oct(fiPmmbfaN2otttc

s

sNgadsgcg

MA

Sialuria in a Portuguese Girl: Clinical, Biochemical,and Molecular Characteristics

Helena Ferreira,* Raili Seppala,† Rui Pinto,‡ Marjan Huizing,† Esmeralda Martins,* Ana Cristina Braga,*Lourenco Gomes,* Donna M. Krasnewich,§ M. Clara Sa Miranda,‡ and William A. Gahl†

*Departamento de Pediatria, Hospital Maria Pia, Porto, Portugal; †Section on Human Biochemical Genetics, Heritable Disorders

olecular Genetics and Metabolism 67, 131–137 (1999)rticle ID mgme.1999.2852, available online at http://www.idealibrary.com on

Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892;‡Instituto de Genetica Medica, Jacinto de Magalhaes, 4150 Porto, Portugal; and §Medical Genetics Branch,

tional

ebruar

az

mehmdaqfiNcodfmpdst(

pcb2r

National Human Genome Research Institute, Na

Received F

Sialuria, a disorder of sialic acid (NeuAc) metab-lism characterized by increased free NeuAc in theytoplasm of cells, is due to failure of CMP-Neu5Aco feedback inhibit UDP-N-acetylglucosamineUDP-GlcNAc) 2-epimerase. We now describe thefth patient in the world with sialuria, a 7-year-oldortuguese girl with developmental delay, hepato-egaly, coarse facies, and urinary excretion of 19mol of free NeuAc/mg creatinine. The patient’s fi-roblasts stored excess free NeuAc in the cytosolicraction, and fibroblast UDP-GlcNAc 2-epimerasectivity was only 26% inhibited by 100 mM CMP-eu5Ac (normal, 79%). The patient’s UDP-GlcNAc-epimerase gene displayed an R266Q mutation innly one allele, consistent with known sialuria mu-ations and with the proposed dominant nature ofhis disorder. Extensive description of sialuria pa-ients will help to define the clinical and biochemi-al spectrum of this disease. © 1999 Academic Press

Key Words: sialuria; UDP-GlcNAc 2-epimerase;ialic acid; feedback inhibition; allosterism.

N-Acetylneuraminic acid (NeuAc), or sialic acid,erves as the terminal negatively charged sugar in-linked glycoproteins. NeuAc is synthesized fromlucose through N-acetylmannosamine (ManNAc)nd reacts with cytidine triphosphate to form cyti-ine monosphosphate (CMP)-Neu5Ac, the charged

ialic acid donor for synthesis of sialylglycoconju-ates (1). The rate-limiting step in NeuAc synthesis,onversion of uridine diphosphate (UDP)-N-acetyl-lucosamine to ManNAc, is catalyzed by UDP-N-

crcs

131

Institutes of Health, Bethesda, Maryland 20892

y 16, 1999

cetylglucosamine (GlcNAc) 2-epimerase. This en-yme is feedback inhibited by CMP-Neu5Ac (2,3).There exist several human disorders of sialic acidetabolism. Sialuria is a very rare metabolic dis-

ase due to failure of CMP-Neu5Ac to feedback in-ibit UDP-GlcNAc 2-epimerase (4–6). This abnor-ality of allosterism causes variable degrees of

evelopmental delay, coarse facies, and hepatomeg-ly, along with daily urinary excretion of gramuantities of free (unbound) NeuAc (7). In culturedbroblasts, the intracellular accumulation of freeeuAc occurs within the cytoplasm (6), in marked

ontrast to the lysosomal storage of sialic acid inther disorders of sialic acid metabolism (8). In Sallaisease (9,10) and its more severe variant, infantileree sialic acid storage disease, or ISSD (11), lysoso-

al free NeuAc accumulates due to defective trans-ort out of lysosomes. In sialidosis (neuraminidaseeficiency) and in galactosialidosis, bound NeuAc istored in lysosomes as sialylglycoconjugates becausehe terminal sialic acid residues cannot be cleaved7,12).

Only four patients with sialuria have been re-orted to date (6,13–17). Recently, the molecularauses of three of these cases were elucidated (18),ased upon the cloning of the human UDP-GlcNAc-epimerase. This gene resembles the gene for theat epimerase (19,20), a bifunctional enzyme, and

odes for a 722-amino-acid protein. The three sialu-ia patients exhibited missense mutations in a spe-ific region of the gene, resulting in the amino acidubstitutions R263L, R266W, and R266Q. These

1096-7192/99 $30.00Copyright © 1999 by Academic Press

All rights of reproduction in any form reserved.

pl

FIG. 1. Clinical characteristics of this patient at age 7 years. (A) Facial features include broad nasal bridge and hypertelorism,eriorbital fullness, long philtrum, and thin upper lip. (B) Ears are prominent and borderline low-set. Posterior hairline is low. (C) Notearge great toes, widely separated from second toes, and 2,3 syndactyly.

mf2o

wo

the 5th

m

utations indicate that the allosteric binding siteor CMP-Neu5Ac resides in the region of codons63–266. Since the second epimerase allele of eachf the three sialuria patients had a normal sequence,

FIG. 2. Growth curves. Weight has increased from

SIA

FIG. 3. Skeletal radiograph. (A) Clefting and mild beaking of theedially.

e have speculated that sialuria is a dominant dis-rder (18). Since the parents of all sialuria patientsre clinically normal, we believe that the affectedndividuals represent new mutations.

to the 75th centile, and height has remained normal.

133A

LURI

ai

thoracolumbar vertebrae. (B) Lateral distal phalanges curve

mic

mgeawcuedrdt1

h.wnthpts2m(er

pachss6ibv7t5h5g

tbeecbttp,Bvwwgi

PC

NLM

1 IRA E

We now describe the clinical, biochemical, andolecular characteristics of the fifth case of sialuria

n the world, with comparison to the four knownases.

MATERIALS AND METHODS

Patient description. This 7 and 5/12-year-old fe-ale (Figs. 1A–1C) is the only child of nonconsan-

uineous Portuguese parents. Pregnancy and deliv-ry at term were normal, with APGAR scores of 7nd 10 at 1 and 5 min, respectively. The birth weightas 2650 g (10%), length 46 cm (10%), and head

ircumference 33 cm (10%). The neonatal period wasnremarkable. During infancy, the patient experi-nced obstructive sleep apnea, confirmed by oxygenesaturation, and tricuspid insufficiency with aight atrial/right ventricular gradient. Psychomotorevelopment was borderline normal, with head con-rol at 3 months, sitting at 5 months, and walking at8 months.At 28 months, the patient had coarse facies, with

ypertelorism (inner canthal distance, 3.5 cm,97%); interpupilary distance, 5.5 cm (.97%); faceidth, 11.5 cm (.97%); convergent strabismus; wideasal bridge; anteverted nostrils; long philtrum andhin upper lip; prominent ears with a low posteriorairline; high-arched palate and normal tongue; hy-oplastic and widely spaced nipples (internipple dis-ance, 14.5 cm (50–75%); large haluces with a widepace between the first and second toes; and partial,3 syndactyly of the toes. She exhibited decreased

TABLE 1Free Sialic Acid Levels in Cultured Fibroblasts

N

Free NeuAc(nmol/mg protein)

X 6 SEM Range

atient 4 17.4 6 7.4 4.1–38.0ontrolsNormals 6 0.2 6 0.1 0–0.5Published normalsa 10 1.1 6 0.1 0–3.2Sialuria 2 33 7,58Published sialuriab 3 143 75–273

a Ref. (11).b Ref. (6).

34 FERRE

uscle mass and subcutaneous fat, weight, 10.2 kg,5%); height, 88.3 cm (25%); and head circumfer-nce, 48 cm (25–50%). The liver was 7 cm below theight costal margin.

S

t

At 32 months, bilateral adenotonsillectomy waserformed, resulting in improved respiratory statusnd growth. The liver remained 4 cm below the rightostal margin. At 4 and 5 years of age, the patientad pneumonias which resolved, and at 6 years oldhe developed a facial nerve palsy. She experiencedeizures with fever on eight occasions between 2 andyears of age, at which time the neurological exam-

nation, EEG, and cerebral MRI were normal. Herehavior was described as hyperkinetic, and a de-elopmental assessment revealed a full-scale IQ of8. She attended normal school with additional tu-oring. Growth curves are shown in Fig. 2. At 7 and/12 years of age, her weight was 26.4 kg (75–90%);eight, 128.5 cm (75–90%); and head circumference,2 cm (50–75%). The facial dysmorphology had pro-ressed slightly, and the hepatomegaly persisted.Over the course of her evaluation, several labora-

ory studies were performed. At age 28 months, thelood count and differential, C-reactive protein,rythrocyte sedimentation rate, coagulation studies,lectrolytes, glucose, creatinine, total cholesterol,reatine kinase, serum protein electrophoresis, totalilirubin, alkaline phosphatase, and g-glutamyl-ranspeptidase were normal. The alanine amino-ransferase was 76 U/L (normal, ,39) and the as-artate aminotransferase was 114 U/L (normal,56). Serologic tests for hepatitis B and C, Epstein–arr virus, toxoplasmosis, syphilis, and cytomegalo-irus were negative. Immunoglobulins A, G, and Mere normal, while IgE was elevated fivefold. Stoolas negative for ova and parasites, as well as patho-enic bacteria. Sweat chloride, antigliadin antibod-es, and cow milk protein RAST were negative. Ab-

TABLE 2Free Sialic Acid Distribution in Fibroblast

Subcellular Fractions

Fraction

(% of total recovered)

Hexosaminidase Free NeuAc

Patient Patienta Sialuriab Controlb

uclear 27 5 5, 7 18ysosomal 69 5 4, 3 21icrosomal 2 2 2, 2 6

T AL.

oluble 1 88 89, 88 54

a Free NeuAc in whole cell homogenate was 17.6 nmol/mg pro-ein.

b Ref. (6).

dw

mlanlrf8c

0imcgdbapm

w(ca(UpmC

dpGtTfpcC

Nideaw(12N

LURI

ominal ultrasound showed an enlarged liver thatas homogeneous in appearance.At 32 months, a-1 antitrypsin and ceruloplas-in were normal, as well as serum ammonia and

actate levels. Plasma and urinary amino acidsnd urinary organic acids were normal, but uri-ary reducing substances were positive. Thin-

ayer chromatography for urinary oligosaccha-

FIG. 4. Inhibition of UDP-GlcNAc 2-epimerase by CMP-eu5Ac. [3H]UDP-GlcNAc serves as substrate, and [3H]ManNAc

s the product of the epimerase. [3H]GlcNAc represents a degra-ation product of [3H]UDP-GlcNAc, and not a product of thepimerase (6). (A) Normal fibroblast epimerase activity. In thebsence of CMP-Neu5Ac, significant ManNAc is formed (left),hile 100 mM CMP-Neu5Ac inhibits ManNac formation by 79%

right). (B) Fibroblast epimerase activity in the current patient.00 mM CMP-Neu5Ac (right) inhibited ManNAc production by6% compared with ManNAc production in the absence of CMP-eu5Ac (left).

SIA

ides was negative for lactose, sucrose, glucose,ructose, galactose, xylose, and ribose, but 30- to0-fold elevated for free sialic acid (19 mmol/mgreatinine compared with normal values of 0.24 –

cpRs

.64 mmol/mg creatinine). Neuraminidase activityn cultured skin fibroblasts was normal. Electron

icroscopy of a skin biopsy showed fibroblastsontaining cytoplasmic inclusions. Skeletal radio-raphs at age 5 years showed minimal changes ofysostosis multiplex, including abnormal verte-ral bodies with minor beaking of T9-L1 (Fig. 3A)nd medial “clawing” of the lateral three distalhalanges (Fig. 3B). Renal ultrasound was nor-al.

Cellular and biochemical studies. Fibroblastsere cultured and fractionated as previously described

6). Free NeuAc was determined by anion-exchangehromatography using a Dionex AS6 Ion-Pack columnnd quantitated by pulsed amperometric detectionDionex Bio LC, Sunnyvale, CA), as described (6).DP-GlcNAc 2-epimerase was assayed according toreviously published procedures (6), and protein waseasured using a bicinchoninic acid kit from Piercehemical Co.

Molecular studies. Standard molecular proce-ures were employed (21). Fibroblast RNA wasrepared using Trizol Reagent (Life Technologies,aithersburg, MD) and converted to cDNA using

he SuperScript Preamplification System (Lifeechnologies). This cDNA served as the template

or PCR amplification. Automated sequencing waserformed using the dRhodamine Terminator Cy-le Sequencing kit (Applied Biosystems, Fosterity, CA).

135A

FIG. 5. Direct sequencing of normal and sialuria fibroblastDNA. Normal sequence (top) shows CGG for codon R266, com-ared with CAG in the patient’s sequence. Heterozygosity for the266Q mutation is illustrated by two peaks on the automatedequencer printout.

ftdnpntvp1ln

pral2rstcfb

d2Nifisnlbs

gchgw

abSdfTtfmfbe

te3bNlfiU(Rv

mm

ADCHDSSGU

e(

1 IRA E

RESULTS

Free NeuAc concentrations in fibroblasts culturedrom the patient averaged 17.4 6 7.4 nmol/mg pro-ein compared with 0.2 6 0.1 nmol/mg protein for sixifferent normal individuals and values of 7 and 58mol/mg protein for fibroblasts from known sialuriaatients, assayed concurrently (Table 1). Both ourormal and affected controls gave values lower thanhose previously reported (5,9), but our patient’salues were 87 times our mean normal value, com-ared with published values for sialuria which were30 times normal (8). Our patient’s bound NeuAcevel was normal (22.4 6 8.0 nmol/mg protein, n 5 3;ormal range 5 22.6 6 7.9 nmol/mg protein, n 5 4).The subcellular distribution of free NeuAc in our

atient’s fibroblasts was also characteristic of sialu-ia. In one experiment, 69% of the hexosaminidase,lysosomal enzyme marker, was recovered in the

ysosomal fraction of the patient’s fibroblasts (Table), indicating intact lysosomes. In the same prepa-ation, 88% of the recovered NeuAc was found in theoluble fraction (Table 2). This cytoplasmic distribu-ion is typical for known cases of sialuria (6), and

TABLE 3Characteristics of Known Sialuria Patients

Case Nos.

1a 2b 3c 4d 5e

ge (years) 3 2 1 6 7elay 11 11 2 11 11oarse facies 1 1 1 1 1epatomegaly 1 11 111 11 11ysostosis multiplex 1 2 2 2 1leep apnea 2 2 2 1 1eizures 1 2 2 1 1rowth (%) 10 10 50 50 75rine S.A. (mmol/mg creat)f 115g 79 h 117 19

a Refs. (13) and (14).b Refs. (15) and (16).c Ref. (6).d Ref. (17).e Current patient.f Normal values, 0.24–0.64 mmol/mg creatinine.g This value was derived from values reported in mg/24 h using

stimates of creatinine excretion based on age and body weight15).

h “Massively elevated” (6).

36 FERRE

ontrasts markedly with the 56–77% recovery ofree NeuAc in the lysosomal fraction of ISSD fibro-lasts (11).Definitive diagnosis of sialuria is achieved by

ih(f

emonstrating reduced inhibition of UDP-GlcNAc-epimerase by CMP-Neu5Ac. In fact, 100 mM CMP-eu5Ac inhibited normal fibroblast epimerase activ-

ty 79%, but inhibited the epimerase of our patient’sbroblasts by only 26% (Fig. 4). A repeat studyhowed 17% inhibition using our patient’s cells (dataot shown). These values compare well with pub-

ished values of 19–27% inhibition of the epimerasey 50 mM CMP-Neu5Ac in the fibroblasts of otherialuria patients (6).Finally, the mutation in the patient’s epimerase

ene was demonstrated by sequencing of fibroblastDNA. A G to A change in codon 266, present in theeterozygous state, gave rise to a glutamine for ar-inine substitution (Fig. 5). Parental specimensere not available for analysis.

DISCUSSION

Since five patients worldwide have now been di-gnosed biochemically with sialuria, this disorder iseginning to acquire a clinical identity of its own.ialuria patients appear to have variable degrees ofevelopmental delay and hepatomegaly, with uni-ormly mild coarseness of facial features (Table 3).wo patients have exhibited sleep apnea and fea-ures of dysostosis multiplex and three have suf-ered seizures. Normal growth has accompanied

assive urinary excretion of free NeuAc. Clinicalollowup of the original French patient, who shoulde approximately 35 years old by now, would bextremely enlightening.The current patient displayed biochemical fea-

ures diagnostic of sialuria. Her urinary free NeuAcxcretion was modest by sialuria standards, but still0–80 times normal. Likewise, her cultured fibro-lasts contained 87 times the normal amount of freeeuAc (Table 1), and fully 88% of this resided in the

ysosomal fraction (Table 2). The diagnosis was con-rmed by demonstrating only 26% inhibition ofDP-GlcNAc 2-epimerase by 100 mM CMP-Neu5Ac

Fig. 4). Molecular analysis revealed a characteristic266Q mutation in the epimerase gene (Fig. 5) pre-iously reported in patient No. 3 (18).Sialuria represents a very rare example of a hu-an disorder in which an enzymatic activity is nor-al, but the enzyme fails to be normally feedback

T AL.

nhibited by an end-product. Another example is theyperinsulinism and hyperammonemia syndrome22), in which glutamate dehydrogenase fails to beeedback inhibited by GTP. In both of these appar-

et

sdioNofsMsridc

1

1

1

1

1

1

1

1

1

1

2

2

LURI

ntly autosomal dominant disorders, the location ofhe mutation defines the enzyme’s allosteric domain.

It remains important to identify and characterizeialuria patients in an effort to better define thisisorder. In this regard, the key laboratory screen-ng test is thin-layer chromatography for urinaryligosaccharides, which will reveal an increased freeeuAc band. At that point, the cytoplasmic location

f free NeuAc in fibroblasts must be verified to dif-erentiate sialuria from a disorder of lysosomal freeialic acid transport, namely, Salla disease or ISSD.olecular diagnosis may prove straightforward,

ince the known sialuria mutations appear to beestricted to a very small area of the epimerase gene,.e., the region of codons 263–266 (18). As for theiagnosis of any rare disease, a high index of suspi-ion is extremely helpful.

REFERENCES

1. Schauer R. Sialic acids: Chemistry, metabolism and func-tions of sialic acids. Adv Carbohydr Chem Biochem 40:131–234, 1982.

2. Kornfeld S, Kornfeld R, Neufeld EF, O’Brien PJ. The feed-back control of sugar nucleotide biosynthesis in liver. ProcNatl Acad Sci USA 52:371–379, 1964.

3. Sommar KM, Ellis DB. Uridine diphosphate N-acetyl-D-glu-cosamine 2-epimerase from rat liver: Catalytic and regula-tory properties. Biochim Biophys Acta 268:581–589, 1972.

4. Thomas GH, Reynolds LW, Miller CS. Overproduction ofN-acetylneuraminic acid (sialic acid) by sialuria fibroblasts.Pediat Res 19:451–45, 1985.

5. Weiss P, Tietze F, Gahl WA, Seppala R, Ashwell G. Identi-fication of the metabolic defect in sialuria. J Biol Chem264:17635–17636, 1989.

6. Seppala R, Tietze F, Krasnewich D, Weiss P, Ashwell G,Barsh G, Thomas GH, Packman S, Gahl WA. Sialic acidmetabolism in sialuria fibroblasts. J Biol Chem 266:7456–7461, 1991.

7. Gahl WA, Krasnewich DM, Williams JC. Sialidoses. InHandbook of Clinical Neurology, Vol. 22 (Moser HW, Ed.).Amsterdam: Elsevier, pp 353–375, 1996.

8. Gahl WA, Schneider JA, Aula PP. Lysosomal transport dis-orders: Cystinosis and sialic acid storage disorders. In TheMetabolic and Molecular Bases of Inherited Disease, 7th ed.

SIA

(Scriver CR, Beaudet AL, Sly WAS, Valle DL, Eds.). NewYork: McGraw–Hill, pp 3763–3797, 1995.

9. Aula P, Autio S, Raivio KO, Rapola J, Thoden CJ, KoskelaSL, Yamashina I. “Salla disease”: A new lysosomal storagedisorder. Arch Neurol 36:88–94, 1979.

2

0. Renlund M, Tietze F, Gahl WA. Defective sialic acid egressfrom isolated fibroblast lysosomes of patients with Salladisease. Science 232:759–762, 1986.

1. Tietze F, Seppala R, Renlund M, Hopwood JJ, Harper GS,Thomas GH, Gahl WA. Defective lysosomal egress of freesialic acid (N-acetylneuraminic acid) in fibroblasts of pa-tients with infantile free sialic acid storage disease. J BiolChem 264:15316–15322, 1989.

2. Beaudet AL, Thomas GH. Disorders of glycoprotein degra-dation and structure: a-Mannosidosis, b-mannosidosis, fu-cosidosis, sialidosis, aspartylglycosaminuria, and Carbohy-drate-deficient Glycoprotein Syndrome. In The Metabolicand Molecular Bases of Inherited Disease, 7th ed. (ScriverCR, Beaudet AL, Sly WS, Valle DL, Eds.). New York:McGraw–Hill. pp 2529–2562, 1995.

3. Fontaine G, Biserte G, Montreuil J, Dupont A, Farriaux JP.La sialurie: Un trouble metabolique original. Helv PaediatrActa 23(Suppl. XVII):1–32, 1968.

4. Montreuil J, Biserte G, Strecker G, Spik G, Fontaine G,Farriaux JP. Description d’un nouveau type de mediturie:La sialurie. Clin Chim Acta 21:61–69, 1968.

5. Wilcken B, Don N, Greenaway R, Hammond J, Sosula L.Sialuria: A second case. J Inher Metab Dis 10:97–102, 1987.

6. Don NA, Wilcken B. Sialuria: Follow-up report. J InherMetab Dis 14:942, 1991.

7. Krasnewich DM, Tietze F, Krause W, Pretzlaff R, Wenger D,Diwadkar V, Gahl WA. Clinical and biochemical studies inan American child with sialuria. Biochem Med Metab Biol49:90–96, 1993.

8. Seppala R, Lehto V-P, Gahl WA. Cloning of human uridine-diphosphate-N-acetylglucosamine 2-epimerase (UDP-Glc-NAc 2-epimerase) and mutation analysis in sialuria. Am JHum Genet 63:A35/184, 1998 (Abstr).

9. Hinderlich S, Stasche R, Zeitler R, Reutter W. A bifunctionalenzyme catalyzes the first two steps in N-acetylneuraminicacid biosynthesis of rat liver: Purification and characteriza-tion of UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase. J Biol Chem 272:24313–24318, 1997.

0. Stasche R, Hinderlich S, Weise C, Effertz K, Lucka L, Moor-mann P, Reutter W. A bifunctional enzyme catalyzes the firsttwo steps in N-acetylneuraminic acid biosynthesis of rat liver:Molecular cloning and functional expression of UDP-N-acetyl-glucosamine 2-epimerase/N-acetylmannosamine kinase. J BiolChem 272:24319–24324, 1997.

1. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: ALaboratory Manual, 2nd ed. Cold Spring Harbor, NY: ColdSpring Harbor Laboratory, 1989.

137A

2. Stanley CA, Lieu YK, Hsu BYL, Burlina A, Greenberg CR,Hopwood NJ, Perlman K, Rich BH, Zammarchi E, Poncz M.Hyperinsulinism and hyperammonemia in infants with reg-ulatory mutations of the glutamate dehydrogenase gene.N Engl J Med 338:1352–1357, 1998.