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Human Molecular Genetics, 2004, Vol. 13, No. 16 1693-1702
DOI: 10.1093/hmg/ddh184
Human Molecular Genetics, Vol. 13, No. 16 © Oxford University Press 2004; all rights reserved

Search for cognitive trait components of schizophrenia reveals a locus for verbal learning and memory on 4q and for visual working memory on 2q

Tiina Paunio1,2,3,*, Annamari Tuulio-Henriksson2, Tero Hiekkalinna1, Markus Perola1, Teppo Varilo1, Timo Partonen2, Tyrone D. Cannon4,5,6, Jouko Lönnqvist2 and Leena Peltonen1,7,8

1Department of Molecular Medicine, 2Department of Mental Health and Alcohol Research, National Public Health Institute, 00300 Helsinki, Finland, 3Department of Psychiatry, Helsinki University Hospital, 00029 HUS, Helsinki, Finland, 4Department of Psychology, 5Department of Psychiatry and Biobehavioral Sciences, 6Department of Human Molecular Genetics, University of California, Los Angeles, CA 90095-1563, USA, 7Department of Medical Genetics, University of Helsinki, 00029 HUS, Helsinki, Finland and 8Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-7088, USA

Received February 27, 2004; Revised May 27, 2004; Accepted June 3, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Research to identify predisposing genes for complex diseases relying solely on clinical diagnosis is probably not ideal. Here, we analyzed genome-wide data for 168 schizophrenia families using neuropsychological variables associated with disease susceptibility, with the aid of SOLAR, a program for variance-component analysis. The linkage signal was greatly accentuated by application of the quantitative traits compared with diagnosis. We found evidence for a locus for verbal learning and memory on 4q21 (Z=3.01, Zmp=3.84 and empiric P=0.031 for delayed memory; Z=2.96, Zmp=3.4 and P=0.026 for verbal learning) and suggestive evidence for visual working memory on 2q36 (Z=2.80, Zmp=2.08 and P=0.093). In addition, some evidence emerged for a locus for recognition memory on 10p13, visual attention on 15q22 and executive function on 9p22 in the complete sample, as well as for delayed memory on 8q12, semantic clustering and intrusions on 1q42 and visual attention on 3p25 in the genealogically distinctive sample subsets. Of the loci linked to schizophrenia in diverse populations, in addition to the earlier mentioned regions, some evidence of linkage was observed for 2q, 6q, 7q, 11q, 13q, 14q, 18q and 22q. Our results reveal initial information on the effect of the loci associated with schizophrenia in multiple studies, and emphasize the value of trait components in the search for susceptibility loci for complex diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The importance of genes in the pathogenesis of schizophrenia (MIM 181500), a mental disorder characterized by psychotic symptoms and deficits in cognitive and social functioning, has been well established in twin, family and adoption studies (1). Molecular genetic studies in various populations suggest both significant locus heterogeneity and allelic diversity (2). Even studies of familiar forms of schizophrenia have failed to identify a direct causal relationship with any single gene. The recently revealed associations between schizophrenia and positional candidate genes, such as neuregulin 1 and dysbindin (3,4, reviewed in 5), are encouraging but do not entirely explain the linkage findings that initially triggered these studies. Furthermore, the assessed risk estimates tend to be low despite the multiple testing effects and the great upward bias in estimating locus-specific effects from genome-wide scans (6).

It appears likely that genetic susceptibility to schizophrenia may be influenced by many different genes affecting a number of functions in the brain. Some of these genes may be inherited in a predisposing configuration without resulting in a clinical phenotype, but nevertheless affecting the functioning of the relevant brain systems. Consequently, the clinical diagnosis may not adequately model the genetic background of the schizophrenia phenotype for genetic linkage studies. Instead, identification of the genes contributing to trait components, such as the set of central nervous system disturbances associated with the illness, may represent a more powerful strategy. Some of these traits can be more objectively defined and quantified than clinical diagnoses and thus transformed into passable parameters for statistical analysis (7,8). Quantitative traits also extract maximal information from the study sample and thus potentially represent a more powerful strategy for gene identification.

To represent a valuable endophenotype in genetic analyses of complex diseases, a trait should be heritable and closely associated with vulnerability to the disorder. The trait should be independent of the expression of the illness so that the unaffected relatives of the patients should show the same phenotype to a degree correlated with their genetic proximity to an affected individual (9). Many neurocognitive traits have been suggested to meet these criteria for promising trait components of schizophrenia, including attention, working memory, and verbal learning and memory (10–15).

In the present study, we investigated a set of quantitative trait variables derived from neuropsychological test data in a genome-wide linkage analysis of Finnish families ascertained for schizophrenia. The results not only confirm and extend previous linkage findings by us and others, but also demonstrate for the first time the benefit of using trait components instead of clinical diagnosis in the genome-wide hunt for genes involved in susceptibility to schizophrenia.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prior to conducting the linkage analyses, we performed preliminary statistical analyses on the variables derived from the neuropsychological test data, including calculation of the descriptive statistics and the correlations between the traits.

The performance characteristics of the index study and control samples appear in Table 1. Because a variance-component based analysis may be particularly vulnerable to high levels of kurtosis in the trait distribution (16), some of the traits were transformed as described in Materials and Methods. For all the variables, the mean performance of the study sample was inferior to that of the control sample (Table 1). Furthermore, the affected probands generally performed worse than their relatives, and the mean performance of the latter was also below that of the general population (data not shown). Correlations between the transformed traits were sought by bivariate correlation analysis of all individuals of the study sample. The strongest correlation was observed among the different indices of memory function: verbal learning and semantic clustering (0.66; P=0.000001), verbal learning and delayed memory (0.69; P=0.000001) and semantic clustering and delayed memory (0.59; P=0.000001) (Table 2).


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  		Table 1. Characteristics of neuropsychological test measurements in the study sample
 

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  		Table 2. Results from bivariate correlation analysis with Kendall's tau-b correlation coefficient
 
Genome-wide search
In the genome-wide search for quantitative trait loci (QTLs) for the neurocognitive functions, our primary interest was the analysis of the complete study sample (Com). However, owing to the potential differences in the genetic backgrounds of families collected from an internal isolate, representing a more restricted gene pool, we also analyzed separately the families from the internal subisolate (IS) and from the rest of the country (AF).

Several quantitative parameters of verbal learning and memory were found to be linked to chromosome 4q13–25 within a 30 cM region (Fig. 1, Tables 2 and 3). The best individual two-point and multipoint LOD score values, as presented by SOLAR (Z and Zmp, respectively), for the Com were obtained for delayed memory to 4q21 (Z=3.01 to D4S2361; Zmp=3.84) (Table 3). The finding that verbal learning and semantic clustering were linked to the same chromosomal region is not surprising, as these traits correlate with each other. Both families from the internal isolate and elsewhere from Finland apparently contributed to these findings, despite slight variations in the relative LOD score values even with nearby locating markers (Table 3). On the basis of variance-component analysis, variation on 4q would account for 33, 33 and 32% of the variation in delayed memory, semantic clustering and verbal learning, with a residual additive genetic component of 12, 17 and 20%, and a random environmental contribution of 38, 33 and 26%, respectively.



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  		Figure 1. Variance-component analysis of verbal learning, semantic clustering and delayed memory on chromosome 4, and visual working memory on chromosome 2 in Finnish families originally ascertained for schizophrenia. The multipoint LOD score plots and two-point LOD score values, derived from analyses with the SOLAR, are shown on the y axis against the cumulative genetic map (x-axis) in these chromosomal regions and traits. Indicated are results for both the complete study sample (Com) as well as for the geographically distinctive subsamples IS (consisting of families from the internal isolate) and AF (families from the rest of Finland).

 

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  		Table 3. Results of the genome-wide QTL linkage analysis
 
Among the measures for attention and working memory, the best evidence of linkage was obtained with visual working memory to chromosome 2q36. A suggestive evidence for linkage was found in the Com at D2S1363 and was contributed to both AF and IS families (Fig. 1 and Table 3). According to variance-component analysis, variation in the 2q would account for 21% of the variation in visual working memory, with a residual additive genetic component of 15% and a random environmental contribution of 49%.

Some evidence of linkage also emerged for executive function on chromosome 9p22, for recognition memory on 10p13 and for visual attention on 15q22 in the Com (Table 3). Families from the IS sample contributed most to the evidence of the linkage signal on 10p13 and 15q22, and from the AF sample to the signal on 9p22. In IS families, semantic clustering showed some evidence of linkage to chromosome 1q42, and in the AF families measure of intrusions was suggestively linked to 1q32 (Table 4). A genome-wide maximum LOD score of 3.05 was obtained in the AF families for delayed memory to D8S1113 on 8q12. Table 4 shows the more detailed results from QTL analysis of the regions of interest based on the present study as well as on the loci that possibly increase susceptibility to schizophrenia in diverse populations (5,17).


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  		Table 4. Detailed QTL linkage results for most interesting chromosomal regions including schizophrenia loci in Finland (18–20) and world-wide (5,17)
 
Compared with the QTL analysis, application of the clinical diagnosis greatly reduced the linkage signal in the loci putatively revealed in the present study (Table 3), reflecting the capacity of the quantitative traits to extract maximal information from the study sample.

Genome-wide P-values
In order to assess the relative significance of the most important findings of the current study, we permutated some of the neuropsychological functions, and recalculated the data for genome-wide scans using the permutated phenotypes. All adjustments, including adaptation of age and gender as covariates, were identical to those used in the original data analysis. The phenotype permutation was confined to the family members having both phenotype and genotype data.

In 1000 permutated genome scans with visual working memory, a maximum two-point LOD score ≥2.80 was found 93 times, which resulted in an empiric P-value of 0.093. For functions related to verbal learning and memory, a maximum two-point LOD score ≥2.96 was found 26 times for verbal learning (P=0.026), a LOD score ≥2.86 was found 143 times for semantic clustering (P=0.143) and a LOD score ≥3.01 was found 31 times for delayed memory (P=0.031). Thus, verbal learning and delayed memory were the functions providing statistically the most significant evidence of linkage in terms of the empiric P-value in the current study.

Analysis of families with respect to linkage to 1q42 and 5q31
On the basis of our previous results, our major interest in the search for schizophrenia susceptibility genes was focused on chromosomes 1q32–42 and 5q31 (18–20). We divided the sample families into those linked or not linked to dichotomized clinical diagnosis of schizophrenia spectrum disorder with markers providing the highest evidence of linkage: D1S2709 (using diagnostic criteria of liability class (LC) 3 and dominant model of inheritance) (19) and FGF1 (using LC 4 and recessive model of inheritance) (20) on the earlier mentioned chromosomal regions. The cut-off LOD score for linkage, at {theta}=0, was determined as 0.20 or more, whereas Z<0 was the criterion for non-linkage. The subsample 1q42/linked contained 21 families with 79 individuals having both genotype and phenotype data; the other subsamples were: 1q42/non-linked had 24 families with 101 individuals, 5q31/linked had 17 families with 71 individuals and 5q31/non-linked had 33 families with 133 individuals. Only three families were linked and nine families were non-linked to both chromosomal regions. These sample subsets were analyzed separately for visual working memory and delayed memory, with the focus on relative changes in the LOD scores on 2q and 4q, respectively.

With the markers D4S2367 and D4S3243, the evidence for linkage of delayed memory was 0.38 and 4.87 (D4S2367) and 0.11 and 4.0 (D4S3243) in families linked versus non-linked to chromosome 5q31, respectively. A similar though somewhat less marked difference of linkage signal was observed in families linked versus non-linked to chromosome 1q42 (Z=1.24 to D4S2367 and Z=3.17 to D4S3243 in the non-linked families). For visual working memory, no such discrepancy in the LOD score values was observed for the markers on chromosome 2q (Fig. 2).



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  		Figure 2. QTL linkage of families ascertained for linkage of dichotomous clinical diagnosis of schizophrenia spectrum disorder to 1q42 and 5q31. The LOD scores on 2q for visual working memory (left) and on 4q for delayed memory (right) were followed. The evidence of linkage of delayed memory to 4q21 was found to be higher in families showing exclusion of linkage to either 5q31 (Z=4.87 with D4S2367) or, to a lesser extent, to 1q42 (Z=3.17 with D4S3243). For the visual working memory QTL, no such difference was observed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Here, we searched for genetic loci contributing to a set of quantitative cognitive functions, by performing QTL-based statistical analyses of genotypes collected from 168 Finnish schizophrenia families. To our knowledge, this is the first published study designed to characterize genome-wide susceptibility loci for cognitive functions representing potential quantitative traits underlying schizophrenia. All traits we analyzed are closely associated with vulnerability to this disorder (10–14). The traits appeared to be heritable in the families, and although the affected probands tended to perform worse than their unaffected relatives, the mean performance of the latter was also below that of the general population (15,21). Because measurable trait components such as these allow for analysis of the whole spectrum of phenotypic features, they have the potential to guide us to the identification of genes more tangible than those underlying the non-quantifiable clinical diagnosis. Genome-wide genotype data on 449 markers provided us a means for defining the susceptibility regions for the various traits without a prior hypothesis regarding the biological background.

We recognize that when analyzing multiple, and to some extent correlating, traits that are further associated with the ascertainment criteria of the sample, approximation of statistical significance is demanding. To make some type of estimate, we assessed the relative significance of the findings for four traits with the highest genome-wide maximum LOD score values by permutating these traits within the families and recalculating the genome-wide scans using the permutated phenotypes. The strongest evidence was obtained for verbal learning (empiric P=0.026), found to be linked to D4S2361 on 4q21 with a LOD score of 2.96. The importance of this region is emphasized by the fact that we found evidence for linkage of several related traits to markers within a 30 cM region on 4q13–25. The best individual two-point LOD score value (3.01) in the Com was obtained for delayed memory to D4S2361. In addition, semantic clustering was linked to the same region. These results imply that the traits associated with verbal memory and strategies of verbal learning could represent useful features in genetic research of schizophrenia, as consistently suggested by previous studies (11,22,23). The long arm of chromosome 4 has been of interest in our schizophrenia study, since the initial finding of linkage to D4S1586 in the genome scan of the original set of the IS families (18). Further, when the families of the isolate were genealogically linked, the best evidence of linkage (Z>3.0) was obtained in close vicinity to the marker showing the best evidence for linkage in the present study (unpublished data). Moreover, the findings in other populations would support the role of the 4q loci in schizophrenia, although the markers giving the best evidence of linkage to the end-state qualitative diagnosis locate at a 30 Mb distance from the QTL, identified here at D4S2361 (24–27).

The present study also revealed linkage of visual working memory on chromosome 2q34–36. The region of linkage is close to a schizophrenia susceptibility locus detected in our recent study, with the highest genome-wide LOD score for families from the isolate (4.4) at D2S427, located only 10 cM telomeric from D2S1363 (20). This locus was also implicated in a genome-wide scan on schizophrenia families from another population isolate, the Micronesian islander population of Kosrae (28). Visual working memory has received consistent support as a highly valid trait component of schizophrenia (10,14). The visual span backward task, used in the present study, monitors for elements of the visual working memory, and recent evidence suggests that this particular task represents a valuable trait component of schizophrenia (12,15,21). For this trait, a recent study detected some evidence of linkage to a marker on 1q42 in Finnish twin pairs discordant for schizophrenia (29). We also found some evidence of linkage of recognition memory to chromosome 10p13, visual attention to 15q22 and executive function to 9p22 in the Com. The region on 10p13 has been implicated in several studies as a susceptibility locus, either for schizophrenia and schizoaffective disorder or for bipolar disorder (30–33). The 15q has also generated some interest in schizophrenia research, particularly as a locus for a neurophysiological deficit (suppression of P50 inhibition) in schizophrenia families and for periodic catatonia (34,35), but the current locus for visual attention is some 20 cM centromeric from findings of studies focused on the vicinity of the gene encoding for the alpha-7 nicotinic cholinergic receptor subunit (ACHRA7). However, there was some evidence of linkage of another trait (perseverations) in the AF sample to the immediate vicinity of the ACHRA7 gene on 15q13. A genome-wide maximum LOD score of 3.05 was obtained in the AF families for delayed memory to 8q12, 27 Mb from neuregulin1, a particularly potential positional candidate gene for schizophrenia (3, reviewed in 5). Since the initial finding of linkage to 1q32 (18), the long arm of chromosome 1 has been the focus of genetic studies of schizophrenia in Finland (36,37). Here, we found evidence of linkage of semantic clustering and verbal learning in the IS sample, and of intrusive recall errors in the AF sample in this particular chromosomal region. Semantic clustering also showed some evidence of linkage in the IS sample to another region, 7q21, of putative interest in Finnish schizophrenia research (36). Of the loci that possibly increase susceptibility to schizophrenia in diverse populations (5,17), in addition to the earlier mentioned regions, some evidence of linkage was observed for markers located on chromosomes 2q11 (genome-wide maximum for auditory verbal attention in the IS sample), 3p25 (genome-wide maximum for visual attention in the AF sample), 6q25, 11q24, 13q34, 14q11 and 18q22,as on 22q12 (genome-wide maximum for perseverations in the Com).

Finally, we explored the statistical evidence for some interplay between the QTLs on 2q and 4q, revealed in the present study, and other loci on 1q and 5q earlier identified in Finnish schizophrenia families (18–20). Interestingly, the evidence of linkage of delayed memory to 4q21 was higher for families showing exclusion of linkage to either 5q31 or, to a lesser extent, 1q42. In sharp contrast, no such difference was observed with linkage of visual working memory to 2q36. Thus, we found some initial evidence of negative interaction between the schizophrenia susceptibility loci on 5q31 and the QTL on 4q21. Alternatively, the finding could indicate that the families linked for example to 5q are descendants of a founder allele and more closely related to each other than to the rest of the sample. Hence, eliminating linked families might have left a more homogeneous subsample with stronger linkage signals on 4q, without there necessarily being any functional interaction between the two loci.

To conclude, we detected evidence of linkage of several quantitatively measurable functions related to verbal learning and memory to chromosome 4q21, and suggestive evidence for visual working memory to chromosome 2q35–36 in Finnish schizophrenia families. Both chromosomes have been implicated earlier via genome-wide scans for genetic susceptibility loci for qualitative diagnosis of schizophrenia in Finland as well as elsewhere. Suggestive evidence was also obtained for linkage of recognition memory to chromosome 10p13, a region implicated by several other schizophrenia study groups world-wide, as well as of executive function to 9p22 and visual attention to 15q22 in the Com. In all these loci the linkage information was greatly accentuated by applying the quantitative traits, compared with dichotomous trait definition, emphasizing the value of trait components in addition to the end diagnosis in the search for susceptibility loci for schizophrenia. The current findings should be highly useful in subsequent studies aimed at physical restriction of the loci of interest and analysis of the positional candidate genes, as well as in understanding eventually their role in the normal function of the brain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Samples
Study participants were drawn from a cohort comprising all individuals born in Finland between 1940 and 1976 and screened for a history of hospitalization, drug prescriptions and work disability due to a psychotic disorder between 1969 and 1998 using the Hospital Discharge Register the Free Medicine Register, and the Pension Register. Information was linked to the National Population Register Centre to construct core families. Details concerning the ascertainment strategy, permission to access the registers, informed consent, contacting the probands and family members, and sample collection are given elsewhere (18,20). All available medical records were collected for consensus diagnoses and performing the Operational Criteria Checklist for Psychotic Illness (38). The restricted clinical study included a neuropsychological test battery and the Structured Clinical Interview for DSM-IV. The final consensus best-estimate lifetime diagnosis was assigned based on all information.

The present study sample was obtained by linking the data derived from neuropsychological tests with the genotypes of genome-wide polymorphic microsatellite markers (20). There were 168 nuclear families with at least two individuals having both interview and molecular genetic data (Table 1). The sample thus comprised 598 individuals: 179 had a diagnosis of schizophrenia (age range 31–73, mean 45.8±8.3; 65 females and 114 males) and 419 were siblings or parents (age range 24–88, mean 52.4±11.9; 218 females and 201 males).

Genotypes were available from a total of 815 individuals of these families. This information was utilized in the subsequent linkage analyses (see later). One hundred and ten families (65%) came from an internal isolate in northern Finland with a high age-corrected lifetime risk of schizophrenia (3.2%) (39) (IS subsample). Although many individual nuclear families of the IS sample can be linked to larger pedigrees (18,20), owing to incompleteness of the data with a significant amount of missing information, analysis of only IS core families was considered to be the most appropriate approach for the current study. The remainder of the families lived elsewhere in Finland (AF subsample). The genealogical search has been reported previously in detail (18,40). The correct family structures were confirmed by Mendelian inheritance analysis of various multiallelic markers within the families (20).

The control twin pairs were originally recruited to represent a control sample without any mental disorder in a study examining the inheritance of neuropsychological dysfunction and structural abnormalities of the brain in twins discordant for schizophrenia (12). A comprehensive neuropsychological test battery, including all the tasks used in the present study, was administered. In the present study, the control data represented that of the general population, as required for the chosen variance-component analyses. The age and sex structures of the sample corresponded well with those of the present study population. To avoid violating the internal independence of the data, every other MZ and DZ co-twin was randomly selected, resulting in a control sample of 56 individuals (26 females).

Neuropsychological test procedures
The neuropsychological tests were administered to all the subjects in the same order (details concerning the examination and scoring process is detailed in literature (15). Auditory attention and verbal working memory were assessed by the Digit Span forward and backward tests, respectively, of the Wechsler Memory Scale-Revised (41). The Visual Span forward and backward subtests were used to assess visual attention and visual working memory (41). Verbal learning and memory were assessed with the California Verbal Learning Test (42), which examines recall and recognition of word lists over a number of trials. The present study reports the following variables: total word recall in five trials (verbal learning), using semantic clusters as a learning strategy (semantic clustering), making recall errors (perseverations and intrusions), recalled words after a 30 min delay (delayed memory) and recognition memory. The Color–Word interference score computed from the Stroop task was used for assessing executive function (43).

Genotyping
DNA was extracted from EDTA-anticoagulated whole blood according to standard procedures. A genome-wide scan was carried out using a set of 449 markers spaced at 7.5±4.05 cM intervals on average (range 0.55–39.75; 80% of intervals were ≤10 cM) across all the human autosomes. The markers, di-, tri- and tetranucleotide repeats, were selected from the CHLC-6 set and supplementary markers were added from the Généthon map. The maps we used incorporated information from the Marshfield meiotic map, the Stanford University G3 RH data and the Human Genome Browser (http://genome.cse.ucsc.edu/index.html; draft assembly issued on January 9, 2001), which is based on sequence data from the Human Genome Project.

Polymerase chain reactions (PCR) were set up with 20 ng genomic DNA. PCR cycling consisted of denaturation at 95°C for 5 min, followed by 30 cycles at 95°C for 30 s, at 55°C for 30 s, and at 72°C for 60 s, concluding with extension at 72°C for 10 min. The gels were run on an Applied Biosystems (ABI) 377 DNA sequencer, using ABI Prims* 377 data collection software. Data were analyzed with the ABI Prims* GeneScan* 2.0.2 with Genotyper 1.1.1.

Statistical analyses
Genotyped markers were checked for incompatibilities by the programs PedCheck (44) and MENDEL (45). The linkage analysis with dichotomous clinical phenotype of four increasingly inclusive LC derived from DSM-IV was performed by using the MLINK program of the LINKAGE package as described (20). LC 1 constituted schizophrenia only, LC 2 added schizoaffective disorder, LC 3 added schizoid, schizotypal or paranoid personality disorder, schizophreniform, delusional and brief psychotic disorder as well as psychosis NUD and LC 4 major affective disorders. Preliminary statistical analyses of the neuropsychological features, including descriptive statistics as well as bivariate correlation analysis, were performed using SPSS software (SPSS Inc, Chicago, USA). SOLAR, an oligogenic variance-component linkage method incorporating the variance-component program FISHER, was applied for the QTL linkage analysis (45,46). As grouping the various neuropsychological functions to fewer items could have discarded valuable linkage information (47), we preferred using the raw test data in the analyses. Variance-component based analysis may be vulnerable to deviation from multivariate normality and, in particular, to high levels of kurtosis in the trait distribution (16). The remaining distributions were therefore transformed to approximate normality (log10-transformed values for perseverations, intrusions and executive function; square root for semantic clustering and cubed for recognition memory). The performance characteristics of the index study and control samples are given in Table 1.

All genetic analyses included an ascertainment correction in which individuals with schizophrenia were labeled as the proband. The mean of the control sample was applied as a constraint, and age and gender terms were included as covariates. Our primary interest was analysis of the Com. However, owing to the potential difference in the genetic background of the 110 families collected from an internal isolate representing a more restricted gene pool, we also analyzed separately these families (IS subsample) and those from the rest of the country (AF subsample).

All data, including the complete list of markers as well as the two-point and multipoint LOD scores in all samples, may be found at our website: http://www.ktl.fi/mols/wwwpub.htm.


   ACKNOWLEDGEMENTS
 
The authors are most grateful to the participating individuals, and to all the team workers involved in the sample collection. Thanks to R. Arajärvi, H. Juvonen, M. Muhonen, J. Suokas, K. Suominen and J. Suvisaari for their participation in the diagnostic procedure. We also wish to thank J. Kaprio for access to the twin data, M. Schreck and P. Haimi for their input in the computational issues, and J. Terwilliger, H. Göring and J. Blangero for their statistical advice. This work was supported in part by Millennium Pharmaceuticals, Inc., and Wyeth-Ayerst Research Division. Support from the Emil Aaltonen Foundation for T.H. is gratefully acknowledged. Dr Peltonen occupies the Gordon and Virginia Distinguished Chair in Human Genetics at the David Geffen School of Medicine, UCLA.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Molecular Medicine, National Public Health Institute, Biomedicum, PL 104, 00251 Helsinki, Finland. Tel: +358 947448751; Fax: +358 947448480; Email: tiina.paunio{at}ktl.fi


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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