Abstracts S17 Niuvanniemi Hospital, FIN-70240 Kuopio, Finland; genes as in Angelman’s and Prader-Willi syndromes. 2Department of Psychiatry, Turku University Central Hos- Skuse (1996) proposed that imprinting of a gene on the pital, Turku, Finland; 3Department of Public Health and maternally derived X chromosome, active only on a General Practice, University of Kuopio, Kuopio, Finland; paternal X, is responsible for the behavioral deficits seen 4Research Institute of Public Health, University of Kuopio, in some Turner’s syndrome patients and differences Kuopio, Finland; 5Department of Pharmacology, Univer- between normal males and females. sity of Turku, Turku, Finland; 6Department of Clinical It is proposed that imprinting of a relevant DNA Physiology, Kuopio University Hospital, Kuopio, Finland. sequence, not a change in the actual sequence, can explain Objective: The dopaminergic system in the human the pattern of increased risk for schizophrenia to first- brain is thought to play a major role in the development degree relatives of patients with schizophrenia, and the of alcohol consumption habits and alcoholism. The DRD2 sharp decline in risk to second degree relatives, as well gene Taq1 ‘A’ polymorphism has been shown to affect D2 as the sex differences in inheritance. It is plausible that receptor availability in post mortem and in vivo measure- an abnormally imprinted X/Y homologous gene can cause ments. Several studies have suggested an association untoward cerebral development and periodic perceptive between Taq1-A1 allele and alcoholism, but this relation- deficits in schizophrenia. Evidence from previous litera- ship has remained controversial. The recent report ture and our own cohort of families with schizophrenia showed that homozygous D −/− knock-out mice lacking 2 are being used to test this hypothesis. D2 receptors consumed about 50% to 60% less ethanol + + than wildtype D2 / mice, and heterozygous mice had an intermediate level of alcohol consumption. Gottesman II, Shields J (1982) Schizophrenia: The Epigenetic Puzzle, Methods: The study population consisted of a random Cambridge University Press, Cambridge. sample of 984 (non-alcoholic) Finnish caucasian males. Skuse D (1998) Imprinting, The X-chromosome and the male brain. Among them 100 subjects reported no alcohol use during The Genomic Imprinting Symposium, October 8–10, 1998, Dur- the last 12 months and were defined as abstainers. The ham, NC. Web page:www. geneimprint.com/ symposium/ presen- self-reported alcohol consumption in pure ethanol tations.hfml (grams/week) was compared between subjects with A1A1 genotype and subjects with A1A2 or A2A2 genotypes. The genotypic distribution of the study population was in Hardy-Weinberg equilibrium. We also adjusted the results EPIGENETIC DIFFERENCES IN GENETICALLY IDENT- for thirteen sociodemographic covariates. ICAL ORGANISMS. Petronis A, Popendikyte V, Paterson Results: There was no significant difference in the overall AD, Gottesman II, Nobrega J, Macciardi F, Kennedy JL.. genotype distribution between the 100 abstainers and the 884 non-abstainers. Among the 884 non-abstainers, the Centre for Addiction and Mental Health, and University of Toronto, 250 College St., Toronto ON M5T 1R8, Can- alcohol consumption of the homozygous A1A1 group was Ȱ about 30% lower than in A1A2 group, and 40% lower ada. E-mail: petronisa cs.clarke-inst.on.ca. than in A2A2 group (p = 0.042, p = 0.041 in an adjusted Phenotypic discordance of monozygotic twins (MZ) multivariate model). Based on the evidence gathered in is one of the most unclear issues in genetics of complex our previous studies we adjusted the results also for traits. Several molecular mechanisms, such as somatic COMT genotype, but it did not attenuate the difference (p mutations, asymmetric inheritance of mitochondria, and = 0.037). skewed X inactivation in female twins, have been sug- Conclusions: Analogously to the findings on DRD2 knock- gested but these are not common and therefore of limited out mice, our homozygous A1A1 men consumed 40% less explanatory value. New opportunities for investigation of ethanol than subjects with A2A2 genotype, and hetero- the molecular substrate of differences in MZ twins arise zygous subjects had an intermediate level of ethanol con- if emphasis is shifted from DNA sequence-based factors sumption. One interpretation of our findings could be that to epigenetic DNA modification. By definition, epigenetics individuals having the A1A1 genotype may not be very refers to modifications in gene expression that are brought sensitive to the dopaminergic effects of ethanol because of about by heritable, but potentially reversible, changes in the low density of D2 receptors responsible for dopamine- DNA methylation and/or chromatin conformation. We mediated feelings of euphoria and reward. investigated DNA methylation patterns in the putative promoter region of human dopamine D2 receptor gene (DRD2) in lymphocyte DNA from six pairs of monozygotic VII. Epigenetics twins concordant and discordant for SCZ. In addition, we investigated methylation in drd2 promoter region of stria- tum tissues from 12 inbred Sprague-Dawley rats. DNA ABERRANT IMPRINTING AS AN EPIGENETIC MECH- samples were subjected to bisulphite modification-based ANISM FOR PSYCHOSIS. Lynn E DeLisi. Department of mapping of methylated cytosines. In the tested region, the Psychiatry, State University of New York, Stony Brook positions of methylated cytosines exhibited three types of 1194. variation i) within an individual (somatic mosaicism of Gottesman (1982) called schizophrenia an “Epigen- epigenetic signals); ii) within the sets of MZ twins and etic Puzzle”. However, at that time he was unaware of the among inbred animals; iii) across individuals belonging to kinds of molecular mechanisms that could be responsible. different sets of MZ twins. To our knowledge, this is the One such is gene imprinting. It is now known that first systematic search for epigenetic variation in psychi- imprinting (inactivation of gene expression by atric research. Epigenetic studies may lead to a better methylation) influences brain growth and development, understanding of molecular mechanisms of phenotypic the development of some diseases, and the suppression of differences in genetically identical (or very similar) transcription of inappropriate DNA sequences. It also can organisms. pathologically effect the expression of some mutated Abstracts S18 SCHIZOPHRENIA AS AN EPIGENETIC PUZZLE – IN X-CHROMOSOME IMPRINTING AND ITS POTENTIAL WHAT SENSE WERE GOTTESMAN & SHIELDS COR- INFLUENCE UPON SEXUAL DIMORPHISM IN NEUROD- RECT?. Crow TJ. POWIC, University Department of Psy- EVELOPMENTAL DISORDERS: NEW EVIDENCE FROM chiatry, Warneford Hospital, Oxford OX3 7JX. STRUCTURAL BRAIN IMAGING. Skuse DH1, Price CJ2, Family, twin and adoption studies each contribute to Jacobs PA3, Bishop DVM4. 1Behavioural Sciences Unit, the conclusion that there is a genetic contribution to the Institute of Child Health, 30 Guilford Street, London etiology of schizophrenia but the nature of the contri- WC1N 1EH 2UK Wellcome Department of Cognitive Neur- bution is obscure. Extent of discordance in MZ twins, ology, Institute of Neurology, Queen Square, London rapid decrease in risk to relatives beyond those of first WC1N 3BG, UK 3Wessex Regional Genetics Laboratory, degree, unequal risk to parents and children relative to Salisbury District Hospital, Salisbury, Wiltshire, SP2 8BJ siblings, and persistence of the condition at high preva- 4Department of Experimental Psychology, University of lence in spite of a substantial fecundity disadvantage all Oxford, South Parks Road, Oxford OX1 3UD. suggest that there are problems in conceiving the genetic Many neurodevelopmental disorders, such as schizo- contribution as a classical Mendelian trait. Gottesman & phrenia, are sexually dimorphic in their clinical course Shields (1982) referred to the problem as an “Epigenetic and cognitive phenotype. Social communication impair- Puzzle”, and offered as a solution that the genes were mul- ments are substantially more common among males than tiple and quantitative in action. females. Male vulnerability has conventionally been attri- Against this solution it can be argued that the facts of buted to neuroendocrine mechanisms. An alternative schizophrenia (the consistency of the brain changes, the explanation will be proposed. Recent evidence for similarity of symptom patterns across populations) sug- imprinting of the X-chromosome comes from observations gest a degree of homogeneity to the disease process that of Turner syndrome, a sporadic disorder of human sits uneasily with the polygenic-threshold theory. How- females in which all or part of one X-chromosome is ever any protagonist of a single gene or even an oligogene deleted. We have postulated the existence of an imprinted locus that is expressed only from the paternally inherited theory now has to face the apparent failure of a large body X and which is associated with superior social communi- of linkage investigations to identify any genes of major cation skills. Such a locus would not be expressed in effect. males, whose single X is always maternal in origin. Its An alternative solution to the problem is to assume effect could be to protect normal 46,XX females from that Gottesman & Shields were exactly correct in their social communication deficits, in the presence of other assumption that schizophrenia is “epigenetic” in the genetic and environmental risk factors. sense in which the term is now used to describe modifi- Evidence that X-linked loci influence specific areas of cations (eg by methylation) of gene expression, as con- brain development will be presented (Price et al, 1999). trasted with differences which relate to variations in the We found X-monosomy as such is consistently associated DNA sequence itself. This solution can be related to an with grey-matter reductions in cognitive brain systems alternative conception of the genetics of schizophrenia – that are mainly associated with social cognition. Turner that the variation relates to the capacity for language and females lacking a paternal X-chromosome (45,Xm) have to the mechanism by which Homo sapiens separated from reduced grey matter density in an area contiguous with a precursor hominid species (Crow, 1996, 1997). It is pro- one such system, suggesting imprinting of the X may have posed that the process of speciation occurred through evolved to influence cortical development in areas already changes within a region of X-Y homology and that these controlled by non-imprinted X-linked loci that are changes were subject to sexual selection. important for social communication. In summary, epigen- Such genes are subject to epigenetic modification (X etic mechanisms may influence the expression of X-linked inactivation) that differs between mammalian species genes involved in the development of distinct brain sys- (Jegalian & Page. 1998) and such inactivation or tems relevant to social cognition. imprinting is relevant to species-related characteristics (Vrana et al, 1998). The sequence of changes in the course of primate TWO INDEPENDENT IMPRINTED EVENTS REGULATE evolution in regions of homology on the Y to regions of FISSION YEAST MATING-TYPE SWITCHING AND the X chromosome may therefore be the basis for the SILENCING. Amar J.S. Klar. Developmental Genetics Sec- mechanism of speciation, and epigenetic modification of tion, Gene Regulation and Chromosome Biology Labora- such sequences is relevant to the intra-specific variation tory, NCI-Frederick Cancer Research and Development that is apparently associated with it. Center, PO Box B, Bldg. 539, Frederick, MD 21702–1201 USA. Several human diseases occur due to defects in gen- Crow TJ. Language and psychosis: common evolutionary origins. omic imprinting, a phenomenon whereby either the Endeavour 1996; 20: 105–109. maternally or the paternally inherited allele of a develop- Crow TJ. Is schizophrenia the price that Homo sapiens pays for langu- mentally important gene is unexpressed (silenced) via a age? Schizophrenia Research 1997b; 28: 127–141. somatically heritable, nonmutational alteration of the Gottesman II & Shields J. Schizophrenia: The Epigenetic Puzzle. CUP, chromosome. We have exploited the fission yeast, Schizo- Cambridge, 1982. saccharomyces pombe, as a model system to understand Jegalian K. Page DC. A proposed mechanism by which genes common the mechanism of such a violation of Mendel’s laws of to mammalian X and Y chromosomes evolve to become X inacti- vated. Nature 1998; 394: 776–780. inheritance. This yeast exists in two interchangeable cell Lambson B, Affara NA et al. Evolution of DNA sequence homologies types called P (plus) and M (minus). These types are between the sex chromosomes in primate species. Genomics 1992; determined by the mat1-P and the mat1-M alleles at the 14: 1032–1040. mat1 locus. The switching occurs due to transposition of Vrana PB. Guan X-J, Tilghman SM. Genomic imprinting is disrupted a copy of one of the silent donor loci, mat2-P or mat3-M, in interspecific hybrids. Nature Genetics 1998; 20: 362–365. to the transcriptionally active mat1 locus. The single cell Abstracts S19 pedigrees have shown that sister cells are nearly always in their skin fibroblasts are not mentally impaired. FMR2 different in their potential to switch such that only one- is poorly expressed in leucocytes and not expressed in in-four grandchildren of a cell ever switches in ෂ85% of lymphoblastoid cell lines. cell lineages. Our genetic and molecular evidence has sug- gested that this pattern is due to site- and DNA strand- specific imprinting such that sister chromatids behave dif- VIII. Affective Disorders ferently1–3. The second imprinting event controls silenc- ing of donor loci, apparently by organizing a repressive chromatin structure which prohibits accessibility of these NEW ANALYSES OF THE NIMH BIPOLAR DATASET loci to transcriptional factors. Remarkably, such an epi- 1Nurnberger JI,. Foroud T, Flury L, Meyer ET, Edenberg genetic event is inherited as a chromosomal marker both H, 2DePaulo JR, Stine OC, 3Blehar MC, Detera-Wadleigh S, 4 in mitosis and meiosis . We have identified several trans- 4Gershon ES, 5Reich T, Goate A., NIMH Genetics Initiative acting factors, including histone deacetylases and chrom- 1Indiana University School of Medicine, 2Johns Hopkins odomain-containing proteins, which are essential for University, 3National Institute of Mental Health, 4Univer- 5–6 silencing . One wonders whether such mechanisms of sity of Chicago, 5Washington University of St. Louis. gene regulation by imprinting may specify brain laterality Bipolar Affective Disorder (BP) clearly aggregates such that the two hemispheres perform different cogni- within families. The genetics of BP is complex, however, tive functions. and not consistent with single major locus inheritance; no specific genes have yet been located and confirmed. A col- 1 Klar, A.J.S. 1987. Nature 326, 466–470. laborative study involving four sites was supported as part 2 Klar, A.J.S. 1990. EMBO 9, 1407–1415. of the NIMH Genetics Initiative. Families included were 3 Dalgaard, J.Z. and A.J.S. Klar. 1999. Nature, in press. 4 Grewal, S.I.S. and A.J.S. Klar. 1996. Cell 86; 95–101. required to have at least two affected subjects with bipolar 5 Ivanova, A.V. M.J. Bonaduce, S.V. Ivanov, and A.J.S. Klar. 1998. Nat. I (BPI) disorder or one with BPI and a second with schi- Genet. 19, 192–195. zoaffective disorder, bipolar type (SA/BP). Probands and 6 Grewal, S.I.S., M.J. Bonaduce, and A.J.S. Klar. 1998. Genetics 150, relatives were interviewed and provided a blood sample 563–576. for transformation and storage at a national data bank. We present results from 540 subjects selected from 97 famil- ies. This group included 282 affected sibling pairs, (BP & FRAGILE SITES, DNA METHYLATION AND MENTAL UP), as well as 412 affected relative pairs. A survey was RETARDATION. Sutherland GR, Gecz J, Gedeon AK, Mul- completed with 319 markers. Analysis was carried out ley JC, Richards RI, Yu S. Department of Cytogenetics and using SIBPAL and Genehunter Plus. A number of candi- Molecular Genetics, Women’s and Children’s Hospital, date areas are supported especially areas on chromosomes Adelaide SA 5006. 6, 10 and 16. A summary of results of linkage analysis on Two types of X-linked mental retardation are associa- individual families from this dataset will be presented. ted with fragile sites on the end of the long arm of the X chromosome: fragile X syndrome with FRAXA and fragile XE mental retardation with FRAXE. Each of these fragile SYSTEMATIC SEARCH FOR SUSCEPTIBILITY GENES IN sites is due to expansion of a CCG repeat in the 5Ј-untrans- BIPOLAR AFFECTIVE DISORDER – EVIDENCE FOR A lated region of the FMR1 and FMR2 genes respectively. DISEASE LOCUS AT 10q26. Cichon S(1), Schmidt-Wolf The coding regions of these genes are intact but once the G(1,2), Pu¨ tzstu¨ ck M(1), Hemmer S (1), Albus M(3), Borrm- number of CCG repeats exceeds a certain threshold the ann, M(3), Lerer B(4), Franzek E(5), Lichtermann D (6), region becomes CpG methylated and transcription ceases. Weigelt B(7), Windemuth C(2), Wienker T(2), Maier W(6), Others have shown that demethylation of the FRAXA Rietschel M(6), Propping P(1), No¨then MM (1). (1) Inst. locus with 5-azadeoxycytidine can restore FMR1 gene Human Genetics, Univ. Bonn, Germany; (2) Inst. Medical transcription and protein production. The factors which Statistics, Univ. Bonn; (3) Mental State Hospital Haar; (4) influence the initiation and maintenance of methylation Hadassah Univ. Hospital, Jerusalem, Israel; Depts. of Psy- are unclear. Methylation is lost in the ovary prior to oog- chiatry, (5) Univ. Wu¨ rzburg, (6) Univ. Bonn, and (7) enesis and re-established early in embryogenesis on Univ. Dresden. alleles with more than approximately 230 CCG repeats in A potential susceptibility locus for bipolar affective the case of FRAXA (males only produce permutation car- disorder on chromosome 10q has recently been suggested. rying sperm and these premutation sized alleles are not As part of a systematic genome scan for bipolar suscepti- methylated). For FRAXE the repeat number at which bility genes, we genotyped 19 evenly spaced microsatel- methylation occurs is more variable and is apparently lite markers covering whole chromosome 10 in a sample between 230 and 280 copies. In exceptional situations in of 75 families (66 families from Germany, one family from all or some of the cells in which the allele size exceeds Italy, 8 families from Israel) comprising 429 individuals. the critical limit, methylation fails to be established in Parametric two-point linkage analysis was performed embryogenesis. In males with FRAXA this greatly ameli- employing a dominant and a recessive genetic model. orates the effect of the mutation and these individuals Two models of affection were used: affection status model usually function intellectually within or close to the nor- (ASM)-I included only individuals with bipolar I (BPI) mal range. Others have claimed that expansion of the phenotype as affected, all other psychiatric diagnoses FRAXA repeat beyond 200 copies inhibits translation were coded as “unknown”; ASM II included all individ- even though in the absence of methylation transcription uals with a diagnosis of BPI, bipolar II (BPII), schizoaffec- occurs. This effect may be cell type specific as FRAXA tive disorder, bipolar type (SA/BP), and unipolar, recur- unmethylated mutations can produce FMR1 protein in rent (UPR). Using a dominant model and ASM II, D10S217 chorionic villi cells and leucocytes. FRAXE mental retar- produced a LOD score of 2.86 at theta=0.05. With the dation is a less severe condition than fragile X syndrome neighbouring marker D10S587, a maximum LOD score of and some males with fully methylated FRAXE mutations 1.48 at theta=0.15 was obtained. In a second step, we