J. Inher. Metab. Dis. 21 416È422(1998)SSIEM and Kluwer Academic Publishers. Printed in the Netherlands(

Identification of a common mutation (R245H)in Sanülippo A patients from the Netherlands

B. WEBER1, J. J. P. VAN DE KAMP2, W. J. KLEIJER3, X.-H. GUO1, L. BLANCH1,O. P. VAN DIGGELEN3, R. WEVERS4, B. J. H. M. POORTHUIS5 andJ. J. HOPWOOD1*

Diseases Research Unit, Department of Chemical &1 L ysosomal Pathology, W omenÏsChildrenÏs Hospital, North of ClinicalAdelaide, Australia ; 2 Department Genetics,

of of ClinicalUniversity L eiden, L eiden ; 3 Department Genetics, University Hospital,ofRotterdam ; 4 Klinisch-Genetisch Centrum, Nijmegen ; 5 Department Pediatrics,

ofUniversity L eiden, T he Netherlands

* Correspondence : L ysosomal Diseases Research Unit, Department of ChemicalPathology, W omenÏs & ChildrenÏs Hospital, 72 King W illiam Road, North Adelaide,South Australia, 5006, Australia

MS received 3.6.97 Accepted 23.11.97

Summary : We have identiÐed a common mutation (R245H) in the sulph-amidase gene of SanÐlippo syndrome type A (mucopolysaccharidosis typeIIIA, MPS IIIA) patients from The Netherlands. Allele-speciÐc oligonucleotidehybridization was used to determine the incidence of this mutation in 45 unre-lated MPS IIIA patients from di†erent regions of The Netherlands. R245H waspresent in 51 alleles, representing 56.7% of the total allelic population. Of 39patients, for whom we have uniform clinical details, 13 MPS IIIA patients whowere homozygous for this common mutation had a more uniform but severeclinical phenotype than the remaining 21 or 5 patients, containing respectivelyone or no R245H alleles. The R245H allele had a higher prevalence in westernrather than eastern regions of The Netherlands.

syndrome or mucopolysaccharidosis type III (MPS III) is inherited as anSanÐlippoautosomal recessive disorder with considerable variation in severity of clinicalphenotype (Neufeld and Muenzer 1995). After a period of normal development,patients can present with symptoms of delayed speech, aggressive behaviour, hyper-activity, sleep disturbance, hirsutism, diarrhoea and coarse facies. Clinical onset inseverely a†ected infants can occur following 2È3 years of apparently normal devel-opment. As mild skeletal pathologyÈtypically severe in other MPS conditionsÈonly develops in older patients, clinical diagnosis is often delayed. This preventstimely and adequate genetic counselling and the option of prenatal diagnosis insubsequent pregnancies, which is reliable in both the Ðrst and second trimesters

416

Common mutation in Dutch MPS IIIA 417

et al As a result, recurrence of a†ected children is a frequent pheno-(Kleijer 1996).menon in MPS IIIA families. More recently, diagnosis has improved owing toincreased awareness, but clinically mild patients can still be missed because of rela-tively less severe clinical presentation and a false negative test by some methodsused for determining heparan sulphaturia and Muenzer(Neufeld 1995).

Currently there are four MPS III subtypes (A, B, C, D) that result from deÐ-ciencies of di†erent enzymes involved in the lysosomal degradation of heparan sul-phate. The incidence of SanÐlippo syndrome (MPS III) has been estimated at1 : 24 000 in The Netherlands de Kamp et al 1 : 324 617 in British(van 1981),Columbia et al and 1 : 45 000 in Australia (Hopwood J. J., unpub-(Lowry 1990)lished observations). The higher incidence in The Netherlands may be the result of anationally higher frequency, or the special e†ort to screen for MPS III patients ininstitutions and a national awareness to identify all cases de Kamp,(van 1979 ; vande Kamp et al MPS IIIA is by far the most common type in northern1981).Europe, whereas MPS IIIB is the most common SanÐlippo syndrome in Greece andItaly and Young et al et al(Whiteman 1977 ; Beratis 1986 ; Michelakakis 1995).Genes coding for the MPS IIIA, B and D enzymes have been cloned et(Robertsonal et al et al et al et1988, 1992 ; Scott 1995 ; Karageorgos 1996 ; Weber 1996 ; Zhaoal Recently 34 di†erent mutations, in the gene coding for the enzyme sul-1996).phamidase involved in MPS IIIA have been described for MPS IIIA patients diag-nosed in Australia, Europe and the United States et al et al(Blanch 1997 ; Bunge

et al et al1997 ; DiNatale 1997 ; Weber 1997).In this paper we report the high incidence of one of the mutations (R245H) in 45

unrelated MPS IIIA patients and the clinical phenotype of patients homozygousand heterozygous for this mutation in 39 patients from The Netherlands.

MATERIALS AND METHODSPatients : The clinically well-deÐned patient group consisted of 39 patients from 39di†erent families, 15 female and 24 male. Eleven died between the ages of 8 and 29years (mean 17 years 10 months) with those still living aged between 3 and 40 years(mean 10 years 6 months). There were 15 equally a†ected siblings. Five patients werefrom consanguineous marriages. Although all patients had clinical phenotypes con-sistent with the diagnosis of SanÐlippo syndrome, there was deÐnite clinical variabil-ity between patients. Full clinical details for six MPS IIIA patients were notavailable. Skin Ðbroblasts from all patients and their a†ected siblings were shown tobe deÐcient in sulphamidase activity et al indicating that they were(Karpova 1996),all MPS IIIA.

DNA preparation : Normal Australian control and The Netherlands MPS IIIAskin Ðbroblasts were cultured and genomic DNA was prepared as previouslydescribed et al(Scott 1992).

Allele-speciÐc oligonucleotide analysis : Oligonucleotides used to PCR-amplifyexon 5 and 6 et al of the sulphamidase gene were(Karageorgos 1996)ns38 (5@-ACCTTGGCAATTAACCTCCTTCCG-3@) and ns87 (5@-CTCAC-CCACATTATGCCGTGACCT-3@). This ampliÐed region was then used for

J. Inher. Metab. Dis. 21 (1998)

418 W eber et al

allele-speciÐc oligonucleotide detection of R245H and wild-type sequence. Allele-speciÐc oligonucleotides used in these hybridization studies were : normal, 5@-

and R245H,GTCGGCCGCATGGACC-3@ ; 5@-GTCGGCCACATGGACCA-3@.Hybridization washing temperature was 65¡C.

RESULTS AND DISCUSSION

A G] A substitution at nucleotide 746 changed an arginine residue to a histidine(R245H). AllelespeciÐc oligonucleotides were used to test for the presence of R245Hin a group of 45 MPS IIIA families from The Netherlands (Table 1).

The R245H mutation was found to be present in 56.7% of all MPS IIIA alleles(51/90 alleles) and absent from 120 alleles from normal individuals. Fourteen R245Hhomozygotes and 23 heterozygotes were found in the group of 45 patients. Only 8patients were observed in which R245H was not present. R245 is not conservedamong sulphatases et al However, the high (56.7%) incidence of(Bond 1997).R245H in the Dutch MPS IIIA population makes possible a study of the relation-ship between genotype and clinical phenotype. Full clinical data were available for27 patients (nine R245H homozygotes, 15 R245H heterozygotes and three without aR245H allele).

Of the 9 patients found homozygous for the R245H allele in the group of 27patients, 3 were from consanguineous marriages. Development of a clinical pheno-type had generally been slow for all 9 patients. However, by age 3È5 years, behav-ioural disturbances and poor or absent speech development were clearly present.There was an obvious regression in 6 patients between 3 and 6 years of age. Patientswere usually admitted to institutions at 5È7 years of age. Usual problems weresevere behaviour and sleep disturbances (in 4/8 and 6/8, respectively), diarrhoea(7/9) and recurrent infections (4/9). Four R245H homozygous patients had had con-vulsions while their a†ected siblings did not appear to have had convulsions. All 9patients had increased head circumference and hepatomegaly. A†ected siblings inthese families had similar increases in head circumference and liver size. Threepatients had more or less severe contractures, especially of hands, feet and elbows.

Five patients died between 8 and 21 years of age (mean 13 years). Eight of theR245H homozygous patients were alive at ages between 3 and 11 years. There wasno clear evidence of intrafamilial variability in time of death, except that one patientdied aged 10 years and his a†ected sibling died aged 16 years.

Except for a broader interfamilial and intrafamilial variability, the clinical pictureof R245H heterozygous patients was comparable to that observed for the R245Hhomozygous patients. In most cases the heterozygous patients usually presentedbetween 3 and 5 years of age. Regression occurred between 3 and 18 years andinstitutionalization ranged from 4 to 21 years. Behavioural disturbance, diarrhoeaand recurrent infections were as frequent as in the R245H homozygote group andsleep disturbances were slightly less. However, there was more discrepancy in clini-cal phenotype between a†ected siblings in the heterozygous group compared to the

J. Inher. Metab. Dis. 21 (1998)

Common mutation in Dutch MPS IIIA 419

Table 1 List of MPS IIIA patients studied

First Second A†ected Age at CurrentPatienta Sex allele allele siblings Provinceb death (years) age (years)

1C f R245H R245H Èc ZH 18 È2C f R245H R245H È Ov È 73 f R245H R245H m ZH È 94C m R245H R245H m ZH 10 È5 m R245H R245H È ZH 8 È6 m R245H R245H f U 21 È7 m R245H R245H È ZH È 108 m R245H R245H È ZH 8 È9 m R245H R245H m/f U È 11

10* m R245H R245H È Dr È 411* f R245H R245H È NB È 912* f R245H R245H È Zld È 313* f R245H R245H È F È 714* f R245H R245H È L È È15 m R245H È È NB È 1616 f R245H È È ZH 16 È17 m R245H È È Gld È 918C m R245H È f Dr È 2219 m R245H È f ZH 21 È20 m R245H È f U 29 È21 m R245H È m/f ZH È 4022 m R245H È m Gld 27 È23 m R245H È ZH È 624 m R245H È È ZH È 1325 m R245H È È ZH È 526 f R245H È È NB È 1927 m R245H È È ZH È 2028 m R245H È È Dr È 929 m R245H È È ZH È 430* f R245H È È Gld È 431* m R245H È m Gld È 332* f R245H È È Gld È 2033* f R245H È È NH È 434* f R245H È È U È 1435* f R245H È È U 536* È R245H È È NH È È37* m R245H È È NB È È38C m È È f Fr 20 È39 f È È f Gld 19 È40 m È È m Ov È 1041* f È È È NB È 842* m È È È Gld È 443* È È È È Gld È È44* È È È È Gld È È45* m È È È NB È È

a C, consanguineous ; *, limited clinical datab Province abbreviations, see Table 2c È, unknown

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420 W eber et al

Table 2 Distribution of R245H alleles in provinces of The Netherlands

Province Population (millions) R245H/m R245H] R245H[

Drente (Dr) 0.5 8.0 4 2Friesland (Fr) 0.7 2.9 2 2Gelderland (Gld) 2.0 2.5 5 13Groningen (Gr) 0.6 È 0 0Limburg (Lb) 1.0 4.0 2 0Noord Brabant (NB) 2.5 1.6 4 6Noord Holland (NH) 2.5 1.2 3 3Overijssel (Ov) 1.0 2.0 2 2Utrecht (U) 1.0 7.0 7 3Zuid Holland (ZH) 3.5 5.7 20 8Zeeland (Zld) 0.4 5.0 2 0

Total 15.7 3.3 51 39

homozygous group. Two patients (and an a†ected sibling) had had convulsions.Slight hearing loss was observed in 4/15 patients (and in an a†ected sibling of onepatient). Three R245H heterozygous patients had a normal head circumference. Thea†ected sibling of one of these patients also had a normal head size, whereas thea†ected sibling of another patient with normal head size was macrocephalic.Hepatomegaly was not present in 3 patients nor in 2 a†ected siblings. Contractureswere seen in half of the patients. Four patients died between 16 and 29 years of age(mean 23 years and 4 months). The 17 living patients were aged between 3 and 40years (mean 12 years and 6 months). There was a discrepancy within R245H hetero-zygous patient families. One patient died aged 6 years and his a†ected sibling at 21years of age. Another patient is still alive at 40 years while his a†ected sibling died at20 years of age.

There were three MPS IIIA patients without an R245H allele. One was from aconsanguineous marriage. All three patients had one other a†ected sibling. The twoa†ected siblings from the consanguineous marriage regressed from age 5 years.There was no history of infections or convulsions. One sibling had a severe hearingloss. Death occurred at 8 and 20 years of age. The second sib-pair are alive at 10and 12 years of age, have no regression and remain at home. Recurrent infections orconvulsions have not been observed. Both have hearing loss. The third sib-pairregressed at 3 years of age ; both had convulsions and were admitted to an instituteat ages 5 and 6 years. They died aged 12.5 and 20 years, respectively.

The R245H allele appears to be unevenly distributed throughout the country witha preponderance observed in the western part of the country, particularly in theprovinces of Utrecht and Zuid Holland which have R245H allele incidence permillion population of 7.0 and 5.7, respectively, compared to an average of 2.1 for therest of the country This suggests an uneven geographical distribution for(Table 2).the R245H allele in The Netherlands and may reÑect a founder e†ect in this regionfor this allele.

J. Inher. Metab. Dis. 21 (1998)

Common mutation in Dutch MPS IIIA 421

ACKNOWLEDGEMENTS

thank Patrick Simons for cell culture assistance. This work was supported byWegrants from the National Health and Medical Research Council of Australia, TheWomenÏs & ChildrenÏs Hospital Research Foundation and a Raymond H. Bryan IVFellowship from the American MPS Society, Inc. B.W. was supported by a long-term fellowship of the Human Frontier Science Program, Strasbourg.

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