not shown). Unlike the unknown structure of the intralysosomal storage products, the ab- normal urinary excretion products have been fully characterised. We have investigated whether fibroblast homogenates of the most ----- Wrro tt severe patient with a-NAGA deficiency (Dl.2)

v N can hydrolyse the core structures of these ~I I-L - -; c urinary compounds: -3 1 C LLw -J NeuAca2-3Galf31 z o [NeuAca2 - 6]GalNAc and Gal,IB - 3GalNAc. 0 u The results show that fibroblasts with oa-NAGA Figure 3 Immunoblot of a-NAGA from patients with ac-NAGA deficiency. Fibroblast deficiency are fully normal in their sialidase homogenates (150 pg protein) were used. For patient codes see Patients and methods and /3-galactosidase activity towards these oli- section. gosaccharides (table 4). 462 Keulemans et al

tected in urine of the Dl patients.24 These putative primary storage products could sub- sequently serve as acceptor for the synthesis of

the 0 linked type oligosaccharides. Addition J Med Genet: first published as 10.1136/jmg.33.6.458 on 1 June 1996. Downloaded from of glycosides to primary lysosomal storage products (resynthesis) has been reported for " 2 and f3-mannosid- osis." Although we have shown an azGalNAc containing lysosomal storage product in fibro- .. i., I.- blasts of all patients (we did not have per-

il 1%, tf fibroblasts from the , mission to investigate I .. I.- Japanese patient), the structure of these com- pounds is still unknown. Studies are in progress to identify these primary lysosomal storage products. A B (. oc-NAGA deficiency is one of the rarest and probably most heterogeneous lysosomal stor- age disorders. At present, only eight patients are known from five families of German (D), Japanese (J), Dutch (NL), and Spanish (E) descent, including the new case reported here (table 5). Clinical variability is common in lysosomal storage disorders but overlapping signs and symptoms of subtypes is the rule. In a-NAGA deficiency, however, infantile and adult patients have no obvious overlap of signs and symptoms. This led us to consider the possibility that the phenotype of the eight patients is not solely determined by the oc- NAGA deficiency. Three observations are I. difficult to reconcile with a simple genotype- E F: phenotype correlation. Figure 5 Intracellular labelling of a-N-acetylgalactosamine containing compounds in (1) At the histological level, prominent fibroblasts from patients with a-NAGA deficiency. Lectin histochemistry, using the vacuolisation, the hallmark oflysosomal storage aGalNAc specific lectin from Helix pomatia, was carried out on culturedfibroblasts from disorders, was observed in dermal endothelial 1 1 (F). a control (A), the patients Dl.2 (B), D2. 1 (C), NL1. (D), El. (E), and El.2 cells of the late onset patients (patient J1.1,35 patients of the El family'6) but was not ob-

Table 4 Degradation of core structures of 0-glycosidic by neuraminidase served in the severely affected patients of the http://jmg.bmj.com/ and fi-galactosidase Dl family.2 This is remarkable since vacuo- Neuraminidase activity* f,-galactosidase activity* lisation is usually prominent in the most severe subtype of lysosomal storage disorders and less MU-NANAt DS1t MU-flGal§ Galfl1-3GalNAc evident, or even absent, in the milder sub- a-NAGA deficiency (D 1.2) 78 28 750 650 types.28 The most striking histopathology in the Control 45 18 560 480 infantile patients (D1) is non-lysosomal, the GM,-gangliosidosis 35 13 2 Not detectable 0.3 1 430 450 presence of "spheroids" in axons. Although formation has been observed on September 30, 2021 by guest. Protected copyright. * Enzyme activities: nmol/h/mg. axonal spheroid t 4-methylumbelliferyl-L-D-N-acetylneuraminic acid. in other lysosomal storage disorders,2930 it is a t DST = disialyltetraose: NeuAca2 - 3Galfl1 -3 [NeuAcoc2 - 6]GaINAc. peculiar phenomenon in the absence of clear § 4-methylumbelliferyl-,B-D-galactoside. lysosomal histopathology and difficult to ex- plain. Discussion (2) Enzyme activity is often undetectable in Deficiency of x-N-acetylgalactosaminidase (cx- the most severe subtype of a lysosomal disorder NAGA) would imply major urinary excretion whereas patients with milder subtypes have products containing terminal aGalNAc res- residual activity. This has conclusively been idues at the non-reducing end; however, this shown for metachromatic leucodystrophy and is not observed. All patients with ac-NAGA Tay-Sachs disease3' and for Pompe's disease.32 deficiency excrete sialylglycopeptides with ter- Such a logical correlation is missing in cx-NAGA minal neuraminic acid and the oa-GalNAc moi- deficiency. We found a stop mutation (E193X), ety is internal, a linked to serine or threonine.78 deleting half the protein, in the mildly affected The structures are identical to those present Spanish patients (E1). A translation product in 0 linked glycoproteins. Using their core could not be detected by immunoblotting, structure (NeuAcc2 - 3Gal,B1- 3[NeuAccx2 -6] metabolic labelling of newly synthesised a- GalNAc and Gal/B1- 3GalNAc) we showed NAGA did not indicate the presence of a trun- that fibroblasts from the severe D1.2 patient cated protein, and enzyme activity was virtually (table 5) can hydrolyse sialic acid and galactose zero. This indicates that these patients have, from these compounds. This indicates that in essence, null alleles and one would expect the sialylglycopeptides from urine are not the the most severe phenotype. In contrast, these primary lysosomal storage products, which patients presented with a mild, late onset dis- will probably be GalNAcc 1- Ser/Thr. Minor ease, whereas the most severely affected amounts of these compounds have been de- patients (Dl) have significant, albeit very low, Human a-N-acetylgalactosaminidase (s-NAGA) deficiency: new mutations and the paradox between genotype and phenotype 463

Table 5 Summary of all patients with s-NA GA deficiency Infantile a-NAGA deficiency Adult a-NAGA deficiency Dl.l* Dl.2* D2. lt NL.It NLI. 2* l.1§ El.1l1 El.211 J Med Genet: first published as 10.1136/jmg.33.6.458 on 1 June 1996. Downloaded from Age Age at onset I y 6mth 7mth I y No symptoms? 28y 14y NIT Present age (age at death) 13y 12y (1-5y) 5y 3-6y 51 y 42y 38y Neurological signs Convulsions during fever + + + + - + + + +++ + Psychomotor retardation + + + + + + ? + ? Hypotonia + + + +++ ? Physical signs Skeletal abnormalities - - - - - Dysmorphism - - - - - + + ? Organomegaly - - - - - Angiokeratoma - - - - - + + + + + + Lymphoedema - - - - - + + + + + + Histology Vacuolisation - - NI - - + + + + + + Neuroaxonal dystrophy + + + +++ NI¶ s-GalNAc lectin staining + + + + + + + + + +++ +++ NA** NA** + + + + + + Mutations Homozygous E325K S16OC/E325K Homozygous R329Wj Homozygous E193X * DI: van Diggelen et al,' Schindler et al.2 t D2. 1: present paper. fNL1: de Jong et al." §J1.1: Kansaki et al.3 11 El: Chabas et al." ¶ NI = not investigated. **NA= material not available. tt Wang et al. 4

residual enzyme activity. A logical correlation through screening. The newly identified mis- was similarly lacking in studies of Wang et all3 sense mutation in the Dutch patients (S160C) comparing the severe German patients (Dl) results in 4% residual activity, the highest level and the Japanese patient J1.1 with adult x- among all the patients with ax-NAGA de- NAGA deficiency.'4 The authors could not ficiency; this may contribute to the mild pheno- detect significant ot-NAGA enzyme activity in type. Only in these patients a small amount of either case. mature a-NAGA protein was found 17 hours (3) After the clinical description of the first after metabolic labelling of the enzyme in cul- patients with a-NAGA deficiency (Dl), many tured fibroblasts, which is in agreement with laboratories, including ours, have screened Se- the significant residual enzyme activity. The itelberger patients (infantile neuroaxonal dys- newly identified patient D2. 1, who had ident- trophy) for a-NAGA deficiency, but a second ical a-NAGA mutations as the patients with patient with combined x-NAGA deficiency and neuroaxonal dystrophy (D1), could in time http://jmg.bmj.com/ neuroaxonal dystrophy has not been found. have provided important information as to the These three observations indicate that factors relation of a-NAGA deficiency and neuro- other than a-NAGA contribute to the pheno- axonal dystrophy, but the patient died at the typic variation of the patients. The simplest age of 1V years of hypoxia during a convulsion explanation would be that the severe infantile without being examined histologically and patients (D 1) have a "double disease", ne- neurologically.

uroaxonal dystrophy in addition to a-NAGA In summary, all the genetic, biochemical, on September 30, 2021 by guest. Protected copyright. deficiency, without a causal relationship. Ac- histological, and clinical data ofall patients with cidental occurrence of two independent mono- ax-NAGA deficiency strongly suggest that a- genic diseases in one patient can be observed, NAGA deficiency is not a single disease entity. particularly in consanguineous families (the The possibility that factors other than a-NAGA parents of the patients D1.1 and D1.2 are play a major role in determining the phenotypic consanguineous). For example, in one of the variation of the eight known patients with oa- contributing laboratories (Rotterdam) four NAGA deficiency deserves serious con- cases are known: glycogenosis II combined with sideration. The future discovery ofnew patients methylmalonic aciduria, neurofibromatosis I with a-NAGA deficiency will eventually estab- with multiple sulphatase deficiency, Niemann- lish which phenotypes are solely caused by a- Pick type C with Hurler disease, and combined NAGA deficiency and ifotherfactors/genes play Zellweger syndrome and sulphite oxidase de- a role in other phenotypes. This will probably ficiency. One of the first patients with ,B-man- take a long time since the discovery of the first nosidosis"3 had mucopolysaccharidosis type III eight patients took almost a decade. It will there- A as a second disease.3334 fore pay to study the association of a-NAGA Ifthe severely affected German patients (D1) deficiency and neuroaxonal dystrophy in an oa- did have a double disease, this phenotype is NAGA knock-out mouse. not representative of "true" ax-NAGA de- ficiency. The phenotype associated with com- plete a-NAGA deficiency would be a mild, late We thank Professor Hans Galjaard for his continuing support and Tom de Vries Lentsch for photography. Dr Akihiko Tsuji onset disease with angiokeratoma manifested is gratefully acknowledged for preparing the antiserum against in patient Jl.1 and the Spanish patients (El). a-NAGA. The authors thank Dr E Rodriguez-Diaz (Hospital Valle del Nal6n, Asturias) and Dr M Aparicio (Hospital Clinicao The Dutch patient NL1.2, aged 3-3 years with- Universitario, Salamanca) for referring the Spanish patients El. out overt signs or symptoms, could be a pre- We thank Dr Steven U Walkley (New York) for stimulating discussions about the relationship between neuroaxonal spher- clinical case of at-NAGA deficiency detected oids and lysosomal storage. 464 Keulemans et al

1 van Diggelen OP, Schindler D, Kleijer WJ, et al. Lysosomal ficiency in two adult siblings. _7 Inher Metab Dis 1994;17: a-N-acetylgalactosaminidase deficiency: a new inherited 724-31. metabolic disease. Lancet 1987;ii:804. 17 van Diggelen OP, Zhao H, Kleijer WJ, et al. A fluorometric 2 Schindler D, Bishop DF, Wolfe DE, et al. Neuroaxonal enzyme assay for the diagnosis of Morquio disease type dystrophy due to lysosomal a-N-acetylgalactosaminidase A (MPS IV A). Clin Chim Acta 1990;187:131-40.

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