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1 Tilburg University Differentiating between low and high susceptibility to schizophrenia in twins van Oel, C.J.; Baaré, W.F.C.; Hulshoff Pol, H.E.; Haag, J.; Balazs, J.; Dingemans, A.; Kahn, R.S.; Sitskoorn, Margriet Published in: Schizophrenia Research Publication date: 2001 Link to publication Citation for published version (APA): van Oel, C. J., Baaré, W. F. C., Hulshoff Pol, H. E., Haag, J., Balazs, J., Dingemans, A.,... Sitskoorn, M. M. (2001). Differentiating between low and high susceptibility to schizophrenia in twins: The significance of dermatoglyphic indices in relation to other determinants of brain development. Schizophrenia Research, 52(3), General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. - Users may download and print one copy of any publication from the public portal for the purpose of private study or research - You may not further distribute the material or use it for any profit-making activity or commercial gain - You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright, please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 16. jan. 2019

2 Schizophrenia Research ) 181±193 Differentiating between low and high susceptibility to schizophrenia in twins: the signi cance of dermatoglyphic indices in relation to other determinants of brain development C.J. van Oel*, W.F.C. BaareÂ, H.E. Hulshoff Pol, J. Haag, J. Balazs, A. Dingemans, R.S. Kahn, M.M. Sitskoorn Department of Psychiatry, University Medical Center Heidelberglaan 100, 3584CX Utrecht, Netherlands Received 9 August 2000; accepted 21 December 2000 Abstract Both the skin and the brain develop from the same ectoderm and it is thought, therefore, that dermatoglyphics are informative for early disturbances in brain development in schizophrenia. This study was aimed at investigating the differences in both digital and palmar dermatoglyphic indices between twins discordant for schizophrenia and control twins. Furthermore, the signi cance of dermatoglyphic indices in relation to other determinants of brain development with regard to the susceptibility to schizophrenia was investigated. Data on dermatoglyphic indices of the hand and the palm were obtained from 21 same-sex discordant and 37 same-sex control twins. For 19 discordant and 25 control twins, there was also data available on brain volumes. Non-genetic intra-uterine circumstances early in pregnancy 10±13 weeks of gestation) are associated with a susceptibility to schizophrenia, since both the twins with schizophrenia and the unaffected co-twins showed more uctuating asymmetry of the nger ridges P, 0.01), and marginally higher absolute nger ridge counts P ˆ 0.06) than control twin pairs. Fluctuating asymmetry of the nger ridges was as important as whole brain and left hippocampal volumes in differentiating twins with a high susceptibility to schizophrenia from those with a low susceptibility. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Schizophrenia; Dermatoglyphics; Susceptibility; Brain development; Placental dysfunction 1. Introduction Structural brain abnormalities such as a distortion of the corpus callosum Foong et al., 2000; Woodruff et al., 1995), septum cavum pellucidum Kirkpatrick et al., 1997; Kwon et al., 1998), and congenital * Corresponding author. Present address: Leiden University Medical Center, Department of Gynaecology/K6-76, Albinusdreef 2, PO Box 9600, 2300 RC Leiden, The Netherlands. Tel: 131 0) ; fax: 131 0) address: c.vanoel@lumc.nl C.J. van Oel). malformations Ismail et al., 1998; Lane et al., 1997) are more common in patients with schizophrenia than in controls. This suggests that schizophrenia is partially due to impaired neurodevelopment, that might be accounted for by both environmental and genetic factors. Neuro-pathological studies indicate that a deviation from normal development might occur before the end of the second gestational trimester. Impaired neuronal migration has been suggested as a cause of certain cytoarchitectonic anomalies described in the medial temporal cortex and hippocampal formation of the brains of some /01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S )

3 182 C.J. van Oel et al. / Schizophrenia Research ) 181±193 schizophrenic patients Jones, 1997). Migration of neurons from the proliferation sites to their destination within the central nervous system peaks in the third to fth months of gestation, although neuronal migration can be detected as early as the second month and slightly after the fth month Volpe, 1995). Furthermore, the lack of gliosis in the brains of schizophrenic patients is generally considered as evidence for an early disturbance in brain development, because gliosis is not observed when neuropathological events occur before the end of the second gestational trimester Bogerts, 1993). Dermatoglyphics have been used to trace the occurrence of a prenatal adverse event during the rst half of the pregnancy. Both the skin and the brain develop from the same ectoderm and it is thought, therefore, that deviations in dermatoglyphics are informative for early disturbances in brain development in schizophrenia. Furthermore, dermatoglyphics are particularly useful as a marker for early developmental brain disturbances, because dermatoglyphics develop during a restricted period of time, i.e. between the 10th and the 16th weeks of gestation w.g.). Dermatoglyphics consist of primary ridges that form patterns such as loops, arches and whorls. Although they are only visible externally from the fth month onwards, the primary ridge formation starts several weeks earlier Holbrook, 1988; Jones et al., 1998). The most commonly reported dermatoglyphic markers are the number of whorl patterns, the total and absolute nger ridge counts TFRC and AFRC, respectively) and the palmar AB ridges. Finger ridge patterns depend on the shape of volar pads. These pads develop between 6.5 and 8 w.g., just after the hand loses its webbed appearance. They appear on each nger tip, on the distal palm between each pair of ngers and on the hypothenar area of the palm. Regression of the volar pads starts between 10 and 12 w.g. The interdigital volar pads arise rst and persist longer than the other volar pads Meier et al., 1987; Samandari, 1973). Early ridge formation is associated with a whorl-type pattern, while late ridge formation seems to be related to arch con guration, and intermediate ridge formation with a loop pattern. Pad elevations have no effect on the number of ridges Babler, 1991). The rst sign of ridge formation is visible in the center of the proximal third of the distal or apical pads as early as 10 w.g. By 11 w.g., more of the apical pads show ridges. Both total and absolute nger ridge counts will re ect papillary ridge formation that occurs between 10.5 and 13 w.g. Babler, 1991; Moore and Munger, 1989).. Total nger ridge count expresses only the size of the pattern, while absolute nger ridge count re ects both size and intensity of epidermal ridge formation and might be more sensitive to non-genetic in uences Schaumann and Alter, 1976). By 15±16 w.g., the entire skin of the hand demonstrates papillary ridges Moore and Munger, 1989). Total AB ridge count would thus mark the time period up to the 16th week of gestation. In addition to ridge counts, two other commonly used dermatoglyphic indices concern the uctuating asymmetry of the nger ridges, and the palmar AB ridges, respectively. These both involve the random deviation from perfect bilateral symmetry, which stems from intra-uterine environmental in uences that in some way perturb the expression of the genetic program Newell-Morris and Wienker, 1991). Currently, little is known about the interrelation of several risk factors or events that lead to the onset of schizophrenia. It may well be that a combination of events is necessary to develop schizophrenia, which means that a susceptibility to schizophrenia is present if at least one risk factor is lacking Khoury et al., 1993). Although studies using dermatoglyphic indices that strongly re ect intra-uterine environmental in uences i.e. AFRC; uctuating asymmetry of the nger ridges and the palmar AB ridges) fairly consistently reported higher levels in schizophrenic patients as compared to normal controls Davis and Bracha, 1996; Markow and Wandler, 1986; Mellor, 1992), it is unclear whether such an intra-uterine event attributes as a minor or major risk factor to the susceptibility to schizophrenia in relation to other presumed genetic and environmental factors on brain development in schizophrenia. In an earlier report on this twin sample Baare et al., 2001), we analyzed differences in intra-cranial, whole brain, frontal, hippocampal, parahippocampal, extracerebral cerebrospinal uid, and lateral and third ventricle volumes between discordant and control twin pairs, and within discordant twin pairs. One of the major ndings was that reduced hippocampal and parahippocampal volumes seem to mark an increased susceptibility to schizophrenia in both MZ and DZ

4 C.J. van Oel et al. / Schizophrenia Research ) 181± twin pairs. A decrement of the medial temporal cortex might indicate impaired neurodevelopment before the end of the second gestational trimester Jones, 1997), but might be also associated with chronic) fetal hypoxia or perinatal asphyxia McNeil, 1987; Tuor et al., 1996). Since the timing of migrational processes partially overlap with the formation of the epidermal ridges, dermatoglyphic markers might be super uous to para)hippocampal volume as a marker of an increased susceptibility to schizophrenia. This study was aimed at the following questions. First, are there any differences in both digital and palmar dermatoglyphic indices between twins discordant for schizophrenia and control twins, and does this apply for mono- as well as dizygotic twins? Second, what is the signi cance of dermatoglyphic indices in relation to other determinants of brain development with regard to the susceptibility to schizophrenia? Twins discordant for schizophrenia are particularly useful to gain insight into the susceptibility to schizophrenia. Since only one twin of a discordant pair developed schizophrenia, this means that the non-affected twin lacks at least one risk factor, but probably has several other risk factors in common with the affected twin Rothman and Greenland, 1998). MZ discordant twins share all their genes, whereas DZ discordant twins share on average 50% of their genes, just like ordinary siblings do. However, only both MZ and DZ twins share much of the pre-and postnatal circumstances to the same degree. Therefore, the study of twins might be more sensitive to both genetic and environmental risk factors in schizophrenia than other types of family studies. 2. Methods 2.1. Subjects The twins took part in a twin and family study between 1994 and Affected twins were recruited by advertisements in national newspapers and in collaboration with psychiatric services. Healthy control twin pairs were recruited by advertisements in similar national newspapers and additionally in regional newspapers. A total of 52 affected twin families responded to the advertisements and received information about the study. Of these families, 32 participated in the study. One twin pair was recruited via a GP. Psychiatric services identi ed 77 twins among their patients. Of these twins, ve twins were already known; two pairs had already participated in the study, the other three pairs participated afterwards. Of the remaining twins, 36 indicated that they were willing to participate. These twins were visited to obtain a summary of their psychiatric history if they were identi ed regardless of their diagnosis. In case of a known diagnosis of schizophrenia or schizoaffective disorder with mainly psychotic symptoms, twins were directly included in the study. Presently, the collaboration with psychiatric services resulted in a total of 27 twin families that were included in the study. In response to advertisements for healthy controls, 105 twins indicated that they were interested and were asked about the presence of psychiatric problems in themselves and their family. This left a total number of 93 twins that could be included in the study if they additionally matched on zygosity, sex, and age with affected twin pairs. Of these 93 twin pairs, six pairs refused to participate at a later stage, because the research protocol took too much time one pair), or because they were anxious of undergoing a MRI-scan one pair), or unknown/other reasons four pairs). One hundred and twelve twins from 60 affected families were included in the study, and underwent extensive psychiatric assessment see Section 2.2). In 51 pairs of twins, at least one twin was diagnosed as suffering from schizophrenia or schizoaffective disorder, schizophrenic subtype. In one DZ discordant twin pair, the affected twin met criteria for bipolar disease and this twin pair was excluded from the further analyses. The affected twin of another discordant DZ twin pair had given his informed consent, but developed lung cancer ICD-10: C34.9) and died. The co-twins of eight twins did not participate in the study: four co-twins refused, one died, and the other three co-twins may participate at a later date. Three control twin pairs were excluded from the study because of alcohol abuse 1 MZ twin pair), major depression 1 MZ twin pair), and schizoid personality disorder 1 DZ twin pair). All participants gave their written informed consent after full explanation of the study aims and procedures.

5 184 C.J. van Oel et al. / Schizophrenia Research ) 181±193 Table 1 Psychiatric diagnoses of the discordant twin pairs n ˆ 21), and age of disease onset years) for twins with schizophrenia 2.2. Diagnostic procedure Patient Diagnosis MZ twins Schizophrenia Disorganized 3 Paranoid 6 Residual 1 Undifferentiated 2 Depressive disorder a 3 Conduct disorder 1 Schizotypal PD 2 Schizoid PD 1 Alcohol abuse 3 Amphetamine abuse 1 Cannabis abuse 2 Diagnosis DZ twins Schizophrenia Disorganized 2 Paranoid 3 Residual 2 Undifferentiated 2 Depressive disorder b 2 Anxiety disorder 1 Paranoid PD 1 Schizoid PD 1 Avoidant PD 1 Alcohol abuse Amphetamine abuse 2 Polysubstance dependent 1 Cannabis abuse 1 Other substance abuse 1 1 Disease onset Mean years) 22.7 sd 5.8 a Co-twin Two single episodes one with psychotic symptoms), one recurrent episode. b Two single episodes. Each subject underwent extensive psychiatric assessment procedures, including the following semi-structured interviews: Comprehensive Assessments of Symptoms and History Andreasen et al., 1992), the Schedule for Affective Disorders and SchizophreniaÐLifetime version Endicott and Spitzer, 1978), the Structured Interview for DSM- IIIR/IV Personality Pfohl, 1989; Pfohl et al., 1995), and the Family Interview for Genetic Studies Nurnberger et al., 1994). In addition to the personal interviews, informant interviews were conducted with someone proposed by the subject himself. If permission was received, informant interviews were conducted for all subjects with psychotic symptoms or with a doubtful diagnosis. Twins of a pair were not interviewed by the same interviewer. Diagnostic interviews were conducted by interviewers with a degree in psychology, who were blind to twin and diagnostic status. They initially received an extensive training, and also participated in additional training sessions. Information from the personal interview, the interview with a signi cant other, and medical records was put together in a case report. Diagnosis was decided upon this narrative by a senior psychiatrist who was blind for twin and group status. The psychiatric diagnosis Table 1) was established according to criteria of the Diagnostic and Statistical Manual, fourth edition DSM-IV). Subjects ful lling criteria for schizoaffective disorder ) also met the Research Diagnostic Criteria RDC) for schizoaffective disorder, schizophrenic subtype. In twins discordant for schizophrenia, the mean length of discordance for schizophrenia was 13.6 sd: 8.99) years at entrance in the study. At the time of participation, four twin pairs 1 DZ and 3 MZ) were less than 4 years discordant for schizophrenia. Since then, however, several years have passed by, and presently only 1 DZ twin pair is less than 4 years discordant for schizophrenia. None of the four unaffected co-twins received a schizophrenia-spectrum diagnose. The probability that any of the discordant pairs would become concordant in the future was presumed to be very low, since most twins will become concordant for schizophrenia within 4 years, and nearly no twins will do so after 8 years of discordance Belmaker et al., 1974). Healthy control twins had no known rst-degree relatives with a diagnosis on Axis I, no history of psychotic disorder in their second-degree relatives, and did not meet criteria for a diagnosis on Axis I or Axis II. All participants gave written informed consent after full explanation of the study aims and procedures. Palm and nger prints were available for 12 MZ ve female; seven male) and 9 same-sex DZ ve female; four male) twins discordant for schizophrenia. The control group consisted of 23 MZ eight female; 15 male) and 13 same-sex DZ seven female; six male) twin pairs. Due to poor quality of either the

6 C.J. van Oel et al. / Schizophrenia Research ) 181± palm three pairs) or the nger prints three pairs), both palm and nger prints were available for 51 pairs of twins. Additional data on brain volumes were available for 44 pairs of these twins 19 discordant, 25 control twin pairs). These twin pairs were also included in an earlier report on structural brain abnormalities in 29 twin pairs discordant for schizophrenia and 29 control twin pairs Baare et al., 2001) Zygosity DNA was extracted from 20 ml blood by standard procedures. Zygosity was determined by DNA ngerprinting using the polymorphic markers, D06S474, D07S1804, D07S1870, D12S811, D13S119, D13S126, D13S788, D20S119, D22S683, DXS1001 and ELN, or D13S317, VWA, D74520, D35158, TH01, TP0X, CSF1P0 and D Dermatoglyhic assessment Rolled, inked nger and palm prints were obtained and scored by two well-experienced professional dactyloscopists. They used a Henry magni er glass, which has a red line that can be placed upon the palm or nger print. The red line is orientated so as to connect the core of the nger print or the palmar triradius with the other palmar) triradius in order to count the intersecting ridges between these two marker points. First pattern type was classi ed, and then the ridges were counted. The raters were blind with respect to the diagnosis and family membership Finger ridge pattern Finger ridge patterns were either classi ed as an arch, tended arch, ulnar loop, radial loop, whorl or combined gure, using the classi cation of Henry Henry, 1937). Total number of whorl patterns was computed for each subject. In addition, pattern asymmetry was assessed by counting pattern matches on their ve pairs of homologous ngers Finger ridges Whorls and combined gures do have two triradii each and thus two ridge counts. For the Total Finger Ridge Count TFRC) only the highest ridge count was used and adds up over the 10 ngers. The Absolute Finger Ridge Count AFRC) was computed by adding up the number of ridges between all the digital triradii and the center of the nger ridge pattern. The highest ridge count of each nger was used to test for directional asymmetry. Fluctuating asymmetry was assessed by totaling the absolute differences between the total ridge counts on the ve pairs of homologous ngers Asy-Digit) AB ridges The epidermal ridges between the index and the middle nger were counted on both sides and add up for the AB total ridge count AB-TRC). Left and right AB ridge counts were compared for directional asymmetry. Fluctuating asymmetry was assessed by computing the absolute difference between the left and right AB ridge count Asy-AB) Brain volumes The acquisition of brain scans and the subsequent segmentation procedures have been extensively described elsewhere Baare et al., 2001). Here we summarize only the most important aspects. MRI images were obtained on a 0.5 Tesla T) Philips Gyroscan scanner for six twin pairs discordant for schizophrenia. The remaining 13 discordant and 25 control twin pairs were scanned on a 1.5 T Philips Gyroscan NT scanner that became available 1.5 years after initiation of the study. To evaluate the effect of the type of scanner used, eight healthy controls were scanned on both the 0.5 and 1.5 T scanner. For the analyses of the volumes of interest VOI), T1-weighted scans 0.5 T: TE ˆ 13 ms; TR ˆ 30 ms; ip angle ˆ 308; 170±180 contiguous coronal slices; mm 3 voxels; 1.5 T: TE ˆ 4.6 ms, TR ˆ 30 ms, ip angle ˆ 308, 170±180 contiguous coronal slices; mm 3 voxels) and dual contrast turbo spin echo DTSE) scans 0.5T : TE ˆ 30 ms; TI ˆ 300 ms; TR ˆ 2720 ms; 36 contiguous slices; mm 3 voxels, frontal and occipital poles not included); 1.5 T: TE1 ˆ 14 ms, TE2 ˆ 80 ms, TR ˆ 6350 ms, 120 contiguous slices; mm 3 voxels) of the whole head were acquired. VOI were automatically intracranial volume, whole brain), semi-automatically lateral and third ventricles) or manually hippocampus, parahippocampal gyrus, total frontal volume) segmented. To correct for scanner type, scaling

7 186 C.J. van Oel et al. / Schizophrenia Research ) 181±193 Table 2 Descriptives for both the twins n, mean, sd) Number of whorls TFRC AFRC Asydigit AB-TRC AsyAB n discordant pairs) Sz-index ) ) ) ) ) ) Sz-co-twin ) ) ) ) ) ) n control pairs) Contr-A ) ) ) ) ) ) Contr-B ) ) ) ) ) ) factors were calculated mean volume 1.5 T /mean volume 0.5 T ) and applied to the 0.5 T VOIs measured in the larger study sample 26 and 96 twins scanned on 0.5 T, respectively 1.5 T) Statistical analyses MANOVA was used to analyze the data on TFRC, AFRC, total ridge count between the palmar AB ridges AB-TRC), and uctuating asymmetry of both the ngers and the AB ridges. First, several variables were considered as possible confounds. Although this involves multiple testing, a P-value of P # 0.05 was chosen as the cut-off value for statistical signi cance of possible confounds, not to erroneously overlook a potential confound. The number of epidermal ridges is often higher on the right than on the left side Schaumann and Alter, 1976), and this so-called `directional asymmetry' might affect the occurrence of uctuating asymmetry. Therefore, we tested whether directional asymmetry in discordant twins differed from that in control twins. Furthermore, we tested for differences in dermatoglyphics between MZ and DZ twins, respectively males and females, to decide upon the confounds that should be controlled for in the further analyses. Diagnostic group discordant vs control twin pairs) was used as a between factor; twin pairs twin with schizophrenia or rstborn control, and unaffected co-twin or second-born control) were analyzed as matched samples. To test for directional asymmetry of the ngers, two additional within factors were speci ed: nger ve levels) and side left, right). Only the latter within factor was used to test for directional asymmetry of the AB ridge. The presence of signi cant differences between discordant and control twin pairs were univariately analyzed. Since this involves multiple testing, a P # 0.01 was chosen as the cut-off value for statistical signi cance, with P reported as nonsigni cant trends. For each of the ve homologous nger pairs, asymmetry of the nger ridge pattern was nonparametrically tested using the Wilcoxon Signed ranks test for matched pairs. Intraclass correlations for TRFC, AFRC, AB-TRC, and uctuating asymmetry of both the ngers and the AB ridges were computed on the standardized residuals controlling for sex). Intraclass correlations re ect the similarity between twin pairs and were computed separately for MZ and DZ twins. Also, both for MZ and DZ twins contingency phi) coef cients were computed to assess the similarity in the number of whorls between the twin pairs. The Fisher r to z transformation Winkler and Hays, 1975) was used to compare correlations to each other. To deal with the problem of multiple testing, P # 0.01 was chosen as the cut-off value for statistical signi cance, with P reported as nonsigni cant trends. To test the relevance of dermatoglyphic indices as a component cause of the susceptibility of schizophrenia in relation to other determinants of brain development in schizophrenia, a discriminant analysis was performed Stevens, 1996). Using P # 0.05, model construction occurred using forward selection of the digital dermatoglyphic indices, sex, and several brain regions of interest: intracranial volume, whole brain volume, total frontal volume, lateral ventricles, third ventricle, total hippocampus, left hippocampus, right hippocampus, and total parahippocampal gyrus. All brain volumes were corrected for sex, and age. Furthermore, intracranial volume was used as a covariant for whole brain, and lateral and third ventricle

8 C.J. van Oel et al. / Schizophrenia Research ) 181± Table 3 Intraclass correlations intraclass correlation on standardized residuals after correction for sex) Number of whorls a TFRC AFRC Asydigit AB-TRC AsyAB MZ Control twins n ˆ 23) b 0.93 b e,d 0.22 d Discordant twins n ˆ 11) 0.90 n ˆ 12) 0.83 e 0.92 f DZ Control twins n ˆ 13) 0.89 n ˆ 13) 0.49 b,g 0.60 b,g 0.00 g 0.18 c 0.00 Discordant twins n ˆ 8) 0.88 n ˆ 9) 0.02 c 0.03 f a Contingency coef cient non-parametric). b MZ vs DZ control twins: P, ˆ c MZ vs DZ discordant twins: P, ˆ d n ˆ 22. e MZ vs DZ discordant twins: P, ˆ f n ˆ 12. g MZ vs DZ control twins: P, ˆ volumes, whereas whole brain volume was a covariant for the other brain volumes. To account for the lack of independence between both twins of a pair, the discriminant analysis was conducted on the average values of the twin pairs. Statistical analyses were performed with SPPS V8.0 for Windows. All tests were two-tailed. 3. Results 3.1. Characteristics On average, discordant twin pairs were 36.3 sd: 11.1) years old; this was not signi cantly different from the mean age 37.5; sd: 14.0) of control twin pairs. The twins suffering from schizophrenia attained a signi cantly F 1,55 ˆ 11.65, P, 0.05) lower educational level mean years of education: 11.2 sd: 2.4 years) than both the non-schizophrenic co-twins mean years of education: 13.3; sd: 2.5 years) and the control twins mean years of education: 13.1; sd: 2.1 years). In twins suffering from schizophrenia, the mean age of disease onset was 22.7 years sd: 5.8 years). Table 2 summarizes the descriptive information on the six dermatoglyphic markers for the discordant and for the control twin pairs Directional asymmetry Discordant twins did not signi cantly differ from control twins in the frequency of nger ridge patterns that did not match with the pattern on the homologous ngers. However, we did nd a signi cant side effect for nger ridge count, F 1,51) ˆ 9.96; P ˆ The number of epidermal ridges on the right hand mean: 66.94; sd: 23.11) was larger than on the left hand 63.38; sd: 22.47). There were no differences between the diagnostic groups, neither were there any signi cant interactions with the group factor. Similarly, we only obtained a signi cant side effect for the AB ridges, F 1,51) ˆ P ˆ There were more epidermal ridges on the left palm than on the right palm mean left: 40.00; sd: 5.00; mean right: 39.38; sd: 5.49). Since directional asymmetry in discordant twins did not signi cantly differ from that in control twins, we did not correct for directional asymmetry in the further analyses Differences between MZ and DZ twins, and sex We did not nd any signi cant differences in dermatoglyphic indices between MZ and DZ twins, nor did we nd any signi cant interaction of zygosity with diagnostic group. Therefore, we did not control for zygosity in the further analyses. Sex was considered as a covariate in the further MANOVAs, because TFRC and AFRC marginally) signi cantly differed between males and females TFRC: F 1,51 ˆ 4.31; P ˆ 0.01; AFRC: F 1,51 ˆ 3.25; P ˆ 0.08). Men had a higher TFRC mean: 141; sd: 43) and AFRC mean: 174; sd: 70) than women TFRC: mean: 120, sd: 40; AFRC: mean: 145, sd:

9 188 Table 4 Absolute volumes values are mean sd) in cm 3 ) NC1 ˆ healthy control twin, rst born; NC2 ˆ healthy control twin, second born; left L) and right R) volumes of interest; ICV, intracranial; WB, whole brain; FL, frontal lobe; HC, hippocampus; PH, parahippocampal gyrus; LV, lateral ventricle; 3V, third ventricle) Structure Monozygotic Dizygotic Discordant twin pairs n ˆ 11) Healthy twin pairs n ˆ 14) Discordant twin pairs n ˆ 8) Healthy twin pairs n ˆ 11) Patient Cotwin NC1 NC2 Patient Cotwin NC1 NC2 ICV ) ) ) ) ) ) ) ) WB L ) ) ) ) ) ) ) ) R ) ) ) ) ) ) ) ) FL L ) ) ) ) ) ) ) ) R ) ) ) ) ) ) ) ) HC L ) ) ) ) ) ) ) ) R ) ) ) ) ) ) ) ) PH L ) ) ) ) ) ) ) ) R ) ) ) ) ) ) ) ) LV L ) ) ) ) ) ) ) ) R ) ) ) ) ) ) ) ) 3V ) ) ) ) ) ) ) ) C.J. van Oel et al. / Schizophrenia Research ) 181±193

10 C.J. van Oel et al. / Schizophrenia Research ) 181± Table 5 Signi cance level of factors included in the model; group centroids and pooled within-groups correlation between discriminating variables and the discriminant function n ˆ 44) Step order 60). No signi cant effect of sex was found for asymmetry of both the nger ridge counts and the palmar AB ridges Mean differences between discordant and control twin pairs Discordant twins signi cantly differed from control twins on only one of the ve dermatoglyphic indices. A signi cant main effect for diagnostic group was found, with increased uctuating asymmetry of the nger ridges in discordant twin pairs as compared to control twin pairs F 1,51 ˆ 8.90; P, 0.01; Table 2). There was a trend towards signi cant differences between discordant and control twin pairs in AFRC F 1,51 ˆ 3.64; P ˆ 0.06). Discordant twin pairs had more epidermal ridges than had control twins Table 2). The difference in TFRC was not signi cant F 1,51 ˆ 2.78; P ˆ 0.10). No signi cant differences were found for AB-TRC and uctuating asymmetry of the AB ridges AB-TRC: F 1,51 ˆ 1.25; P ˆ 0.27; Asy_AB: F 1,51 ˆ 0.26; P ˆ 0.61) Intrapair differences P-value Group centroids Correlation Schizophrenic twins Control twins 0.85 Whole brain Fluctuating Asymmetry Left hippocampus Third ventricle The intraclass correlations are summarized in Table 3. No signi cant intrapair differences were found Table 6 Classi cation table Classi ed n Correct Wrong Discordant twin pairs %) %) control twin pairs %) %) Correctly classi ed: 79.6% between affected and control twins. The total and absolute nger ridge counts of both MZ discordant and control twin pairs resembled each other nearly) signi cant more than those of respectively DZ discordant TFRC: z ˆ 2.38, P, 0.05; AFRC: z ˆ 3.22, P, 0.01) and control twin pairs TFRC: z ˆ 3.41, P, 0.01; AFRC: z ˆ 3.21, P, 0.01). This was also true for the palmar ridge count AB_TRC) in control twins z ˆ 2.20, P, 0.05) A model differentiating affected twin pairs from control twin pairs Table 4 presents absolute volumetric data for the current subset of twin pairs. Use of the discriminant analysis could determine if a composite of the variables strengthened the differentiation between the two groups of twins while eliminating the potential redundancy of the variables. Table 5 summarizes the results of the discriminant analysis. The discriminant analysis yielded a model that consists of four variables that correctly classi ed 79.55% of the cases into the appropriate group Table 6). These variables were whole brain volume wb), the uctuating asymmetry of the nger ridges Asydig), and the volumes of the left hippocampus hv-l) and the third ventricle 3v). Together, these four variables signi cantly discriminated between discordant and control twins group means: discordant Table 7 Pooled within-group correlations between uctuating asymmetry of the nger ridges and absolute brain volumes left L) and right R) volumes of interest; ICV, intracranial; WB, whole brain; FL, frontal lobe; HC, hippocampus; PH, parahippocampal gyrus; LV, lateral ventricle; 3V, third ventricle) Brain structure Side Fluctuating asymmetry of nger ridges ICV WB L 0.09 R 0.10 FL L R HC L 0.32 R 0.11 PH L 0.11 R 0.07 LV L R V 20.15

11 190 C.J. van Oel et al. / Schizophrenia Research ) 181±193 twins: 21.12; control twins, 0.85; X 2 4): , P, 0.001). In addition to the uctuating asymmetry of the epidermal nger ridges, whole brain and left hippocampus, but not the third ventricle, were equally strongly correlated with the discriminant function Table 5). The pooled within-group correlations Table 7) of the uctuating asymmetry of the nger ridges with the left hippocampus, the third ventricle and whole brain were 0.32, and 0.10, respectively. 4. Discussion This twin study compared digital and palmar dermatoglyphic indices between 21 pairs of twins discordant for schizophrenia and 36 pairs of control twins, and whether these differences applied for MZ as well as DZ twins. Furthermore, we investigated the signi cance of dermatoglyphic indices in relation to other determinants of brain development, i.e. brain volumes, with regard to the susceptibility to schizophrenia. Males had signi cantly higher total nger ridge counts TFRC), and marginally signi cantly higher absolute nger ridge counts AFRC) than females, and sex was therefore used as a covariate in the further analyses. We did not nd an in uence of zygosity on any of the dermatoglyphic indices. In addition, no differences were found between the twins with schizophrenia and their unaffected co-twins. Interestingly, only the two digital markers that most strongly mark the occurrence of intra-uterine environmental in uences uctuating asymmetry of the nger ridges and the AFRC) were nearly) signi cantly higher in the discordant twins than in control twins. None of the dermatoglyphic markers of the hand palms were signi cantly different between the two groups. Finally, the uctuating asymmetry of the epidermal nger ridges, along with whole brain, left hippocampus and third ventricle signi cantly discriminated discordant twins from control twins. The model correctly classi ed 80% of the twins into the appropriate group. Affected families were characterized by high levels of uctuating asymmetry, larger volumes of the third ventricle, and smaller whole brain and left hippocampal volumes. Our results suggest that an adverse intra-uterine event happened towards the end of the rst gestational trimester, between the 10th to 13th week of gestation. Like others Davis and Bracha, 1996), we did not nd differences in the frequencies of whorl-type patterns of epidermal ridges on the ngers, and therefore an adverse event is unlikely to have occurred prior to the 10th week of gestation. The upper limit can be inferred from our ndings of increased uctuating asymmetry of the nger ridges in discordant compared to control twins, combined with the absence of differences in hand palm indices. After the 13th week of gestation, the epidermal ridges only develop in the hand palms Babler, 1991; Moore and Munger, 1989). In contrast to our results, Davis and Bracha 1996) found no evidence of developmental differences before the 13th week of gestation. They compared discordant and concordant MZ twins to MZ control groups from literature, and found that only the palmar dermatoglyphic indices of the discordant twins showed deviations from normal development. They therefore concluded that an adverse intrauterine event occurred between the 13th and the 15th week of gestation. These different ndings might be partially explained by a different sample selection. By design, the twins in the study of Davis and Bracha did not have a known history of prenatal insults Bracha et al., 1992), whereas we included twins regardless of their pregnancy and/or birth history. Unlike our ndings, Davis and Bracha 1996) found signi cantly larger intra-pair differences in discordant twins as compared to control twins. This might possibly be related to a difference in placentation. In twins, increased intra-pair differences in uctuating asymmetry of the AB-ridges are associated with monochorionic placentation or fused placentas Bogle et al., 1994), since close implantation sites results in greater intra-uterine competition between co-twins than the implantation sites leading to completely separate placentas Buzzard et al., 1983; Corey et al., 1979). In contrast to the MZ twin sample of Davis and Bracha, our twin sample consists of both MZ and DZ twins, and since DZ twins always have two placentas, this might partially explain the absence of intra-pair differences in our study. From our data, one might conclude that intra-uterine circumstances early in pregnancy are associated with a susceptibility to schizophrenia, since both the twins with schizophrenia and the unaffected co-twins showed more uctuating asymmetry of the nger

12 C.J. van Oel et al. / Schizophrenia Research ) 181± ridges, and marginally higher AFRC than control twin pairs. This increased susceptibility is invoked by a non-genetic event, since the intra-class correlations for uctuating asymmetry of the nger ridges were low, and none of these intra-class correlations were signi cantly different from zero, which typically indicates a lack of genetic in uence. The AFRC is also thought to re ect non-genetic in uences, because it combines both size and intensity of ridge formation Schaumann and Alter, 1976). Increased uctuating asymmetry of the nger ridges in schizophrenic patients, as compared to controls has also been reported by others Markow and Gottesman, 1989; Mellor, 1992), although not always Davis and Bracha, 1996). The results of the stepwise discriminant analysis indicated that the uctuating asymmetry of the nger ridges signi cantly contributed to a composite index that could be useful in differentiating twins with a high susceptibility to schizophrenia from those with a low susceptibility. Fluctuating asymmetry seemed to be as important as whole brain and left hippocampus volumes in differentiating between a high and low susceptibility to schizophrenia, since these indices were equally strongly associated with the discriminant function. The uctuating asymmetry of the nger ridges was most strongly correlated with left hippocampal volume. Interestingly, the development of the epidermal nger ridges partially coincides with migration and areal differentiaton of the hippocampus Bayer et al., 1993; Kostovic et al., 1989; Kostovic et al., 1993; Moore and Munger, 1989). Moreover, during this period of co-development, by the 11th week, the corpus luteum degenerates and the placenta assumes its role Larsen, 1997). This may be considered as an important intra-uterine event in the etiology of schizophrenia, since placental insuf ciency has been mentioned as one of the possible etiological mechanisms in schizophrenia Hultmanetal.,1999).Inananimalmodel which induced reduced placental blood ow during the second half of gestation, Mallard et al. 1999) particularly found reduced hippocampal volume and enlarged lateral ventricles in growth-restricted animals. These structural alterations are similar to those seen in schizophrenia Elkis et al., 1995; Nelson et al., 1998; Van Horn and McManus, 1992). However, placental insuf ciency might not fully explain reductions in hippocampal volumes as found in schizophrenia. The hippocampus is also particularly sensitive for perinatal hypoxia Tuor et al., 1996). Indeed, reduced left and right hippocampal volumes were found to be associated with increased rates of labor and delivery complications, particularly prolonged labor, in MZ twins with schizophrenia as compared to their unaffected co-twins McNeil et al., 2000). The main limitation of the present study is that we only used data of discordant mono- and dizygotic twins. It has been suggested that schizophrenia in discordant pairs result from a stronger in uence of environmental factors, as compared to concordant pairs, who might have a higher genetic liability to schizophrenia Markow and Gottesman, 1989). In view of the higher concordance rate in MZ than DZ twins, this resulted in a more selected sample of MZ than DZ affected twins. Since we were interested in the usefulness of dermatoglyphic markers to trace environmental in uences that might attribute to an increased risk of schizophrenia, the model that differentiates schizophrenic families from control families might be less valid in a group with a high genetic liability for schizophrenia. One should be therefore cautious in generalizing the current ndings. Indeed, Fananas et al. 1990) found that the lowest total nger and a-b ridge counts were found in schizophrenic patients with a negative family history for schizophrenia. Balgir et al. 1993) reported comparable results. However, Fananas et al. 1996) could not replicate the earlier nding in a later, but smaller sample. This suggests that the relationship between a higher liability for schizophrenia and increased epidermal ridges might be a rather weak one. In summary, non-genetic intra-uterine events early in pregnancy w.g.) are associated with an increased susceptibility to schizophrenia, since both the twins with schizophrenia and the unaffected cotwins showed more uctuating asymmetry of the nger ridges, and marginally higher AFRC than control twins. Fluctuating asymmetry of the nger ridges, whole brain and left hippocampal volumes equally contributed to a composite index that could be useful in differentiating twins with a high susceptibility to schizophrenia from those with a low susceptibility. Fluctuating asymmetry of the nger ridges was most strongly correlated with left hippocampal volume. The development of the epidermal nger ridges partially coincides with migration and areal differentiation of the hippocampus, and fully overlaps

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