© 2001 Oxford University PressHuman Molecular Genetics, 2001, Vol. 10, No. 26 2961–2972
Dissecting a population genome for targeted screening
of disease mutations
Tomi Pastinen1, Markus Perola1,2, Jaakko Ignatius3,4, Chiara Sabatti2, Päivi Tainola1,
Minna Levander1, Ann-Christine Syvänen1,5 and Leena Peltonen1,2,4,*
1Department of Molecular Medicine, National Public Health Institute, Biomedicum, 00250 Helsinki, Finland,
2Department of Human Genetics, University of California, Los Angeles, CA 90095-7088, USA, 3Department of Clinical
Neurophysiology, Helsinki University Hospital (Jorvi Hospital), 02740 Espoo, Finland, 4Department of Medical
Genetics, University of Helsinki, 00250 Helsinki, Finland and 5Molecular Medicine, Department of Medical Sciences,
S-751 85 Uppsala, Sweden
Received August 6, 2001; Revised and Accepted October 10, 2001
Compared to mixed populations, population isolates such as Finland show distinct differences in the
prevalence of disease mutations. However, little information exists of the differences on the prevalence of differ-
ent disease alleles in regional populations with different history of multiple bottlenecks. We constructed a
DNA-array and monitored the prevalence of 31 rare and common disease mutations underlying 27 clinical
phenotypes in a large population-based study sample. Over 64 000 genotypes were assigned in 2151 samples
from four geographical areas representing early and late settlement regions of Finland. Each sample was
analyzed in duplicate and a total of 142 000 array-derived genotyping calls were made. On average one in
three individuals was found to be a carrier of one of the 31 monitored mutations. This should remove fears of the
stigmatizing effect of a carrier-screening program monitoring multiple diseases. Regional differences were found in
the prevalence of mutations, providing molecular evidence for the deviating population histories of regional
subisolates. The mutations introduced early into the population revealed relatively even distribution in differ-
ent subregions. More recently introduced rare mutations showed local clustering of disease alleles, indicating
the persistence of population subisolates and the effect of multiple bottlenecks in molding the population gene
pool. Regional differences were observed also for common disease alleles. Such precise information of the carrier
frequencies could form the basis for targeted genetic screens in this population. Our approach describes a
general paradigm for large-scale carrier-screening programs also in other populations.
DNA variants in over 1000, mostly monogenic, traits have
been identified (1). Identification of mutations has not resulted
in immediate DNA diagnosis due to the high heterogeneity of
disease alleles. Isolated populations provide special advantages
for DNA diagnostics and carrier-screening programs due to a
limited spectrum of disease mutations; one test having high
diagnostic specificity and sensitivity (2,3). Targeted screening
of ethnically restricted disease mutations in the appropriate
population subgroups has demonstrated its efficiency in
disease prevention (4). However, for most populations,
rational design of genetic-screening programs requires large
population-based pilot studies to monitor for the diversity and
prevalence of specific disease mutations.
DNA-array technology is a promising approach to monitor
large numbers of sequence variants in one assay (5–8). If a
moderate number of variants in a large number of samples are
to be analyzed, a custom-made, spotted oligonucleotide arrays used
for enzymatic allele discrimination provides a high-throughput
system (8). Such arrays are flexible and can be tailored for
population-specific carrier screening of several disease variants.
Information on the frequency and regional distribution of
individual disease mutations as well as the validation of array-
based assays must exist before such tests can be implemented
population-wide as a standard component of the health care
We constructed a DNA-array for the detection of 31 mutations
in a study sample of 2400 individuals from different geographical
*To whom correspondence should be addressed at: UCLA Department of Human Genetics, Gonda Neuroscience and Genetics Research Center,
Room 6506, 695 Charles E. Young Drive South, Box 708822, Los Angeles, CA 90095-7088, USA. Tel: +1 310 794 5631; Fax: +1 310 794 5446;
Tomi Pastinen, Montreal Genome Centre, Montreal, H3G 1A4 Quebec, Canada
2962 Human Molecular Genetics, 2001, Vol. 10, No. 26
regions of Finland to monitor for the regional differences in the
prevalence of disease alleles. The majority of the mutations
included on the array, ‘the Finnish Chip’, are recessive disease
mutations enriched in the Finnish population, belonging to the
‘Finnish disease heritage’. In addition, we incorporated to the
array some disease mutations common in most Caucasian
populations, like mutations of α-1-antitrypsin, factor V and the
hereditary hemochromatosis gene.
We did not include on the array dominant disease mutations
like those of hereditary breast and ovarian cancer (BRCA1 and 2),
familial non-polypotic colon cancer or familial hypercholes-
terolemia due to their rarity. Recent studies in cancer patients
(9,10) or population-based surveys (11) demonstrate the low
prevalence of these mutations in the Finnish population
The microarray-based primer extension assay proved to be
highly robust and provided reliable genotyping results at a
relatively low cost. Interestingly, we found distinct regional
differences in the carrier frequency of both rare and common
disease mutations within this genetically homogeneous,
isolated population, the findings providing support for multiple
historical population bottle necks. The population-wide
carrier frequency of disease alleles detected by this panel of
31 mutations was 1:3, illustrating the prediction that if a
wider panel of disease mutations were to be included on the
array, every Finn would be a carrier of at least one of the
tested disease mutations. This should alleviate fears of the
stigmatizing effect of carrier-screening programs monitoring
numerous disease mutations.
Mutations screened with the DNA array
The mutation-screening panel on the DNA-array comprised
the known major mutations of Finnish diseases (12), a total of
19 mutations in 16 different genes. Most mutations show a
strong founder effect and the coverage of the mutation detection
varied between 74 and 99% for different diseases. The
common Caucasian mutations consisted of 10 mutations in
nine genes including two common polymorphisms of factor V
(13) and the prothrombin gene (14). A list of the mutations
included on the array as well as the corresponding diseases and
OMIM symbols are provided in Table 1. A pair of allele-specific
detection primers for each of the mutations was spotted on
derivatized microscopic glass-slides (8). Eighty replicate
arrays were spotted onto each slide and custom-made reaction
chambers were used to analyze up to 80 separate samples per
slide. This facilitated the monitoring of 2480 mutations (31
genotypes for 80 individuals) on a single microscopic glass
slide (Fig. 1). The allele-specific primer extension reactions
provided reliable discrimination between the obtained genotypes
We determined the carrier frequencies of disease mutations
by genotyping 2151 anonymous DNA samples from four
geographical regions (Fig. 2). One rural sample was collected
from southern Botnia, representing the ‘early settlement’
region, which was inhabited some 2000 years ago. Another
rural sample was collected from North Karelia, representing
the ‘late settlement’ northeastern region, permanently
inhabited after the 16th century. The two urban community
samples were from Helsinki and from the city of Oulu on the
northwestern coastline. Both of these cities have been targets
for internal population movement during the past eight to nine
decades. However, according to Y chromosomal haplotype
analysis, the influence from early immigration from Western
Europe is much less evident in Oulu region than in southern
Finland (15). Further, immigration to Helsinki, the capital, has
been much more excessive than to Oulu. As positive controls,
212 samples from carriers of the disease mutations were allo-
cated blindly among the study samples.
Quality of the data from the arrays
Each sample was amplified by PCR and assayed in duplicate
on separate microarrays. The genotyping results for each
sample were read and the genotypes were independently
assigned from the two arrays. Moreover, the genotypes were
called on coded samples, without knowledge of the sample
status to serve as quality control of the array-based screening
procedure. The reference methods were systematically used to
verify the genotyping result if a sample indicated a carrier
status, if the results from the duplicate assays were discrepant
or if the signal intensities on a microarray were too low to
assign the genotype. For samples with low signal intensity on
the array, the reference method was performed only once, and
the sample was omitted from further analysis if this reference
assay failed. In the cases of conflicting duplicate genotype
calls and for every carrier genotype sample the reference
method was used to confirm the genotype.
Since each sample was analyzed in duplicate, a total of
142 000 array derived genotyping calls were made. None of
the heterozygous controls was assigned as normal homo-
zygotes. Of the 71 000 genotypes generated, 95% were called
successfully. In 5% of the samples the signal intensities were
too low to make a reliable genotype call, and the reference
methods were used to assign the genotypes. Less than 0.1% of
the samples gave conflicting results in the duplicate array-
based assay, due to inconsistencies resulting from the spotting
of the primer-arrays. The final assignment of genotype in the
study samples was made in 99.15% of cases (64 076 out of
64 625 genotypes were assigned). The genotyping results are
summarized in Tables 2 and 3.
Carrier frequencies of the ‘Finnish disease heritage’
A particularly informative approach to the analysis of the
regional prevalence of the Finnish mutations is to divide them
based on the time of their assumed introduction into the population.
Recent introduction of a disease allele is characterized by the
geographically restricted occurrence of patients, a genealogical
history revealing ancestors in the same communities and a
demonstration of linkage disequilibrium over a wide genetic
interval flanking the disease mutation (12,16). Representative
examples of such mutations in our screen include vLINCL
(17), EPMR (18), Salla disease (19), CCD (20) and two X-linked
retinoschisis (RS) (21) mutations (Table 1). Figure 3 demon-
strates the carrier frequencies of five young mutations in the
four regional samples. Notably, all these mutations show
evidence of local clustering of disease alleles, indicating the
persistence of the subisolates. A striking example of such
clustering is the high frequency (1:44) of the vLINCL mutation
Human Molecular Genetics, 2001, Vol. 10, No. 26 2971
9. Syrjäkoski, K., Vahteristo, P., Eerola, H., Tamminen, A., Kivinummi, K.,
Sarantaus, L., Holli, K., Blomqvist, C., Kallioniemi, O.P., Kainu, T. et al.
(2000) Population-based study of BRCA1 and BRCA2 mutations in 1035
unselected Finnish breast cancer patients. J. Natl Cancer. Inst., 92, 1529–1531.
10. Aaltonen, L.A., Salovaara, R., Kristo, P., Canzian, F., Hemminki, A.,
Peltomäki, P., Chadwick, R.B., Kaariainen, H., Eskelinen, M., Jarvinen, H.
et al. (1998) Incidence of hereditary nonpolyposis colorectal cancer and
the feasibility of molecular screening for the disease. New Engl. J. Med.,
11. Vuorio, A.F., Turtola, H., Piilahti, K.M., Repo, P., Kanninen, T. and
Kontula, K. (1997) Familial hypercholesterolemia in the Finnish north
Karelia. A molecular, clinical, and genealogical study. Arterioscler.
Thromb. Vasc. Biol., 17, 3127–3138.
12. Peltonen, L., Jalanko, A. and Varilo, T. (1999) Molecular genetics of the
Finnish disease heritage. Hum. Mol. Genet., 8, 1913–1923.
13. Bertina, R.M., Koeleman, B.P., Koster, T., Rosendaal, F.R., Dirven, R.J.,
de Ronde, H., van der Velden, P.A. and Reitsma, P.H. (1994) Mutation in
blood coagulation factor V associated with resistance to activated
protein C. Nature, 369, 64–67.
14. Poort, S.R., Rosendaal, F.R., Reitsma, P.H. and Bertina, R.M. (1996) A
common genetic variation in the 3′-untranslated region of the prothrombin
gene is associated with elevated plasma prothrombin levels and an
increase in venous thrombosis. Blood, 88, 3698–3703.
15. Kittles, R.A., Perola, M., Peltonen, L., Bergen, A.W., Aragon, R.A.,
Virkkunen, M., Linnoila, M., Goldman, D. and Long, J.C. (1998) Dual
origins of Finns revealed by Y chromosome haplotype variation. Am. J.
Hum. Genet., 62, 1171–1179.
16. Varilo, T. (1999) The age of the mutations in the Finnish heritage: a
genealogical and linkage disequilibrium study. Academic Dissertation.,
University of Helsinki, Finland.
17. Savukoski, M., Klockars, T., Holmberg, V., Santavuori, P., Lander, E.S.
and Peltonen, L. (1998) CLN5, a novel gene encoding a putative
transmembrane protein mutated in Finnish variant late infantile neuronal
ceroid lipofuscinosis. Nat. Genet., 19, 286–288.
18. Ranta, S., Zhang, Y., Ross, B., Lonka, L., Takkunen, E., Messer, A.,
Sharp, J., Wheeler, R., Kusumi, K., Mole, S. et al. (1999) The neuronal
ceroid lipofuscinoses in human EPMR and mnd mutant mice are
associated with mutations in CLN8. Nat. Genet., 23, 233–236.
19. Verheijen, F.W., Verbeek, E., Aula, N., Beerens, C.E., Havelaar, A.C.,
Joosse, M., Peltonen, L., Aula, P., Galjaard, H., van der Spek, P.J. et al.
(1999) A new gene., encoding an anion transporter, is mutated in sialic
acid storage diseases. Nat. Genet., 23, 462–465.
20. Hoglund, P., Haila, S., Socha, J., Tomaszewski, L., Saarialho-Kere, U.,
Karjalainen-Lindsberg, M.L., Airola, K., Holmberg, C., de la Chapelle, A.
and Kere, J. (1996) Mutations of the down-regulated in adenoma (DRA)
gene cause congenital chloride diarrhoea. Nat. Genet., 14, 316–319.
21. Huopaniemi, L., Rantala, A., Forsius, H., Somer, M., de la Chapelle, A.
and Alitalo, T. (1999) Three widespread founder mutations contribute to
high incidence of X-linked juvenile retinoschisis in Finland. Eur. J. Hum.
Genet., 7, 368–376.
22. Kestilä, M., Lenkkeri, U., Mannikko, M., Lamerdin, J., McCready, P.,
Putaala, H., Ruotsalainen, V., Morita, T., Nissinen, M., Herva, R. et al. (1998)
Positionally cloned gene for a novel glomerular protein—nephrin—is
mutated in congenital nephrotic syndrome. Mol. Cell, 1, 575–582.
23. Ikonen, E., Baumann, M., Gron, K., Syvänen, A.C., Enomaa, N., Halila, R.,
Aula, P. and Peltonen, L. (1991) Aspartylglucosaminuria: cDNA encod-
ing human aspartylglucosaminidase and the missense mutation causing
the disease. EMBO J., 10, 51–58.
24. Hastbacka, J., de la Chapelle, A., Mahtani, M.M., Clines, G., Reeve-Daly, M.P.,
Daly, M., Hamilton, B.A., Kusumi, K., Trivedi, B., Weaver, A. et al.
(1994) The diastrophic dysplasia gene encodes a novel sulfate transporter:
positional cloning by fine-structure linkage disequilibrium mapping. Cell,
25. de la Chapelle, A. and Wright, F. (1998) Linkage disequilibrium mapping
in isolated populations: the example of Finland revisited. Proc. Natl Acad.
Sci. USA, 95, 12416–12423.
26. Kelsell, D.P., Dunlop, J., Stevens, H.P., Lench, N.J., Liang, J.N., Parry, G.,
Mueller, R.F. and Leigh, I.M. (1997) Connexin 26 mutations in hereditary
non-syndromic sensorineural deafness. Nature, 387, 80–83.
27. Zelante, L., Gasparini, P., Estivill, X., Melchionda, S., D’Agruma, L.,
Govea, N., Mila, M., Monica, M.D., Lutfi, J., Shohat, M. et al. (1997)
Connexin 26 mutations associated with the most common form of
non-syndromic neurosensory autosomal recessive deafness (DFNB1) in
Mediterraneans. Hum. Mol. Genet., 6, 1605–1609.
28. Kere, J., Estivill, X., Chillon, M., Morral, N., Nunes, V., Norio, R.,
Savilahti, E. and de la Chapelle, A. (1994) Cystic fibrosis in a
low-incidence population: two major mutations in Finland. Hum. Genet.,
29. Guldberg, P., Henriksen, K.F., Sipila, I., Guttler, F. and de la Chapelle, A.
(1995) Phenylketonuria in a low incidence population: molecular
characterisation of mutations in Finland. J. Med. Genet., 32, 976–978.
30. Visakorpi, J.K., Palo, J. and Renkonen, O.V. (1971) The incidence of Pku
in Finland. Acta Paediatr. Scand., 60, 666–668.
31. TIBD Consortium (1995) Isolation of a novel gene underlying Batten
disease, CLN3. Cell, 82, 949–957.
32. Järvelä, I., Mitchison, H.M., Munroe, P.B., O’Rawe, A.M., Mole, S.E. and
Syvänen, A.C. (1996) Rapid diagnostic test for the major mutation
underlying Batten disease. J. Med. Genet., 33, 1041–1042.
33. Beutler, E., West, C. and Gelbart, T. (1997) HLA-H and associated
proteins in patients with hemochromatosis. Mol. Med., 3, 397–402.
34. Jeffrey, G.P., Chakrabarti, S., Hegele, R.A. and Adams, P.C. (1999)
Polymorphism in intron 4 of HFE may cause overestimation of C282Y
homozygote prevalence in haemochromatosis. Nat. Genet., 22, 325–326.
35. Cargill, M., Altshuler, D., Ireland, J., Sklar, P., Ardlie, K., Patil, N., Shaw, N.,
Lane, C.R., Lim, E.P., Kalyanaraman, N. et al. (1999) Characterization of
single-nucleotide polymorphisms in coding regions of human genes.
Nat. Genet., 22, 231–238.
36. Bernardi, F., Faioni, E.M., Castoldi, E., Lunghi, B., Castaman, G.,
Sacchi, E. and Mannucci, P.M. (1997) A factor V genetic component
differing from factor V R506Q contributes to the activated protein C
resistance phenotype. Blood, 90, 1552–1557.
37. Faioni, E.M., Franchi, F., Bucciarelli, P., Margaglione, M., De Stefano, V.,
Castaman, G., Finazzi, G. and Mannucci, P.M. (1999) Coinheritance of
the HR2 haplotype in the factor V gene confers an increased risk of venous
thromboembolism to carriers of factor V R506Q (factor V Leiden). Blood,
38. Nei, M. (1972) Genetic distance between populations. Am. Nat., 106, 283–292.
39. Hacia, J.G. and Collins, F.S. (1999) Mutational analysis using oligonucleotide
microarrays. J. Med. Genet., 36, 730–736.
40. Wang, D.G., Fan, J.B., Siao, C.J., Berno, A., Young, P., Sapolsky, R.,
Ghandour, G., Perkins, N., Winchester, E., Spencer, J. et al. (1998)
Large-scale identification, mapping, and genotyping of single-nucleotide
polymorphisms in the human genome. Science, 280, 1077–1082.
41. Cho, R.J., Mindrinos, M., Richards, D.R., Sapolsky, R.J., Anderson, M.,
Drenkard, E., Dewdney, J., Reuber, T.L., Stammers, M., Federspiel, N.
et al. (1999) Genome-wide mapping with biallelic markers in
Arabidopsis thaliana. Nat. Genet., 23, 203–207.
42. Pastinen, T., Kurg, A., Metspalu, A., Peltonen, L. and Syvänen, A.C.
(1997) Minisequencing: a specific tool for DNA analysis and diagnostics
on oligonucleotide arrays. PCR Methods Appl., 7, 606–614.
43. Hacia, J.G., Sun, B., Hunt, N., Edgemon, K., Mosbrook, D., Robbins, C.,
Fodor, S.P., Tagle, D.A. and Collins, F.S. (1998) Strategies for mutational
analysis of the large multiexon ATM gene using high-density oligonucleotide
arrays. Genome Res., 8, 1245–1258.
44. Fan, J.B., Chen, X., Halushka, M.K., Berno, A., Huang, X., Ryder, T.,
Lipshutz, R.J., Lockhart, D.J. and Chakravarti, A. (2000) Parallel genotyping
of human SNPs using generic high-density oligonucleotide tag arrays.
Genome Res., 10, 853–860.
45. Gerry, N.P., Witowski, N.E., Day, J., Hammer, R.P., Barany, G. and
Barany, F. (1999) Universal DNA microarray method for multiplex
detection of low abundance point mutations. J. Mol. Biol., 292, 251–262.
46. Norio, R., Nevanlinna, H.R. and Perheentupa, J. (1973) Hereditary
diseases in Finland; rare flora in rare soul. Ann. Clin. Res., 5, 109–141.
47. Vesa, J., Hellsten, E., Verkruyse, L.A., Camp, L.A., Rapola, J.,
Santavuori, P., Hofmann, S.L. and Peltonen, L. (1995) Mutations in the
palmitoyl protein thioesterase gene causing infantile neuronal ceroid
lipofuscinosis. Nature, 376, 584–587.
48. The Finnish–German APECED Consortium (1997) An autoimmune
disease, APECED, caused by mutations in a novel gene featuring two
PHD-type zinc-finger domains. Nat. Genet., 17, 399–403.
49. Romppanen, E.L. and Mononen, I. (2000) Detection of the Finnish-type
congenital nephrotic syndrome by restriction fragment length
polymorphism and dual-color oligonucleotide ligation assays.
Clin. Chem., 46, 811–816.
50. Gasparini, P., Rabionet, R., Barbujani, G., Melchionda, S., Petersen, M.,
Brondum-Nielsen, K., Metspalu, A., Oitmaa, E., Pisano, M., Fortina, P.
et al. (2000) High carrier frequency of the 35delG deafness mutation in
2972 Human Molecular Genetics, 2001, Vol. 10, No. 26
European populations. Genetic Analysis Consortium of GJB2 35delG.
Eur. J. Hum. Genet., 8, 19–23.
51. Crystal, R.G. (1989) The α 1-antitrypsin gene and its deficiency states.
Trends Genet., 5, 411–417.
52. Varpela, E., Saris, N.E. and Lokki, O. (1973) The prevalence of serum α
1-antitrypsin deficiency in Finland. Duodecim, 89, 818–824.
53. Heliö, T., Färkkilä, M., Halme, L., Karlsson, M., Palotie, M. and
Kontula, K. (1998) Genetic background of hereditary hemochromatosis
and DNA-diagnostics [Finnish]. Duodecim, 114, 1404–1409.
54. Tuomainen, T.P., Kontula, K., Nyyssonen, K., Lakka, T.A., Helio, T. and
Salonen, J.T. (1999) Increased risk of acute myocardial infarction in
carriers of the hemochromatosis gene Cys282Tyr mutation: a prospective
cohort study in men in eastern Finland. Circulation, 100, 1274–1279.
55. Lahermo, P., Savontaus, M.L., Sistonen, P., Beres, J., de Knijff, P., Aula, P.
and Sajantila, A. (1999) Y chromosomal polymorphisms reveal founding
lineages in the Finns and the Saami. Eur. J. Hum. Genet., 7, 447–458.
56. Beaudet, A.L. (1990) Carrier screening for cystic fibrosis. Am. J. Hum.
Genet., 47, 603–605.
57. Guthrie, A. and Susi, A. (1963) A simple phenylalanine method for
detecting phenylketonuria in large populations of newborn infants.
Pediatrics, 32, 338–343.
58. Romppanen, E.L., Mononen, T. and Mononen, I. (1998) Molecular
diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by
oligonucleotide ligation assay. Clin. Chem., 44, 68–71.
59. Vartiainen, E., Puska, P., Jousilahti, P., Korhonen, H.J., Tuomilehto, J.
and Nissinen, A. (1994) Twenty-year trends in coronary risk factors in north
Karelia and in other areas of Finland. Int. J. Epidemiol., 23, 495–504.
60. Kaprio, J. (1994) Lessons from twin studies in Finland. Ann. Med., 26,
61. Syvänen, A.C., Ikonen, E., Manninen, T., Bengtstrom, M., Soderlund, H.,
Aula, P. and Peltonen, L. (1992) Convenient and quantitative determination
of the frequency of a mutant allele using solid-phase minisequencing:
application to aspartylglucosaminuria in Finland. Genomics, 12, 590–595.
62. Kure, S., Takayanagi, M., Narisawa, K., Tada, K. and Leisti, J. (1992)
Identification of a common mutation in Finnish patients with nonketotic
hyperglycinemia. J. Clin. Invest., 90, 160–164.
63. Torrents, D., Mykkanen, J., Pineda, M., Feliubadalo, L., Estevez, R.,
de Cid, R., Sanjurjo, P., Zorzano, A., Nunes, V., Huoponen, K. et al.
(1999) Identification of SLC7A7, encoding y+LAT-1, as the lysinuric
protein intolerance gene. Nat. Genet., 21, 293–296.
64. Raymond, M., and Rousset, F. (1995) GENEPOP (version 1.2):
Population genetics software for exact tests and ecumenicism. J. Hered.,
65. Guldbrandtsen, B., Tomiuk, J. and Loeschcke, V. (2000) POPDIST, ver-
sion 1.1.1: a program to calculate population genetic distance and identity
measures. J. Hered., 91, 178–179.
66. Conover, W. (1971) Practical Nonparametric Statistics. John Wiley &
Sons, New York.
67. Hollander, M. and Wolfe, D. (1973) Nonparametric Statistical Inference.
John Wiley & Sons, New York, pp. 15–22.