Original article 159

Linkage disequilibrium analyses in the Costa Ricanpopulation suggests discrete gene loci for schizophreniaat 8p23.1 and 8q13.3Consuelo Walss-Bassa, Ana Patricia Monterod, Regina Armase,Albana Dassoria, Salvador A. Contrerasa, Wei Liua, Rolando Medinaa,Douglas Levinsonf, Mariana Pereirad, Ivannia Atmellad, Lisa NeSmitha,Robin Leachb, Laura Almasyc, Henriette Raventosd and Michael A. Escamillaa,b

Linkage studies using multiplex families have repeatedly

implicated chromosome 8 as involved in schizophrenia

etiology. The reported areas of linkage, however, span a

wide chromosomal region. The present study used the

founder population of the Central Valley of Costa Rica

and phenotyping strategies alternative to DSM-IV

classifications in attempts to further delimitate the

areas on chromosome 8 that may harbor schizophrenia

susceptibility genes. A linkage disequilibrium screen

of chromosome 8 was performed using family trios of

individuals with a history of psychosis. Four discrete

regions showing evidence of association (nominal P values

less than 0.05) to the phenotype of schizophrenia were

identified: 8p23.1, 8p21.3, 8q13.3 and 8q24.3. The region

of 8p23.1 precisely overlaps a region showing strong

evidence of linkage disequilibrium for severe bipolar

disorder in Costa Rica. The same chromosomal regions

were identified when the broader phenotype definition of

all individuals with functional psychosis was used for

analyses. Stratification of the psychotic sample by

history of mania suggests that the 8q13.3 locus may be

preferentially associated with non-manic psychosis.

These results may be helpful in targeting specific areas

to be analyzed in association-based or linkage

disequilibrium-based studies, for researchers who

have found evidence of linkage to schizophrenia on

chromosome 8 within their previous studies. Psychiatr

Genet 16:159–168 �c 2006 Lippincott Williams & Wilkins.

Psychiatric Genetics 2006, 16:159–168

Keywords: association, Costa Rica, linkage disequilibrium, mania, psycho-sis, schizophrenia

Departments of aPsychiatry, bCellular and Structrual Biology, University of TexasHealth Science Center at San Antonio, cSouthwest Foundation for BiomedicalResearch, San Antonio, Texas, dCenter for research in Cellular and MolecularBiology, University of Costa Rica, San Jose, Costa Rica, eLangley PorterPsychiatric Institute, University of California at San Francisco, San Francisco,California and fDepartment of Psychiatry, Stanford University, Stanford, California,USA.

Correspondence and requests for reprints to Michael Escamilla, 7703 Floyd CurlDrive, San Antonio, TX 78229-3900, USATel: + 1 210 562 5111; fax: + 1 210 562 5114;e-mail: escamillam@uthscsa.edu

Sponsorship: This work was supported in part by National Institute of HealthGrants R01-MH61884 and K01 MH01453 (M.A.E.), the NARSAD YoungInvestigator Award (M.A.E.), the Howard Hughes Medical Foundation, the Friendsof Psychiatry (UTHSCSA Department of Psychiatry), the International Center forGenetic Engineering and Biotechnology Project CRP/COS98-01 (H.R.), and aNational Institute of Health Minority Supplement Award 3 R01-Mh61884 toC.W.B.

IntroductionSchizophrenia (SC), a devastating mental illness that

affects around 1% of the population worldwide, is among

the most costly and debilitating of the psychiatric

disorders (Kessler et al., 1995; Wyatt et al., 1995). Although

genetic factors are known to play an important role, the

identification of such genes, and the molecular mechan-

isms of the disease, have remained elusive. This can be

due to the fact that numerous genes of minor effect are

involved in its etiology (Pulver et al., 2000), together with

environmental factors (Howes et al., 2004). Furthermore,

different combinations of disease susceptibility genes are

also expected. The complex inheritance and genetic

heterogeneity would explain the difficulties in replicating

linkage studies of SC. Nevertheless, through the use of

narrower and more precise phenotypes, certain broad

chromosomal regions have shown, in more than one

independent study, evidence of linkage at levels of

significance considered suggestive or significant by

Lander and Kruglyak (1995). The small arm of chromo-

some 8 (8p) is one such region (Pulver et al., 1995;

Kendler et al., 1996; Blouin et al., 1998; Shaw et al., 1998;

Brzustowicz et al., 1999; Gurling et al., 2001).

Most of the early studies searching for SC susceptibility

genes were carried out using large multiplex families. In

family linkage studies, mapping signals generally span

many centi-Morgans resulting in broad areas of possible

linkage (up to 20 cM or larger). The fact that earlier

studies predominantly used multiplex mapping, together

with the use of different sample sizes, analytic and family

ascertainment strategies, phenotype designation methods

0955-8829 �c 2006 Lippincott Williams & Wilkins

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

and inaccurate marker maps could account for the wide

area of positive linkage results spanning chromosome

8p. It is also possible that the chromosome 8p region

may potentially harbor several predisposition genes.

Supporting the latter possibility, several candidate genes

in the 8p region have recently been reported to be

associated with SC (Stefansson et al., 2002; Gerber et al.,2003; Katsu et al., 2003; Yang et al., 2003; Nakata and

Ujike, 2004).

One potentially useful method, both to map at a genome

level and to map at narrow regions where SC suscept-

ibility genes are located on chromosome 8, is to perform

systematic linkage disequilibrium (LD) analyses. The

present study performed an LD screen for SC genes on

chromosome 8 using a sample of individuals with a history

of psychosis, descendant from the founding population of

the Central Valley of Costa Rica (CVCR). The CVCR

population is an ideal one for performing LD analysis at

the genome screening level because of its structure,

origin and well documented genealogical information

(Escamilla et al., 1999; Ophoff et al., 2002; Mathews et al.,2004). A subset of the present day CVCR inhabitants are

descended from a small group of founders dating back to

the first Spanish settlement in 1569, approximately 20

generations ago (Escamilla et al., 1996). This type of

isolated expansion is ideal for detecting founder haplo-

types in genetic illnesses; compared with the few other

isolated populations in the world, such as Finland, regions

of the Netherlands and French Canada, and the Amish,

Costa Rica’s expansion has been the greatest and from

the fewest number of founders (Escamilla et al., 2001).

Gene mapping studies in non-psychiatric disorders in this

population have also led to significant findings, leading to

eventual identification of disease genes for inherited

deafness (Leon et al., 1992) and familial telangiectasia

(Uhrhammer et al., 1995).

Chromosome 8 is of special interest for those studying

psychiatric disorders (especially those involving psycho-

sis) in the CVCR. A genome-wide LD screen of severe

bipolar disorder (BP) conducted in this population

reported the most significant finding to be on a well

delimited region on chromosome 8p (Ophoff et al., 2002).

Patients with severe BP, such as those studied in the

CVCR, often have psychotic episodes (Escamilla et al.,2002) and share clinical and epidemiological features of

SC (Boteva and Lieberman, 2003). In addition to

investigating whether evidence for specific SC loci could

be found on chromosome 8 in the CVCR population, we

also wished to investigate whether SC patients would

show LD to the same region that has been shown to be in

LD with severe BP in this population (Ophoff et al.,2002). Other loci have been found in common between

SC and severe BP in samples from this population

(Freimer et al., 1996; Escamilla et al., 1999, 2001; DeLisi

et al., 2002; Walss-Bass et al., unpublished observation)

and other populations (Gershon and Badner, 2001).

Although formal criteria for defining SC have been

developed and the diagnosis using current classification

systems is highly reliable (Nurnberger et al., 1994),

naturalistic studies of BP and SC have suggested that

alternate classifications may better define the underlying

types of psychotic disorder (Kendler et al., 1998; Swann etal., 2001). To further try to understand the nature of the

phenotypes showing LD to various loci on chromosome 8

in the CVCR, an alternative phenotyping strategy was

also used. First, individuals with a history of psychosis

[regardless of DSM-IV (Diagnostic and statistical manual

of mental disorders – fourth edition) category] were

analyzed for evidence of LD on chromosome 8. Next, it

was hypothesized that the presence or absence of mania

in the course of chronic psychotic illness might better

define homogeneous subtypes of psychotic disorder, in a

manner that is obfuscated when using DSM-IV classifica-

tions. Division of the ‘psychotic disorder’ group into two

more genetically homogeneous groups, if successful,

should result in (i) distinct genetic loci showing LD to

each of the two subtypes, and (ii) more power to detect

loci (i.e. a smaller sample of a more homogeneous

phenotype should have as much power to detect LD as

a larger sample that is not as genetically homogeneous).

The results of this study support a locus for SC at the

same location as a locus for severe BP in the CVCR

population, on chromosome 8p23.1. Supportive evidence

for other possible SC loci at two other chromosome 8

regions is also reported. Finally, we report a unique locus

on 8q, which demonstrates LD to ‘non-manic’ psychotic

disorders.

MethodsAll study participants (154 probands and 289 relatives)

were recruited in accordance with the principles of the

Declaration of Helsinki and with approval from the

Institutional Review Boards of the University of Costa

Rica and the University of Texas Health Science Center

at San Antonio. Probands were recruited independently

from one another from psychiatric hospitals and clinics in

the CVCR. All probands had disease onset by the age of

40 years, a history of at least one psychiatric hospitaliza-

tion, a discharge diagnosis of SC, bilineal Spanish

surnames and a current residence in the CVCR.

Furthermore, we performed genealogical analysis to

determine the ancestry of all participants in our study

(Table 1, section A). Two hundred and eighty-nine

parents and first-degree relatives of probands were

recruited where possible to permit determination of the

genetic phase and to allow for family-based LD analyses.

These additional participants were not diagnosed or

assessed for psychiatric disorders. The breakdown of

160 Psychiatric Genetics 2006, Vol 16 No 4

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family pedigrees recruited for our study is shown in Table

1, section B).

As in previous LD studies of BP in this population

(Escamilla et al., 1996, 1999), each proband was

interviewed by a bilingual psychiatrist who was blind to

the previous history of the participant, using the

Diagnostic Interview for Genetic Studies (Nurnberger

et al., 1994). Medical records (inpatient and outpatient)

were also abstracted for each proband. An interview

with a close relative, using a Spanish version of the

Family Interview for Genetic Studies was also completed

for each proband. All 154 probands were diagnosed

using a best-estimate diagnostic process like the one

described in previous CVCR studies (Escamilla et al.,1996, 1999). The process arrives at a lifetime consensus

diagnosis or diagnoses using the DSM-III-R, DSM-IV

and RDC systems. For each proband, the best estimators

also diagnosed whether manic and/or depressive syn-

dromes (as defined by the DSM-IV) had been present

during the course of the disorder. Each proband was also

evaluated at the best estimate for a history of psychosis,

as defined by a history of hallucinations, delusions,

disorganized thinking or behavior (symptoms 1–4 of

category A of DSM-IV SC). All probands had a history

of psychosis.

Best-estimate consensus diagnoses of the 154 probands

recruited for this study are shown in Table 1 (section C).

All 154 probands had a history of psychosis. Seventy-two

of the 154 probands had both a history of psychosis and at

least one manic syndrome during the course of their

illness and 82 had never met full criteria for a manic

syndrome (DSM-IV criteria). Of the 72 participants who

had histories of both psychosis and mania, 14 had final

consensus DSM-IV diagnoses of bipolar type I disorder

(BPI), 19 were diagnosed with schizoaffective bipolar

disorder (SABP), and 39 were diagnosed with SC. When

probands had both manic syndromes and psychotic

episodes, and the psychoses lasted more than 2 weeks

in the absence of mood syndromes, the DSM-IV allows a

diagnosis of either SC or SABP to be given. In deciding

between SC and SABP for such cases, using DSM-IV

criteria, SABP was operationalized as a case in which the

affective syndromes (manias and depressions) had been

present for 30% or more of the entire illness. This

operationalization is consistent with the Family Interview

for Genetic Studies and the Diagnostic Interview for

Genetic Studies, which query this item, and with current

classifications used in the NIMH Genetics Initiative

studies of Schizophrenia (Levinson and Escamilla,

personal communications).

Genotyping

A genomewide survey of background LD in the CVCR

population showed that LD extended to distances of 2–

7 cM in 310 of 1012 adjacent marker pairs (Service et al.,1999). We therefore expected that, if there is a founder

mutation in the CVCR that influences SC, using a marker

density of 5 cM and the sample described above, we

would have reasonable power, as a first pass, to identify

loci most likely to be in LD with genes influencing our

diagnoses of interest.

DNA from a total of 443 people, of which 154 were

diagnosed probands, was genotyped. Marker order,

heterozygosity and sex-averaged distance from the p-

terminal are listed in Fig. 1. Genotyping was initially

performed using 30 fluorescently labeled microsatellite

markers of the highest heterozygosity available, spaced at

an average of 5 cM. Follow-up genotyping was then

performed using closely spaced microsatellite markers

positioned in the areas where positive evidence of

association was initially obtained (Table 2). Standard

polymerase chain reaction (PCR) was performed using

the ABI 877 automated thermocycler (Applied Biosys-

tems, Foster city, CA, USA) or the PE 9700 PCR

instrument (Applied Biosystems, Foster city, CA, USA).

Amplified fragments were analyzed on the ABI 377

sequencer (Applied Biosystems, Foster city, CA, USA)

and genotypes were assigned using GENESCAN and

GENOTYPER software. Each genotype was scored

separately by two individuals, who were blind to the

diagnosis of the participants. Genotypes were compared

using a software program, discrepancies were discussed

and final genotypes agreed upon. Genotypes were

checked for violations of Mendelian inheritance by the

PEDSYS program INFER. A total of four families were

discarded from statistical analyses owing to recurrent

Mendelian discrepancies.

Table 1 Diagnostic and family structure information

Proband information n

A. AncestryAt least four great-grandparents born in the CentralValley of Costa Rica

152a

All known ancestors from the Guanacaste area ofCosta Rica

2

Total 154B. Family structure

Both parents sampled 45At least one parent sampled 62No parents, but one or more siblings sampled 13No relatives available 34Total 154

C. DSM-IV diagnosis of probandsSchizophrenia 105Schizoaffective bipolar 19Schizoaffective depressed 6Bipolar disorder I 14Major depressive episode with psychosis 6Psychosis not otherwise specified 4Total 154

aNine of the probands had incomplete information to ascertain complete ancestryof the great-grandparents. DSM-IV, Diagnostic and statistical manual of mentaldisorders – fourth edition.

LD analysis of chromosome 8 in Costa Rica Walss-Bass et al. 161

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Fig. 1

II

IV

I

Region

Ideogram

8p23+38p23+2

8p23+1

8p22

8p21+2

8p11+18q11+1

8q12+38q13+18q13+28q13+3

8p21+1

8p12

8p11+238p11+228p11+21

8q11+21

8q11+22

8q11+23

8q12+18q12+2

8q21+118q21+128q21+13

8q21+2

8q21+3

8q22+18q22+2

8q22+3

8q23+18q23+2

8q23+3

8q24+118q24+12

8q24+13

8q24+21

8q24+228q24+23

8q24+3

8p21+3

D8 S 264

D8 S 277

D8 S 1825

D8 S 503

D8 S 542

D8 S 516D8 S 1721

D8 S 520D8 S 552D8 S 549

D8 S 261

D8 S 282D8 S 1786D8 S 1734

D8 S 1771

D8 S 1820D8 S 505

D8 S 532D8 S 285D8 S 260

D8 S 1841D8 S 1767

D8 S 1117D8 S 543D8 S 1795

D8 S 279

D8 S 1144D8 S 1705

D8 S 1757

D8 S 275D8 S 271D8 S 270D8 S 1762

D8 S 1784D8 S 1779

D8 S 514D8 S 1799

D8 S 284

D8 S 256D8 S 272

Heterozygosity

D8 S 1837

D8 S 1743D8 S 1836

0.73

8.34

15.38

16.19

16.1917.00

17.00

20.6126.4331.73

37.0441.5542.8545.41

46.26

50.0556.04

60.8764.7571.00

79.3682.26

83.5085.2387.54

87.54

91.4694.61

96.21

97.28

98.88102.6

103.69

112.42118.15123.54

130.00133.64

143.82148.12

154.02

156.59162.94

165.93

0.83

0.73

0.710.610.74

0.600.71

0.770.800.61

0.790.70

0.740.86

0.670.770.750.80

0.830.78

0.820.79

0.680.68

0.740.66

0.88

0.760.840.80

0.750.770.79

0.770.68

0.760.760.84

0.830.83

0.80

0.810.83

0.84

D8 S 258

Marker pter (cM)

III

Chromosome 8 schizophrenia susceptibility loci in Costa Rica. Boxed numbers to the left of the chromosome ideogram represent chromosomalareas with positive association in our present study. Markers are positioned according to genetic distance (cM) from the p-terminal. Marker positionsand heterozygosity are from Marshfield.

162 Psychiatric Genetics 2006, Vol 16 No 4

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Statistical analysis

Potential LD was evaluated between markers and a

putative disease locus by the T1 and T3 tests of

association implemented in the program CLUMP (Sham

and Curtis, 1995). Both tests evaluate whether particular

alleles at a locus are overrepresented on disease chromo-

somes, compared with non-disease chromosomes. The T3

statistic is most powerful when one allele at a particular

marker is associated with the disease, while the T1

statistic looks at whether allele frequencies are statisti-

cally different from one group to another. The T1 and T3

CLUMP tests thus offer compensatory strengths and

weaknesses when used in the search for genes in a

complex disease. At the time this study was originally

designed, newer family-based association tests that can

accommodate multiple affected family members, such as

FBAT (Laird et al., 2000), were not available. Our design

therefore focused on one affected individual (i.e. the

proband) per family and used the CLUMP test as our

primary method of analysis. Current and future studies in

this population sample will diagnose all individuals within

these families and will probably use FBATor other similar

analyses to assess the association between markers and

phenotypes of interest.

To determine whether the association detected by the

CLUMP analyses was due to LD, the likelihood-ratio test

of LD (measured by the parameter l), implemented in

DISLAMB (Terwilliger, 1995), was performed. The

likelihood-ratio test of LD has been previously used to

Table 2 Results of T1 and T3 CLUMP analyses (P values) for schizophrenia and psychosis in the Central Valley of Costa Rica

Markers Schizophrenia Psychosis

Initial screen Follow-upmarkers

pter (n = 105) All (n = 154) Mania (n = 72) No mania (n = 82)

cM T1 T3 TI T3 T1 T3 T1 T3

D8S264 0.730 NS NS NS NS NS NS NS NSD8S277 8.340 NS NS NS NS NS 0.0839 NS NS

D8S1825 15.38 NS NS 0.082 NS 0.008 0.034 NS NSD8S542 16.19 0.036 0.008 0.090 0.031 0.019 0.084 0.063 0.027

D8S503 16.19 0.019 0.044 NS NS NS NS NS NSD8S516 17.00 NS NS 0.092 NS NS NS NS NSD8S1721 17.00 NS NS NS NS NS NS NS NS

D8S520 20.61 NS NS NS NS NS NS NS NSD8S552 26.43 NS NS NS NS NS NS NS NSD8S549 31.73 NS NS NS NS NS NS NS NSD8S261 37.04 NS NS NS NS NS NS NS NS

D8S258 41.55 0.049 0.080 0.055 NS NS NS NS NSD8S282 42.85 0.092 0.048 NS 0.028 NS NS 0.057 0.033

D8S1786 45.41 NS NS NS NS NS NS NS NSD8S1734 46.26 NS NS NS NS NS NS NS NSD8S1771 50.05 NS NS NS NS NS NS NS NSD8S1820 56.04 NS NS NS NS NS NS NS NSD8S505 60.87 NS NS NS NS NS NS 0.068 NSD8S532 64.75 NS NS NS NS NS NS NS NSD8S285 71.00 0.059 0.062 0.0699 0.065 NS NS 0.071 0.026

D8S260 79.36 NS NS NS NS NS NS NS NSD8S1841 82.26 NS NS NS 0.030 NS 0.0789 NS 0.088D8S1767 83.51 NS NS NS NS NS NS NS NSD8S1117 85.23 0.098 NS 0.010 0.076 0.039 NS 0.034 0.047

D8S543 87.54 0.023 0.049 0.0280 0.035 NS NS 0.002 0.009

D8S1795 87.54 NS NS NS NS NS NS NS NSD8S279 91.46 NS NS NS NS NS NS NS NSD8S1144 94.61 NS NS NS NS NS NS NS NS

D8S1705 96.21 NS NS NS NS NS NS 0.001 0.012

D8S1757 97.28 NS NS NS NS NS NS NS NSD8S275 98.88 NS 0.071 NS NS NS NS NS 0.041

D8S271 102.6 NS NS NS NS NS NS NS NSD8S270 103.6 NS NS 0.098 NS NS NS NS NSD8S1762 112.4 NS NS NS NS NS NS NS NSD8S1784 118.1 NS NS NS NS NS NS NS NSD8S1779 123.5 NS NS NS NS NS NS NS NSD8S514 130.0 NS NS NS NS 0.089 NS NS NSD8S1799 133.6 NS NS NS NS NS NS NS NSD8S284 143.8 NS NS NS NS NS NS NS NSD8S256 148.1 NS NS NS NS NS NS NS NSD8S272 154.0 NS NS NS NS NS NS NS NSD8S1837 156.5 NS NS NS NS NS NS NS NSD8S1743 162.9 NS NS NS NS NS NS NS NSD8S1836 165.9 0.026 0.039 0.015 0.010 NS NS 0.070 0.051

Only P values less than 0.1 are listed. Genetic distances for each marker from the pter are from Marshfield. Numbers in bold are those with P < 0.05. All, all diagnosis witha history of psychosis; NS, non-significant.

LD analysis of chromosome 8 in Costa Rica Walss-Bass et al. 163

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

assess LD of BP disorder in the Costa Rican population

(Escamilla et al., 1999). In that study, simulation tests

were performed to determine the power to detect LD

using different sample sizes and phenocopy rates. It was

found that with a sample of 90 patients and their

relatives, assuming a phenocopy rate of 50%, a suggestive

level of association (Pr 0.05) could be detected 82% of

the time. Increasing the sample size to 200 improved the

power to detect LD to 99% of the time. Given that the

sample size in our present study is 105 SC patients from

the same Costa Rican population, and that parental

alleles were available in the majority of the individuals

(through either direct genotyping or inference), we felt

that by establishing an a priori level of significance of

P = 0.05 we would be able to detect LD over 82% of the

time, if 50% of the individuals have the same mutation.

Alleles from chromosomes of ‘affected’ persons were

compared with non-transmitted parental chromosomes,

using the CLUMP and DISLAMB tests modified to allow

analysis of DNA from affected individuals whose parents

were not available (Escamilla et al., 1999). Whenever

possible, if both parents were not available, additional

siblings of the affected individual were genotyped to

permit inference of the parental alleles.

Initially, the phenotype of interest studied was SC, as

defined by the DSM-IV by best-estimate consensus. To

maximize the power of analysis and because of the fact

that the population sample contained several individuals

whose parents were not available for analysis, the sample

group used for the control chromosomes included non-

transmitted chromosomes from the parents of probands

of all diagnoses. The allele frequency distribution (for

each marker tested) from this group was not statistically

different from that of the parents of SC probands only. It

was therefore felt that the sample of parents of all

diagnosis was from the same population pool as the

sample of parents of SC probands only. To address the

possibility of population stratification, however, LD

analysis of the markers that showed positive results in

the CLUMP tests was performed using the more

conservative transmission disequilibrium test of Spielman

et al. (1993), which uses only affected subjects with both

parents available for genotyping.

Given the symptomatic and syndromal overlaps between

SC, BP and other psychotic disorders, an alternate

phenotyping strategy was also pursued. A more general-

ized ‘psychosis’ (all psychotic disorders) phenotype was

analyzed for evidence of LD. Subsequently, this sample

was divided into those with and those without a history of

mania (full manic syndrome or episode, by consensus

diagnosis). The ‘psychosis’ sample included all 154

probands, all of who had a history of psychosis (by best-

estimate consensus). Forty-seven percent of these

individuals had a history of full mania (‘manic psychosis’

sample), while 53% had a psychotic disorder with no

previous history of mania. The control sample of non-

transmitted chromosomes remained the same for each

subset.

ResultsResults from the T1 and T3 CLUMP analyses are shown

in Table 2. Markers listed in the first column are those in

the initial 5 cM screen, while markers listed in the second

column are follow-up markers in the regions where

evidence of association was initially seen. For simplicity

purposes, only P values less than 0.10 are listed [others

are listed as non–significant (NS)] and P values less than

0.05 are displayed in bold.

From the initial 5 cM screen, evidence of association

(Pr 0.05) with the SC phenotype (105 affected

individuals) was obtained at four markers, representing

four distinct chromosomal regions (see Fig. 1 for position

of markers along the chromosome): region I: 8p23.1

(D8S542), region II: 8p21.3 (D8S282), region III: 8q13.3

(D8S543) and region IV: 8q24.3 (D8S1836). The

strongest evidence of association was obtained at

D8S542 for the SC category (P = 0.008 for T3). Of the

markers that suggested positive association to the SC

phenotype, D8S542 and D8S543, of regions I and III,

respectively, also showed evidence of association by the

more conservative transmission disequilibrium test ana-

lysis (P = 0.032 and 0.033, respectively). When the

phenotype of ‘psychosis’ was analyzed (154 individuals),

the same four markers showed evidence of association at

Pr 0.05. No additional markers showed evidence of

association to the ‘psychosis’ phenotype.

Follow-up markers

Additional, follow-up markers (markers in close proximity to

markers that tested positive on the initial screen) were

tested for regions I, II and III. In each case, at least one

additional marker, closely linked to the screening markers in

those regions, gave additional evidence of association to SC

or to all psychosis categories (D8S503 in region I, D8S258 in

region II, and D8S1841 and D8S1117 in region III).

Analyses of psychoses with and without a history

of mania

Stratification of the total ‘psychosis’ sample by the

presence or absence of mania yielded two additional

regions, which had not passed our a priori threshold for

association to the ‘SC’ or the ‘all psychosis’ phenotypes

(Table 2). These two additional loci, 8q12.1 (D8S285)

and 8q21.13 (D8S1705 and D8S275), showed possible

association to the ‘no history of mania’ psychosis

phenotype (n = 82) at levels of significance of P < 0.05.

Regions I, II, III and IV, which had shown evidence of

possible association to the ‘all psychosis’ and ‘SC’

164 Psychiatric Genetics 2006, Vol 16 No 4

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phenotypes, all showed evidence of association at

Pr 0.05 to this phenotype (psychosis without mania)

as well. The strongest evidence of association to this

phenotype was at 8q21.13 (P = 0.001 at D8S1705).

For the ‘psychosis with mania’ group (n = 72), only

regions I and III showed association at levels less than

Pr 0.05. Region I showed the strongest evidence of

association to the psychosis with mania phenotype

(P = 0.008 at D8S1825 and P = 0.019 at D8S542).

Results from the DISLAMB analysis are shown in Table 3

for all diagnostic categories. For simplicity purposes, only

l values greater than 0 are displayed. P values less than

0.05 are displayed in bold. Except for region IV, at 8q24.3,

evidence of LD (l> 0) was obtained in the same regions

that displayed positive association to SC and psychosis

(either stratified or overall). Only two regions, however,

met the criteria for LD at a significance level of P < 0.05:

region I (D8S503), at 8p23.1 was seen to be in LD with

SC (P = 0.0270). In region III, at 8q13.3, markers

D8S1841 and D8S543 were in apparent LD with SC

(P = 0.0398 and 0.0191 respectively), while markers

D8S1117 and D8S543 appeared to be in LD with overall

psychosis (P = 0.0230 and 0.0240, respectively). Inter-

estingly, when the psychotic sample was stratified to

include only non-manic individuals, all three of the region

III markers displayed P values less than 0.05 (P = 0.0350,

0.01176 and 0.0044 for markers D8S1841, D8S1117 and

D8S543, respectively).

Table 3 Results of DISLAMB analysis (k and P values) for schizophrenia and psychosis in the Central Valley of Costa Rica

Markers Schizophrenia Psychosis

Initial screen Follow-upmarkers

pter (n = 105) All (n = 154) Mania (n = 72) No mania (n = 82)

cM l P l P l P l P

D8S264 0.730 NS NS NS NS NS NS NS NSD8S277 8.340 NS NS NS NS NS NS NS NS

D8S1825 15.38 NS NS NS NS NS NS NS NSD8S542 16.19 0.0464 0.4009 NS NS 0.0502 0.3592 NS NS

D8S503 16.19 0.1465 0.0270 0.0900 0.3935 NS NS 0.1155 0.1887D8S516 17.00 NS NS NS NS NS NS NS NSD8S1721 17.00 NS NS NS NS NS NS NS NS

D8S520 20.61 NS NS NS NS NS NS NS NSD8S552 26.43 NS NS NS NS NS NS NS NSD8S549 31.73 NS NS NS NS NS NS NS NSD8S261 37.04 NS NS NS NS NS NS NS NS

D8S258 41.55 NS NS NS NS NS NS NS NSD8S282 42.85 0.1147 0.3363 0.1103 0.2909 NS NS 0.1490 0.1144

D8S1786 45.41 NS NS NS NS NS NS NS NSD8S1734 46.26 NS NS NS NS NS NS NS NSD8S1771 50.05 NS NS NS NS NS NS NS NSD8S1820 56.04 NS NS NS NS NS NS NS NSD8S505 60.87 NS NS 0.1310 0.1931 0.1928 0.0727 0.0221 0.4390D8S532 64.75 NS NS NS NS NS NS NS NSD8S285 71.00 0.1407 0.1856 0.1530 0.1324 0.3338 0.2638 0.1693 0.1005D8S260 79.36 NS NS NS NS NS NS NS NS

D8S1841 82.26 0.2073 0.0398 0.1850 0.0550 NS NS 0.2250 0.0350

D8S1767 83.51 0.1780 0.2196 0.1890 0.1505 0.2016 0.1968 0.1785 0.2583D8S1117 85.23 0.1780 0.0850 0.2096 0.0230 0.1661 0.1857 0.2454 0.0176

D8S543 87.54 0.2009 0.0191 0.1805 0.0240 NS NS 0.2565 0.0044

D8S1795 87.54 NS NS NS NS NS NS NS NSD8S279 91.46 NS NS NS NS NS NS NS NSD8S1144 94.61 NS NS NS NS NS NS NS NS

D8S1705 96.21 NS NS NS NS NS NS NS NSD8S1757 97.28 NS NS NS NS NS NS NS NS

D8S275 98.88 0.1614 0.1101 0.1192 0.2806 0.1614 0.1686 NS NSD8S271 102.6 NS NS NS NS NS NS NS NS

D8S270 103.6 NS NS NS NS NS NS NS NSD8S1762 112.4 NS NS NS NS NS NS NS NSD8S1784 118.1 NS NS NS NS NS NS NS NSD8S1779 123.5 NS NS NS NS NS NS NS NSD8S514 130.0 NS NS NS NS NS NS NS NSD8S1799 133.6 NS NS NS NS NS NS NS NSD8S284 143.8 0.1119 0.0982 NS NS NS NS NS NSD8S256 148.1 NS NS NS NS NS NS NS NSD8S272 154.0 NS NS NS NS NS NS NS NSD8S1837 156.5 NS NS NS NS NS NS NS NSD8S1743 162.9 NS NS NS NS NS NS NS NSD8S1836 165.9 NS NS NS NS NS NS NS NS

Only l values more than 0 and P values less than 0.1 are listed. Genetic distances for each marker from the pter are from Marshfield. Numbers in bold are those withP < 0.05. All, all diagnosis with a history of psychosis; NS, non-significant.

LD analysis of chromosome 8 in Costa Rica Walss-Bass et al. 165

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

DiscussionThe current study utilized an LD approach to screen for

possible SC loci in the CVCR, using an a priori threshold

of P < 0.05. The results of this study suggest two discrete

loci (8p23.1 and 8q13.3) that are most likely to harbor SC

susceptibility genes on chromosome 8, in the Costa Rican

population, as well as two additional loci on chromosome

8 meriting further investigation for psychosis-related

phenotypes. Evidence for both association and LD at

these loci are not at the levels typically required to prove

significant evidence of linkage at the genome screen

level. In addition, as the statistical tools used in this study

(CLUMP T1, CLUMP T3 and DISLAMB tests) are not

independent of each other, corrections for multiple

testing were not applied. Given the size of the sample

involved in this study, and the anticipated complexity of

SC at the genetic level, strongly significant evidence of

LD will require larger samples, additional (haplotype-

based) analyses or additional statistical tools. This study

presents an approach to identifying genes for complex

disorders within a founder-type population (Escamilla etal., 1996), by using a genome screening approach with

previously defined levels of significance (based on the

sample size and power calculations) to identify regions

showing possible LD. The intention of the study is to

identify possible gene loci, which will then be followed

up by more intensive study to identify predisposition

genes within the population of interest.

Several studies have reported linkage of SC to chromo-

some 8p, beginning with a study by Pulver et al. (1995),

conducted in pedigrees drawn from the northeast United

States. Since then, numerous studies using different

populations have reported linkages of SC to 8p (Kendler

et al., 1996; Blouin et al., 1998; Kaufmann et al., 1998; Shaw

et al., 1998; Brzustowicz et al., 1999; Gurling et al., 2001).

Most of the studies point to the region of 8p22-21,

although in two studies, the region of possible linkage

extends as far as 8p23-11 (Kaufmann et al., 1998; Gurling

et al., 2001). In addition, a genome wide screen for severe

BPI in the CVCR population showed evidence for a

possible BPI gene locus on 8p (Ophoff et al., 2002). Given

these interesting findings from previous studies, this

study analyzed a new set of individuals with a history of

psychosis from the CVCR, predominantly those with SC,

in order to identify possible discrete genetic loci for

psychotic disorders along chromosome 8, using LD

screening techniques.

This study identified four distinct chromosomal regions

showing possible association with putative SC genes on

chromosome 8: region I at 8p23.1, region II at 8p21.3,

region III at 8q13.3 and region IV at 8q24.3. Expansion of

the phenotype, to include all available individuals with

any history of psychosis (SC, BPI, schizoaffective bipolar,

schizoaffective depressed, psychosis not otherwise speci-

fied, major depression with psychosis), resulted in the

same four regions showing association. Only region I

(D8S542) showed stronger evidence of association (by an

order of magnitude) when the narrower phenotypic

definition (SC) was used. Except for region IV, all regions

in which association was detected in the current study

overlap with regions previously reported to be in linkage

with SC (Kendler et al., 1996; Blouin et al., 1998;

Kaufmann et al., 1998; Brzustowicz et al., 1999; Gurling

et al., 2001). The regions suggested by these earlier

studies cover wide areas, expanding over up to 50 cM,

leading to difficulties in fine mapping and gene

identification. The existence of several susceptibility

genes in a relatively small chromosomal region may

increase the power to find positive linkage signals in the

area, which may be the reason why chromosome 8 has

been continuously reported to be associated with SC.

When LD techniques are used in isolated populations,

finer localization of susceptibility loci may be achieved

and more than one gene can be identified in a given

chromosomal region. The current results suggest there

may indeed be more than one SC gene locus on

chromosome 8, even in a relatively homogeneous

population such as the CVCR. One should interpret the

findings of the current study with caution however, given

the low thresholds set for identifying possible SC loci.

The strongest evidence of association with SC and/or all

psychosis in the current study was obtained in the area of

region I. Three back-to-back markers in our study

(D8S1825, D8S542 and D8S503) gave positive evidence

of association in this region, and D8S503 also showed

evidence of LD (by DISLAMB) to the SC phenotype.

Marker D8S503 is the same marker that yielded the most

significant finding of association in a genome-wide LD

screen of BPI in the CVCR population (Ophoff et al.,2002). Furthermore, it is interesting that the evidence of

association in this area, at marker D8S1825, appears to

increase by an order of magnitude when the affected

sample includes only cases with a history of mania. These

results, together with the previous findings of linkage of

this region with SC (Kaufmann et al., 1998; Gurling et al.,2001) and BP (Ophoff et al., 2002), indicate that the

Region I loci may be associated with psychosis (in

particular psychotic disorders which have shown a history

of mania at some point), rather than to SC or BP

specifically.

The present results provide evidence of association with

SC and/or psychosis in the region of 8p21.3 (Region II,

D8S258 and D8S282). Stratification of the affected

psychotic sample showed segregation of association at

this locus, which appeared to be preferentially associated

with non-manic psychosis.

The current study provides substantial evidence for a SC

locus in Region III (from 82.26 to 87.54 cM on the

166 Psychiatric Genetics 2006, Vol 16 No 4

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Marshfield map). Three markers in this region pass the a

priori thresholds (Pr 0.05) for association and LD

(D8S1841, D8S1117 and D8S543). The level of evidence

for LD at these markers appears to increase when the

phenotype of ‘psychotic disorder’ without a history of

mania is analyzed, suggesting this locus may be relevant

primarily for non-manic psychotic disorders. Two addi-

tional markers, approximately 10 cM centromeric to

D8S543, D8S1705 and D8S275, which did not appear

to be associated with the SC or overall psychosis

phenotypes, also appeared to be associated with non-

manic psychosis. Whether these markers represent

association to yet another susceptibility gene on chromo-

some 8 or are associated to the same putative gene as

markers D8S543 and D8S1117 remains to be determined.

In the case of Region IV, the only reports of association

with psychotic disorders near this area come from linkage

studies with BP (Cichon et al., 2001; Dick et al., 2003).

These studies obtained positive linkage in the region of

8q24, approximately 17 cM away from marker D8S1836,

the marker yielding positive association in the current

study. As Region IV in our current study has only one

marker showing possible association to SC and no marker

showing evidence of LD at Pr 0.05, the evidence for a

SC locus in the CVCR is the least compelling of the four

regions. Additional markers, tightly linked to D8S1836,

need to be analyzed in order to evaluate whether this

region merits further study with regard to SC or other

psychotic phenotypes.

In the present study, evidence of association at a P value

of < 0.05 was not found in the region where the

neuregulin 1 gene is located (Stefansson et al., 2002).

Evidence of association (by CLUMP) and LD (by

DISLAMB) for marker D8S505, which is approximately

0.4 cM from neuregulin 1, however, was found at

significance levels slightly above 0.05. Furthermore, the

likelihood-ratio test revealed l values > 0 in this region.

As described in the methods section, this initial screen

has substantial power to detect loci (at P < 0.05) only for

loci in which 50% or more of the participants have

inherited a SC predisposition gene in common from a

specific CVCR ancestor. SC susceptibility genes shared

by less than 50% of the participants may still show

evidence of LD (and association) in this sample, but will

not necessarily pass the ‘0.05’ threshold. Given the prior

evidence (Stefansson et al., 2002) for the involvement of

neuregulin 1 in SC, and the results found for D8S505 in

the current study, further investigation of the neuregulin

1 gene region in this population is warranted.

In conclusion, this report presents the results of a LD

analysis of chromosome 8 using a founder population and

detailed phenotype analysis to identify discrete regions

most likely to harbor genes for psychotic disorders on

chromosome 8. The least stringent screen in this study

(5 cM screen) identified four distinct chromosomal

regions showing evidence of association to the phenotype

of SC in the CVCR. When more stringent evidence is

required (more than one closely linked marker showing

evidence of association at P < 0.05, at least one of those

markers showing evidence of association using the more

conservative transmission disequilibrium test, and at least

one of those markers showing evidence of LD at P < 0.05,

the present study identifies two distinct regions, 8p23.1

and 8q13.3, as the strongest candidates for further

attempts at identifying SC genes in the CVCR. The

8q13.3 locus may contain two discrete genes related to

non-manic psychotic disorders. In addition, three other

loci, including the neuregulin 1 locus, merit further study

within this population, in order to determine whether

preliminary evidence of association to SC in this sample

reflects actual SC gene loci or false positives. The

statistical significance of our results should be considered

relative but not absolute, as the P values are not corrected

for multiple testing nor confirmed by simulation. Future

analyses of this and other ‘founder’ populations, with

more finely spaced markers, utilization of newer statis-

tical techniques (such as FBAT) that permit multiple

affected individuals to be included in family-based

association analyses, as well as expansion of the sample

and comparison with genome wide results, should be

undertaken to further prioritize regions for future study.

The results of the present study also suggest that the use

of the mania syndrome to refine the psychosis phenotype

may provide a means of improving the methodology used

to identifying genes that underlie the psychotic spectrum

of psychiatric illnesses.

AcknowledgementsWe are indebted to the patients and family members who

participated in this study. We also thank the hospitals and

clinics of Costa Rica (in particular, the Instituto Nacional

de Psiquiatria in Pavas and the Hospital Calderon Guardia

in San Jose) and their excellent clinical staffs for making

this study possible. We also acknowledge the contribu-

tions of the following clinical and statistical researchers to

the completion of this project: Dr Israel Katz (San

Francisco General Hospital, San Francisco, California),

Drs Sophia Vinogradov and Carol Mathews (University of

California at San Francisco), Dr Jean Jacques Garbarz

(Department of Mental Health, San Francisco, Califor-

nia), Dr Rodolfo Salazar-Fonseca (University of Costa

Rica), Drs Rigoberto Castro and J. Saenz (Instituto

Nacional de Psiquiatria de Costa Rica), Drs Alec Miller

and Charles Bowden (University of Texas Health Science

Center at San Antonio) and Maureen O’Connell (South-

west Foundation for Biomedical Research. We thank the

following for their assistance with laboratory work (DNA

extraction, marker testing and creation of cell cultures)

critical to the success of the study: Reinaldo Pereira

LD analysis of chromosome 8 in Costa Rica Walss-Bass et al. 167

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

(University of Costa Rica), Yazmin Ench and Julie

Wisdom (University of Texas Health Science Center at

San Antonio).

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