Original Paper 1610105

Eur J Hum Genet 1993;1:296-300

Marja Mietala* Prospects of Carrier Screening of Kristiina Grön* Ann-Christine Syvänenb Aspartylglucosaminuria in Finland Leena Peltonenh Pentì Aula* a Department of Medical Genetics, University of Turku, and b Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland Abstract The frequency of carriers of the AGUfìii mutation, the pre­ dominant mutation causing aspartylglucosaminuria in Fin­ land, was determined in a population sample comprising 553 newborns from a delivery hospital in southern Finland, and 607 from a hospital in northern Finland. The AGUpin point mutation was identified from cord blood samples using the PCR-based, solid-phase minisequencing method. Nineteen Key Words carriers of the AGUpin mutation were detected, 8 (1:69) in the Aspartylglucosaminuria sample from the southern and 11 (1:55) from the northern Carrier screening population, respectively. The solid-phase minisequencing Mutation detection method proved to be rapid and convenient for the detection of PCR the AGUfìii mutation, and can readily be applied in large-scale Solid-phase minisequencing carrier screening at the population level.

Introduction between 1 and 4 years of age usually as a delay in speech and/or motor development. At the Aspartylglucosaminuria (AGU) is a reces- adult age, AGU patients are severely mentally sively inherited lysosomal storage disease, en­ retarded with characteristic coarse facial fea­ riched in the Finnish population [1], The dis­ tures. Their life span is shortened to 30-40 ease is caused by deficient activity of aspartyl- years [3]. glucosaminidase (AGA) resulting in the accu­ Over 200 AGU patients have been identi­ mulation of glycoasparagines in the lysosomes fied in Finland in contrast to approximately [2], AGU is clinically characterized by severe 40 cases in the rest of the world. The disease is mental retardation, susceptibility to infec­ somewhat more frequent in northern and tions and various connective tissue manifesta­ eastern Finland, the estimations of the carrier tions. The first signs of the disease present frequency ranging from 1:30 to 1:80 based on

Received. March 8,1993 Maija Hietala ©1993 Revision received: June 30,1993 Department of Medical Genetics S. Karger AG, Basel Accepted: July 8,1993 University of Turku 1018-4813/93/ Kiinamyllynkatu 10 0014-0296S2.75/0 FIN-20520 Turku (Finland) the prevalence of the disease [3, 4]. So far the G NORMAL heterozygotes have been identified by re­ —-C c-~:> duced AGA activity in lymphocytes [5]. How­ G MUTATED ever, the high individual variation in AGA 1 1 -A> --C-— activity and the laborious technique make the G -O enzyme assay less suitable for large-scale car­ —G— C -o rier screening. 2.

The mutation responsible for the AGU dis­ -C- —-C— - ease in Finland has recently been identified. G —G -G------The predominant mutation (AGUpin) is a -C e point mutation (G > C) at base pair position 3.

488 of the AGA cDNA, causing a cysteine to -G- ■<>4. serine substitution at amino acid position 163 ■—-c...... <2>—f of the AGA protein. The Cysi63 -» Ser mu­ DNÂ DN 4. poi ol tation is always linked to another point muta­ C* G* tion only 5 bp apart which changes arginine at IC position 161 to glutamine. This mutation does <3 ff G* not influence the enzyme activity, and its bio­ -C- C logical significance is still unknown [6]. Nine­ 5. ty-eight percent of the AGU alleles in the Fin­ IC* |G* nish population carry these two mutations [7]. 6. So far, only 5 Finnish compound heterozygous AGU patients have been identified with the AGUpin mutation in one allele and another yet O BIOTIN y- STREPTAVIDIN unknown mutation in the other [7], DETECTION STEP PRIMER A simple DNA test has been developed for * 3H LABELED NUCLEOTIDE the detection of the AGUpin mutation [7]. This solid-phase minisequencing test is based on amplification of the DNA fragment spanning Fig. 1. Principle of solid-phase minisequencing: 1 = the mutation using one biotinylated PCR amplification with one biotinylated primer; 2 = affini­ primer, collection of the amplification product ty capture on a streptavidin-coated support; 3 = déna­ turation (removal of the unbiotinylated strand); 4 = on a streptavidin-coated matrix, and primer- annealing of a detection step primer and elongation guided single nucleotide incorporation at the with one labeled nucleotide; 5 = dénaturation; 6 = site of the mutation [7, 8] (fig. 1). The result of eluted radioactivity measured in a liquid scintillation the test is obtained as an objective numeric val­ counter. ue which unequivocally defines the genotype. The aim of the present study was to evalu­ ate the applicability of the solid-phase minise­ Materials and Methods quencing technique for large scale screening for AGUpin carriers and to determine the car­ A total of 1,160 cord blood samples were collected rier frequency in the representative popula­ from newborns at delivery hospitals in southern (Tur­ ku University central hospital, 553 samples) and tion samples from northern and southern northern (Lappi central hospital, 607 samples) Fin­ parts of Finland. land. The blood sampling was combined with that of the routine hypothyroidism screening.

297 Table 1. AGUFì„ carriers in newborn cord blood p > 0.50). The range of G/C values in samples samples from Northern and Southern Finland from normal individuals was 0.0020-0.093 and in samples from heterozygotes 0.51-1.1. Study G/C ratios population In preliminary experiments we were not <0.10 0.50-1.1 able to successfully apply the present sample (normal) (carrier) treatment by lysis on blood samples collected Northern (n = 607) 596 11 without an anticoagulant (after removal of the Southern (n = 553) 545 8 serum for hypothyroidism assay). Therefore a Total (n= 1,160) 1,141 19 separate EDTA-blood sample had to be col­ lected for the AGUpin assay. Six samples (0.5%) were rejected because of inefficient PCR amplification, most likely due to partial coagulation of the samples. Fifty microliters of the EDTA-blood samples were treated with a rapid cell lysis method [9]. One tenth of the lysed sample was amplified by the PCR and geno- typed by the solid-phase minisequencing method using Discussion reagents from the AffiGene AGUpm kit (Orion Phar- maceutica, Espoo, Finland). The PCR and minise­ The solid-phase minisequencing technique quencing detection primer sequences and assay condi­ proved to be a simple and reliable test for tions have been described previously [7]. The genotype of each sample is defined by the ratio of incorporated detection of carriers of the AGUpin mutation. 3H-labeled dGTP to that of *3H-dCTP * (G/C ratio). The technique lends itself to automatization and therefore would easily be applicable to large-scale screening programs. The carrier Results frequency of AGU in defined populations has so far not been studied. It is well known that The solid-phase minisequencing method the gene causing AGU and those causing the was applied to determine the frequency of the other recessively inherited diseases enriched AGUpin allele in a population sample of 1,160 in the Finnish population [10] are unevenly newborns. A sample is typed as homozygous distributed among the population. Regional normal when the G/C ratio obtained in the differences of the prevalence of AGU, as well test is below 0.1, as heterozygous (AGUpin as the analysis of the birthplaces of the grand­ carrier) when the ratio ranges from 0.5 to 1 parents of today’s patients have indicated and as homozygous AGU patient when the clustering of the AGU gene in the northern ratio is above 10 [7], The clear differences in and eastern part of the population. A carrier G/C values from each group allow unequivo­ frequency of 1:40 was estimated from the cal identification of different genotypes. We prevalence of AGU cases among the mentally identified 19 AGUpin heterozygotes and no retarded patients in the northern district of AGUpin homozygotes. Eight of the heterozy­ the country [11], A carrier frequency in the gotes were from southern and 11 of them from same order of magnitude was recently ob­ northern Finland, giving carrier frequencies tained using the minisequencing assay in a of 1:69 (95% confidence interval 1:217-1:41) quantitative fashion. Aliquots of pooled leu­ and 1:55 (95% Cl 1:133-1:35), respectively kocytes derived from transfusion blood of (table 1). These frequencies did not differ sta­ 1,350 donors were analyzed simultaneously tistically significantly from each other (0.24; with a standard mixture of normal and

298 Hietala/Grön/Syvänen/Peltonen/Aula Prospects of Carrier Screening of AGU AGUfìii DNA. The G/C ratios obtained in tailed cost-benefit calculation but it is evident this analysis corresponded to a carrier fre­ that the total costs of the AGU carrier screen­ quency of 1:36 [7], The blood donors were ing program in the pregnant population in this mainly from the capital area of the country. country would be less than the life-long costs The present study revealed slightly lower of the AGU cases prevented by the program. carrier frequencies than expected, 1:55 and Furthermore, AGU is a disease associated 1:69 in the northern and southern population with severe, nontreatable mental retardation. sample, respectively. It is worth noting, how­ Whether screening for AGU carriers in the ever, that the differences between these re­ whole or in a restricted fraction of the popula­ sults and those mentioned above fall within tion will be put into practice remains to be the same range of significance. The small sam­ seen. A tempting vision for the future is to ple sizes may contribute to the results and, as combine the AGUpm carrier test with similar stated above, considerable differences in the mutation detection of one or more other Fin­ regional subpopulations can be expected. nish genetic diseases in order to increase the Prospects of AGU carrier screening on a efficiency of the program. Recent discoveries population basis in Finland are favored by by linkage studies of the gene loci for infantile several facts. Firstly, the prevalence of the dis­ neuronal ceroid lipofuscinosis, INCL [12], ease is relatively high, with 5-7 new cases myoclonic epilepsy [13] and diastrophic dys­ identified every year in a population of 5 mil­ plasia [14] provide realistic hope that the gene lion, secondly, one mutation is responsible for defects in these diseases will soon be charac­ 98% of the mutant alleles in the population, terized. Specific DNA assays detecting a pa­ and finally, a reliable and simple PCR-based nel of mutations can then be designed and DNA test is available for carrier detection at consequently applied to simultaneous screen­ low cost. Total costs of a screening program ing for carriers of the Finnish inherited dis­ are of course much higher than costs of the eases. laboratory assay alone. Information and counselling, sample collection and delivery as well as report of the result with subsequent actions in cases of identified carriers must be Acknowledgements included in the analysis of the total costs of We thank Ilona Carlsson for technical assistance. the program. We have not performed a de­ This study was supported by the Academy of Finland.

References

1 Autio S: Aspartylglucosaminuria. 3 Aula P, Autio S, Raivio KO, Rapola 5 Aula P, Raivio KO, Autio S: Enzy­ Analysis of thirty-four patients. J J: Aspartylglucosaminuria; in Du­ matic diagnosis and carrier detec­ Ment Defic Res Monogr Ser I, rand P, O’Brien JS (eds): Genetic tion of aspartylglucosaminuria using 1972. Errors of Glycoprotein Metabolism. blood samples. Pediatr Res 1976; 10: 2 Pollitt RJ, Jenner FA: Aspartylgly- Berlin, Springer, 1982, pp 122-152. 625-629. cosaminuria. An inborn error of me­ 4 Autio S, Visakorpi JK, Järvinen 6 Ikonen E, Baumann M, Grön K, Sy- tabolism associated with mental de­ H:Aspartylglucosaminuria (AGU). vänen AC, Enomaa N, Halila R, fect. Lancet 1968;ii:253. Further aspects on its clinical pic­ Aula P, Peltonen L: Aspartylglucos­ ture, mode of inheritance and epide­ aminuria: cDNA encoding human miology based on a series of 57 pa­ aspartylglucosaminidase and the tients. Ann Clin Res 1973:5:149- missense mutation causing the dis­ 155. ease. EMBOJ 1991;10:51-58.

299 7 Syvänen AC, Ikonen E, Manninen 9 Kawasaki ES: Sample preparation 12 Järvelä I, Schleutker J, Haataja L, T, Bengtström M, Söderlund H, from blood, cells and other fluids; in Santavuori P, Puhakka L, Männin­ Aula P, Peltonen L: Convenient and Innis MA, Gelfand DH, Sninsky JJ, en T, Palotie A, Sandkuijl LA, Ren­ quantitative determination of the White TJ (eds): PCR Protocols. A lund M, White R, Aula P, Peltonen frequency of a mutant allele using Guide to Methods and Applications. L: Infantile neuronal ceroid lipofus­ solid-phase minisequencing: Appli­ San Diego, Academic Press, 1990, cinosis (INCL, CLN1) maps to the cation to aspartylglucosaminuria in pp 146-152. short arm of chromosome 1. Ge­ Finland. Genomics 1992; 12:590— 10 Norio R, Nevanlinna HR, Perheen- nomics 1991;8:170-173. 595. tupa J: Hereditary diseases in Fin­ 13 Lehesjoki A-E, Koskimemi M, Sis- 8 Syvänen AC, Aalto-Setälä K, Harju land; rare flora in rare soil. Ann Clin tonen P, Miao J, Hästbacka J, Norio L, Kontula K, Söderlund H: A prim­ Res 1973;5:109-141. R, de la Chapelle A: Localization of er-guided nucleotide incorporation 11 Aula P, Renlund M, Raivio O, Kos- a gene for progressive myoclonus assay in the genotyping of apolipo- kela S-L: Screening of inherited epilepsy to chromosome 21q22. protein E. Genomics 1990:8:684- oligosaccharidurias among mentally Proc Natl Acad Sci USA 1991;88: 692. retarded patients in northern Fin­ 3696-3699. land. J Ment Defic Res 1986;30: 14 Hästbacka J, Kaitila I, Sistonen P, 365-368. de la Chapelle A: Diastrophic dys­ plasia gene maps to the distal long arm of chromosome 5. Proc Natl Acad Sci USA 1990;87:8056-8059.

Hietala/Grön/Syvänen/Peltonen/Aula Prospects of Carrier Screening of AGU

Announcement

European Alliance of Genetic Support The EAGS is an umbrella organization of national groups (EAGS) and supemational patient support groups in Europe, helping people and families who suffer from genetic/ During its annual meeting held in Barcelona, May congenital disorders. At present the alliance is com­ 7-8, 1993, the members of the EAGS issued the fol­ prised of the organizations listed below. lowing statement: Further information can be obtained from the fol­ ‘On behalf of families with genetic and congenital lowing alliances of genetic support groups: disorders the EAGS calls for equal access to full infor­ mation, early diagnosis at accredited centers and the The Netherlands: VSOP (phone: 31-2155-28155) maintenance of confidentiality and freedom of choice United Kingdom: GIG (phone: 44-865-226324) Marfan (phone: 44-252-547441) for all, within the legal framework of each country. Ireland: IDO (phone: 353-1-4784-333) The EAGS also strongly opposes the patenting of Germany: EAMDA (phone: 49-731-302-3230) the human genome or any part of it as such.’ Denmark: I.C.F.(M.)A. (phone: 45-8667-4422)

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