The genetics of schizophrenia

Schizophrenia Elsevier

SCHIZO

Research,

157

4 (1991) 151-111

00117

The genetics of schizophrenia Current

knowledge

and future directions*

Ming T. Tsuang,‘y2 Mark W. Gilbertson’,

and Stephen V. Faraone’

‘Psychiatry Service Brocktoni West Roxbury Veterans Administrution Medical Center and Section ;?r Psychiatric Epidemiology and Genetics, Harvard Medical School Department of Psychiatry, Brockton, MA, U.S.A., ‘Program in Psychiatric Epidemiology, Harvard Schoo1.y of Medicine and Public Health, Boston. MA, U.S.A., and jDepartment of Clinical Psychology. University of Florida, Gainesville, FL, U.S.A. (Received

19 February

1990, accepted

27 February

1990)

Multiple research paradigms have provided evidence for a substantial genetic component in the etiology of schizophrenic disorders. This article reviews the major research strategies which have been employed in the examination of the genetic hypothesis in schizophrenia. Family studies have provided overwhelming support regarding familial transmission but cannot clearly resolve issues related to genetic-versusenvironmental mechanisms. Twin and adoption studies, however, offer consistent evidence for a substantial genetic component and indicate environmental familial factors to be much less important. Quantitative modeling studies represent more specific attempts to identify the genetic mechanism and mode of inheritance responsible for the familial distribution of schizophrenia. To date, however, these quantitative models have not unequivocally supported a specific mode of genetic transmission. For instance, relevant studies provide little support for the mechanism of single major locus inheritance. Furthermore, although a mechanism involving two, three, or four loci cannot be ruled out, there is no compelling support for such models. The multifactorial polygenic model has received the most support and indicates that genetic factors play a greater role than environmental factors in familial transmission. A mixed genetic model including both a multifactorial component and a single major locus cannot be ruled out. Finally, studies of linkage analysis offer a more powerful technique used for testing the hypothesis of a single pathogenic gene, but the results of linkage analysis in schizophrenia are still preliminary and inconsistent. Evidence for a chromosome 5 gene locus has been provided in some studies but not replicated in others. The important implications of genetic-phenotypic heterogeneity and methodological deficiencies are discussed with respect to limitations on the interpretability of these studies and directions for future research. Key words: Genetics;

(Schizophrenia)

INTRODUCTION

Knowledge progresses through a series of dynamic phases which as a whole bring the investigator Correspondence 10: M.T. Tsuang, Psychiatry Service (116A), Brockton-West Roxbury Veterans Administration Medical Center, 940 Belmont Street, Brockton, MA, 02401, U.S.A. * Preparation of this article was supported in part by the Veterans Administration’s Health Services Research and Development Program and National Institute of Mental Health Grants 1 ROlMH41879901 and 1 R37MH43518-01.

0920-9964/91/$03.50

(3 1991 Elsevier Science Publishers

closer to a specific understanding of the phenomenon of interest. The complex etiology of schizophrenic disorders and the role played by familial/ genetic factors illustrate this process well. The focus of this discussion will be to outline major research strategies and lines of evidence which bear upon the hypothesis that schizophrenia is an inherited disorder. Accordingly, four ‘stages’ in the development of a genetic transmission model will be reviewed: (1) family studies (i.e., to what extent do schizophrenic disorders aggregate in families?); (2) twin/adoption studies (i.e., if schizophrenia is

B.V. (Biomedical

Division)

158

familial, to what extent can this pattern be explained by genetic factors as opposed to environmental/cultural factors?); (3) quantitative modeling studies (i.e., if schizophrenia is genetically transmitted, what is the mode of inheritance?); and (4) linkage studies (i.e., which genes are most specifically implicated and where do they reside in the human genome?).

FAMILY

STUDIES

TABLE

I

Risks to relatives Relation

Risk (96)

First-degree Parents

relatives 4.4

Brothers and sisters Neither parent schizophrenic One parent schizophrenic

8.5 8.2 13.8

Fraternal Fraternal

5.6 12.0

Identical

The logic of the family study approach is straightforward: if schizophrenia is an inherited disorder, relatives of schizophrenic patients should manifest a higher incidence of schizophrenia than is found in the general population. Further, for relatives of schizophrenics, the risk should increase as the number of genes they share with the patients increases. Therefore, first-degree relatives (children, siblings, and parents) of schizophrenic patients should demonstrate a higher incidence of schizophrenia than more extended generations of relatives (e.g., grandchildren, uncles, nephews). The risk figures for the general population as well as various relatives of schizophrenics are reported in Table 1. As can be seen in the table, risk figures conform to the predicted familial pattern described above. The values reported in Table 1 come from a number of earlier European family studies. Recent studies using more rigorous research methods and narrower, criterion-based definitions of schizophrenia are also consistent with the genetic hypothesis. However, they report risk figures which are somewhat lower than those seen in Table 1. Tsuang et al. (1980), for example, reported the risk of schizophrenia to first-degree relatives of schizophrenics to be 3.2% compared with 0.6% for relatives of non-psychiatric controls. Guze et al. (1983) reported comparable figures of 3.6% and 0.56%, respectively. In both studies, the increased risk for schizophrenia among relatives of schizophrenics remained statistically significant despite the low prevalence figures. Contemporary studies report risk estimates approximately one-third of those obtained by these earlier European studies, which employed a broader definition of schizophrenia. Diagnostic practices appear to play a

ofschizophrenics

twins of opposite sex twins of same sex twins

Children Both parents

57.7 schizophrenic

Second-degree relatives Uncles and aunts Nephews and nieces Grandchildren Half brothers/sisters First cousins General

(third-degree

population

12.3 36.6

2.0 2.2 2.8 3.2 relatives)

2.9 0.86

Note; unless otherwise noted, figures are based on Slater and Cowie (1971); data mainly derived from pooled data of ZerbinRudin (1967), with only cases of definite schizophrenia counted. From Genes and the Mind (p. 71) by M.T. Tsuang and R. Vandermey, 1980, Oxford University Press, Oxford, England. Copyright 1980 by Oxford University Press. Reprinted by permission.

strong role in differences noted in the reported prevalence figures. The figure of 3.2% obtained by Tsuang et al. (1980), for example, when using stringent Washington University criteria, increases to 3.7% when DSM-III criteria are applied. It increases to 7.8% if the schizophrenia category is broadened to include atypical schizophrenics. Thus, as Tsuang et al. (1984) have noted, the risk figures for schizophrenia based on contemporary criteria are similar to the figures obtained by the earlier European studies when atypical cases are included. The exclusion of atypical schizophrenia cases from family studies may also explain why two recent family studies failed to find familial transmission in schizophrenia. Pope et al. (1982) found no cases of schizophrenia among first-degree relatives of their schizophrenic probands. Abrams and Taylor (1983) found the risk for schizophrenia to be only 1.6% among 128 first-degree relatives of schizophrenics. Although certain methodological problems may explain these results (Kendler, 1983;

159

Weissman et al., 1983) it is also possible that they are due to diagnostic practices. Despite the caveats noted, contemporary figures as well as those reported in Table 1 suggest that the risk for schizophrenia in the first-degree relatives of schizophrenic patients exceeds the observed rate in the general population by 5-10 times. There would appear to be little doubt then that schizophrenia manifests as a familial disorder.

TWIN

STUDIES

The familial aggregation of schizophrenic disorders does not, of course, resolve the genetic hypothesis. This is because resemblance among relatives in a given pedigree may be attributed to either genetic or environmental factors. Shared environmental exposure and cultural transmission of behavioral attributes provide equally plausible interpretations of the familial data reviewed above. A major goal of epidemiological twin and adoption studies is to provide the next step in determining the likelihood of genetic versus environmental mechanisms. Twin studies appear to overwhelmingly support the genetic hypothesis. All of the 11 studies reviewed by Gottesman and Shields (1982) reported that individuals who have a schizophrenic monozygotic (MZ) twin are over four times more likely to develop schizophrenia than individuals who have a dizygotic (DZ) twin. The logic of twin research rests upon the assumption that both MZ and DZ twins share a relatively common environment (current evidence supports this assumption), but differ significantly in the degree to which they share genes. While MZ twins are genetically identical, DZ twins share on the average only one-half of their genes. Therefore a higher concordance rate for schizophrenia in MZ twins than in DZ twins is most parsimoniously explained by genetic mechanisms. In support of this pattern, the pooled sample of 550 MZ and 776 DZ twin pairs reported in the review by Gottesman and Shields revealed concordance rates of 57.7% and 12.8%, respectively. Kendler’s (1983) review of twin research reached similar conclusions. His summary of nine studies including 401 MZ and 478 DZ twin pairs reported

concordance rates of 53% and 15%, respectively. Of additional interest are those MZ twins in which only one member is schizophrenic. The psychiatric adjustment of nonschizophrenics who have a schizophrenic MZ twin has been of considerable interest, particularly in light of evidence that the schizophrenic genotype may manifest itself in a variety of phenotypes along a ‘spectrum’ from mild to severe. In seven of the studies reviewed by Gottesman and Shields (1982) 354 MZ twins provided data relevant to this question. In 11.7% of the cases where the twins were discordant for schizophrenia, the nonschizophrenic twin was diagnosed as having a schizoid personality. In 9.8% the nonschizophrenic twin had some other disorder (e.g., alcoholism, psychopathy, suicidal tendencies, character disorder). Further evidence that nonschizophrenic twins of schizophrenic MZ twins are predisposed to schizophrenia comes from Fischer’s (1971) Danish twin studies. These studies examined the 71 offspring of monozygotic twin pairs who were discordant for schizophrenia. The investigators reported risk factors for the development of schizophrenia in both the offspring of the schizophrenic member and the nonschizophrenic member. Because MZ twins have the same genes, the genetic hypothesis predicts their risk figures to be identical. Fischer’s results are consistent with this prediction. The offspring of the schizophrenic twins had a 9.6% risk of developing schizophrenia. The risk for the offspring of the nonschizophrenic twin members was 12.9%. These risks are essentially the same and consistent with the genetic hypothesis. Taken together, the reviewed twin studies strongly argue for a genetic component to the familial transmission of schizophrenia. An additional measure of genetic contribution based upon a multifactorial summary statistic for concordance rates in both MZ and DZ twins is heritability of liability. Heritability is a measure of the proportion of variation attributable to genetic factors. The major twin studies of schizophrenia are consistent in estimating the heritability of liability for schizophrenia to be rather substantial, at between 60&90%. While evidence clearly suggests a major role for genetic factors in the familial aggregation of schizophrenia, it is important to note that concordance rates for MZ twins and estimates for heritability of liability do not reach 100%. That is,

160

while genetic factors would appear substantial, they do not appear to represent the entire picture.

ADOPTION

STUDIES

By far the most convincing evidence for a genetic contribution to schizophrenia is that provided by a series of adoption studies. The adoption study design effectively removes the possibility of postnatal environmental interaction between the adopted child and biological relatives. There are several types of adoption study designs. One approach has been to study children who were adopted away from a schizophrenic biological parent. This was the case with the pioneering study of Heston (1966). He compared 47 adopted children whose biological mothers were schizophrenic with a control group of 50 adopted children whose biological mothers were not schizophrenic. The results of the study were clear: 11% of the children with schizophrenic biological mothers were themselves schizophrenic; none of the children in the control group were schizophrenic. Thus, Heston demonstrated that schizophrenia was transmitted independently of the postnatal environment created by a schizophrenic mother. These findings were constructively replicated in the Danish adoption studies of Rosenthal and colleagues (1968, 1975). Here also the risk of schizophrenia was higher for the adopted away offspring of schizophrenic biological parents than for the adoptees of biological parents with no known psychiatric disorder (7.7% versus 0%). The same was true of the risks for the ‘spectrum’ disorders of borderline schizophrenia, schizoid personality, and paranoid personality (25.6% versus 14.9%). Researchers in the Danish adoption studies also used an alternative adoption study design that assessed the risk for schizophrenia among the biological and adoptive relatives of identified schizophrenic and control adoptees (Kety et al., 1975, 1978). The biological relatives of schizophrenics manifested an increased risk for schizophrenia in comparison with the adoptive and nonbiological groups of relatives examined. Thus, it is clearly the biological, not the adoptive, relation that appears to be associated with the familial transmission of schizophrenia.

The sample analyzed by Kety and his colleagues (1978) also contained an important subsample: the parental half siblings of the adopted individuals. Half siblings share 25% of their genes, a substantial genetic relationship. Because paternal half siblings are related through a common father, they do not share a common prenatal, perinatal, or neonatal environment. The results of Kety indicated that the biological paternal half-siblings of schizophrenic adoptees are at greater risk for both schizophrenia and uncertain schizophrenia than the biological paternal half-siblings of control adoptees. Because these results rule out in utero influences, birth traumas, and early mothering experience, they provide compelling evidence for the substantial influence of genetic factors in the transmission of schizophrenia. Adoption studies provide an advantage over twin studies in that the comparative incidence of schizophrenia in biological and adoptive relatives allows for a more specific analysis of such factors as cultural transmission and environmental exposure. In particular, the vertical cultural transmission (VCT) of illness from parents to offspring has been explored. VCT can take two forms: direct VCT in which offspring ‘learn’ schizophrenic behavior through direct modeling of a parent with schizophrenia; and indirect VCT whereby ‘schizophrenogenic’ behavior (which does not itself resemble schizophrenic symptoms) on the part of nonschizophrenic parents contributes to schizophrenic symptoms in offspring. The pattern of risk for schizophrenia observed in family studies has not supported a substantial role for either direct or indirect VCT. In particular, cross-fostering adoption studies (i.e., normal control adoptee with schizophrenic adoptive parents) have failed to provide evidence consistent with direct cultural transmission. Wender et al. (1977) for example, found no increased incidence of schizophrenia in the adopted away offspring of normal parents who were subsequently raised by schizophrenic parents. In reviewing this body of literature, Kendler (1988) reports the genetic heritability of schizophrenia to be between 0.60 and 0.70, which is quite similar to estimates based on twin studies, as we reported earlier. In contrast, he suggests that environmental familial factors appear to account for less than 20% of the variance in liability to schizophrenia.

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QUANTITATIVE

MODELING

STUDIES

Methodological issues Acceptance of the hypothesis that genes contribute substantially to the causation of schizophrenia has led to the next phase of research, which has focused upon attempts to determine how the schizophrenic genes are transmitted from generation to generation. From the data already reviewed, it is clear that a classic Mendelian model of inheritance will not adequately describe this genetic transmission. For example, if schizophrenia was caused by a fully penetrant dominant gene (penetrance is defined as the probability that an individual with the genotype will actually express the trait of interest), we would expect that 50% of the offspring of one schizophrenic parent would become schizophrenic. The observed value is much lower, about 12% (cf. Table 1). If schizophrenia were caused by a fully penetrant recessive gene, we would expect 100% of the children with two schizophrenic parents to be schizophrenic. The observed value is 36.6%. Thus, more complex models are needed to describe the genetic transmission of schizophrenia, and quantitative or mathematical modeling studies provide a strategy for doing so. Such studies are discussed briefly below; for details see the review by Faraone and Tsuang (1985). The statistical procedures used to model genetic transmission fall into two categories: prevalence analysis and pedigree segregation analysis. The major difference between the two approaches involves the form in which the family data enter the analysis. In prevalence analysis, the data are reduced to a matrix that specifies the prevalence of schizophrenia in relatives of schizophrenic and control probands. This form of analysis loses important information about the familial pattern in a given sample because it does not treat probands with multiple schizophrenic relatives differently than those with only one such relative. The method of pedigree segregation analysis uses all such information available in the pedigree to test genetic hypotheses. Pedigree segregation analysis also has greater statistical power than prevalence analysis and also allows for bias corrections such as nonrandom sampling and variable age-of-onset parameters. Prevalence analysis, however, does have one advantage over pedigree segregation

analysis in that it offers an overall statistical goodness of fit measure to determine how well the model predicts the observed data. The mode of inheritance has substantial implications for etiological research and clinical practice. A conclusive demonstration that a single major locus (SML) is involved in schizophrenia would hold the promise that a relatively direct biochemical pathway from genotype to phenotype accounts for the pathophysiology of the disorder. If a multifactorial polygenic (MFP) model describes the mode of transmission, the search for a simple biochemical pathway is likely to be less fruitful. The polygenic model relegates genetic factors to an amorphous pool of small, indistinguishable components that contribute additively to the pathophysiology of the disorder. Because none of these components are either necessary or sufficient for pathogenesis, the MFP model predicts that complex multivariate designs will be required to establish reliable physiological differences between schizophrenics and controls. Understanding the genetic mechanism in schizophrenia will help researchers develop sampling selection rules to maximize the yield of etiological research. Likewise in clinical practice, knowledge of the mode of inheritance will facilitate genetic counseling and have important implications for helping afflicted individuals. Single major locus models Single major locus models propose that the pair of genes present at a single locus is responsible for the transmission of schizophrenia. The major problem facing SML proponents is how to explain the nonMendelian distribution of the disorder. To this end, more complex SML models have been developed that include one or all the following modifications to the classic Mendelian model: (a) reduced penetrance of the pathogenic gene, (b) the existence of phenocopies (i.e., the environmental induction of schizophrenia in individuals without the schizophrenic genotype), and (c) the addition of an environmentally related liability-threshold construct (i.e., liability is determined by a combination of genotype and environmental influence; it is proposed that, if liability exceeds a certain threshold, the individual will develop schizophrenia). Results of SML analyses have been quite contradictory. Many of these studies are hampered by

162

methodological weaknesses in their data collection and statistical procedures, however. In general, these models accurately predict the general population prevalence, the prevalence in offspring of schizophrenics, and the incidence in siblings of schizophrenia. Collectively, however, prevalence and pedigree segregation analyses do not support the SML model of inheritance for schizophrenia. In addition, all studies that provide statistical tests of model adequacy reject the model. Those that cannot rule out the model note that the risk to MZ twins and the offspring of two schizophrenics are underpredicted by it. In order to account for these results, the SML model must appeal to a rather substantial level of environmental heritability (i.e., the proportion of phenotypic variability attributable to environmental factors). However, this makes the SML model genetically less appealing. Although the negative statistical results seem compelling, the rejection of the genetic model may merely indicate that some of the nongenetic assumptions of the model (e.g., the age of onset distribution, random mating) are wrong. Given this possibility, it is worth noting that the best fitting models from studies providing no statistical tests are reasonably consistent. These studies suggest that if an SML model is involved in the transmission of schizophrenia the pathogenic gene is probably recessive with reduced penetrance. Multiple locus models

The failure to find an SML model that unequivocally accounts for the familial transmission of schizophrenia has led to the testing of polygenic models. These models assume that genes found at more than one locus are responsible for the familial pattern of schizophrenia. The models that have been proposed can be conveniently divided into two general classes. Limited loci polygenic (LLP) models propose a relatively limited number of loci, whereas multifactorial polygenic (MFP) models propose a large, unspecified number of loci. The application of quantitative models involving several pairs of genes segregating at several loci is hampered by the fact that there are 100 possible two-loci models that can describe the transmission of a dichotomous trait. If the trait is partitioned into a trichotomy (e.g., schizophrenia, schizotypal disorder and unaffected) there are 2634 transmisqsion possibilities. In practice, investigators have

chosen one or more models on the basis of either their own hypotheses about schizophrenia or their determination of which models are biologically meaningful. As with the SML studies, so far the data collection and statistical procedures are not rigorous enough to allow the formulation of strong conclusions. However, two trends are evident. First, the studies that compared the SML and LLP models agree that there are no dramatic differences between their abilities to account for the familial transmission of schizophrenia. Further, the LLP models are similar to the SML model in predicting that the majority of individuals with the schizophrenic genotype will not develop schizophrenia. For example, the best two-loci model of Elston and Namboodiri (1977) predicts that 87% of individuals with schizophrenic genotypes will never develop schizophrenia. Such results are not consistent with the degree of concordance between monozygotic twins unless one appeals to unlikely environmental explanations of twin similarity. Unlike LLP models, MFP models do not specify the number of loci involved in schizophrenia. Instead they assume that there are many interchangeable loci; genes at these loci have small, additive effects on the individual’s liability for schizophrenia. This model assumes that all people have some unobservable liability or predisposition to develop schizophrenia. Gottesman and Shields (1967) and Hanson et al. (1977) noted five points in favour of MFP models. First, like other MFP disorders, schizophrenia is found with varying severities. Second, the risk for schizophrenia is greater for persons with many schizophrenic relatives than for persons with few schizophrenic relatives. Third, the risk to a person increases as a function of the severity of a schizophrenic relative’s illness. Fourth, nonschizophrenic individuals from the schizophrenia spectrum can be conceptualized as having a liability close to but not exceeding the threshold for schizophrenia. Finally, MFP disorders are expected to respond slowly to natural selection. One must’note that the MFP model is more adequate than the SML model if concordance in MZ twins is substantially greater than the concordance in DZ twins. Thus, the twin studies of schizophrenia are consistent with the MFP model. To date, all tests of the MFP model have used prevalence analyses. The results have, as in previ-

163

ous models, suffered from methodological weaknesses and contradictory findings. Nevertheless, the MFP results are more promising than the SML results (Gottesman and Shields, 1967; Rao et al., 198 1; McGue et al., 1983). In particular, pathanalytic MFP studies support the hypothesis that schizophrenia is to a large extent a disorder with a mostly genetic multifactorial etiology. As outlined earlier, under this model, genetic factors account for 60-70% of the familial pattern of schizophrenia. Environmental factors are important to a much lesser degree. Overall, the results suggest that the MFP model deserves serious consideration. These results cannot rule out as yet, however, the possibility of a mixed model in which an SML component and a MFP component both exist. The mixed model The mixed model approach assumes that the liability to develop a disorder can be due to an SML component, an MFP component, a common environmental effect for siblings, and/or a random residual environmental effect. Only a small number of reported studies is currently available, and these studies provide somewhat equivocal results. As an example, the mixed model was applied to a methodologically sound family study data set by Risch and Baron (1984). In diagnosing the relatives, four phenotypic classes were used: schizophrenia; definite schizotypal personality disorder (SPD-D); probable schizotypal personality disorder (SPD-P); and unaffected. Two sets of analyses were performed. The first set defined SPD-D, SPD-P, and unaffected as one phenotypic class. The second set maintained the distinction among the four phenotypes. In the first set of analyses, the mixed model converged to a single major locus solution. In other words, the MFP heritability was estimated to be zero. Further, when no MFP effect was assumed, Mendelian transmission probabilities could not be rejected, but environmental familial transmission was. As a further test, the authors examined the ability to predict MZ twin concordance rates, population incidence, and the prevalence of schizophrenia among children with zero, one, or two schizophrenic parents. The dominant model substantially overpredicted MZ concordance and prevalence among children having one schizophrenic parent. The prevalence among children of

two nonschizophrenic parents was substantially underestimated. The predictive ability of the recessive model was much better, resulting in only a slight underestimation of population incidence and prevalence among children having one schizophrenic parent. The incorporation of SPD-D and SPD-P into the analyses modified the results. The hypothesis of no MFP effect could not be rejected, nor could the hypothesis of no SML effect. Under the assumption of no MFP effect, neither dominant nor recessive inheritance could be rejected, but the hypothesis of familial environmental transmission could be. Under the assumption of no SML, the MFP heritability was estimated to be 0.79. In predicting MZ twin concordance, population incidence, and prevalence in children, the assumption of dominance resulted in predictions highly discrepant from observed data. The recessive mixed model provided the most accurate predictions. Latent trait model An innovative approach relevant to quantitative modeling has been suggested by Matthysse (1985) and recently tested in clinical samples (Matthysse et al., 1986; Holzman et al., 1988). Its foundation lies in a statistical technique known as latent structure analysis. The model assumes the existence of a ‘latent trait’ which is not directly observable and, depending upon its site of involvement in the brain, can cause schizophrenia or other specific phenotypic manifestations. It is hypothesized that the latent trait displays Mendelian transmission while the ‘manifest’ traits (e.g., schizophrenia, schizotypal personality disorder, etc.) do not necessarily conform to such a genetic pattern. Therefore, the object of latent structure analysis is to determine whether the latent trait is genetically transmitted, to specify the mode of transmission, and to define how the manifest traits are related to or dependent upon the underlying latent trait. It requires the ability to define other ‘manifest’ traits, in addition to schizophrenia, which appear to be genetically related. Matthysse and colleagues (1985, 1986) have focused primarily upon smooth pursuit eye movement (SPEM) dysfunctions as a biological marker of schizophrenia and have suggested that schizophrenia and SPEM dysfunctions may be transmitted as independent phenotypic manifestations of a single latent trait. An advantage to the addi-

164

tional study of SPEM dysfunction lies in the fact that they are considerably more common than schizophrenia. SPEM dysfunctions consist of a variety of eye tracking dysfunctions including saccadic intrusions in smooth pursuit, and have been reported by these authors to occur in 5ll85% of schizophrenics and in 45% of their biological firstdegree relatives. (This contrasts with a prevalence of approximately 8% in the general population.) Applying the latent structure model to two divergent samples (i.e., a ‘Chicago-Boston’ sample composed of the parents of psychotic probands, and a ‘Norwegian’ sample composed of the offspring of discordant MZ and DZ twins), the authors obtained strikingly similar and consistent results. Their findings suggest that SPEM dysfunctions and some schizophrenias may be considered expressions of a single underlying trait that is transmitted by an autosomal dominant gene. Hence, it is the ‘latent trait’ which conforms to an SML model of transmission and not schizophrenia per se. It is assumed that the latent trait is related to a meaningful biological substrate which is currently not understood or observable, but which mediates both the expression of schizophrenia and SPEM dysfunctions. While further work is required in this area, the findings of Matthysse, Holzman, and colleagues provide the potential for significant enhancement of quantitative modeling techniques as well as the molecular genetics techniques to be discussed below. Summary The results of genetic modeling studies tend to favor MFP models over SML and LLP models. However, this conclusion must be considered tenuous for several reasons. Most important, the data used for most of these studies were collected with methodological procedures of unknown reliability and validity. With a few exceptions, the quality of these data sets is compromised by non-blind interviewers, unspecified diagnostic criteria, and collection of family-history data from informants. Each of these problems adds sources of variability to the data that may have obscured the effect of a single major locus. Therefore, more rigorous family studies are needed before the SML hypotheses can be discarded. Additionally, the MFP model receives its strongest support from the path analysis studies. How-

ever, because the path analysis approach relies on prevalence data, it may be less powerful than pedigree segregation analyses which rejected the SML model. Reduced power decreases the likelihood of finding true differences and therefore increases the likelihood of erroneously accepting the null hypothesis. Thus, power is particularly important given that in the path analytic approach the MFP model corresponds to the null hypothesis. Finally, new and innovative approaches such as the latent trait model will require more extensive exploration.

LINKAGE

STUDIES

Biological background The most recent and perhaps most exciting area to emerge in psychiatric genetics is that of linkage studies and molecular genetics. This next phase offers the potential of an even more powerful method of establishing genetic etiology in schizophrenia than the statistical methods of pedigree segregation and path analysis. In linkage studies, interest turns to the identification of actual gene locations in the transmission of schizophrenic disorders. The structure of human chromosomes and the well-described series of genetic events involved in human reproduction have enabled human geneticists to develop experimental tests for the detection of single genes that contribute to psychiatric and other disorders. Given the relatively new and recent nature of linkage technology, this discussion will focus on the methodology of the approach and will briefly review a small number of recent findings. To understand more fully how tests of linkage can detect major genes, it is necessary to briefly review a few basic facts of human cytogenetics. At the molecular level, a gene is a sequence of the deoxyribonucleic acids (DNA). DNA consists of a sequence of deoxyribose sugar-phosphate groups each attached to one of four nitrogen bases: adenine (A), thymine (T), guanine (G), and cytosine (C). The sequence of base pairs in a gene determines the sequence of amino acids and hence the protein the gene will create. Although gene size is highly variable, the number of bases contained in a single typical gene is of the order of 1000. Individual genes are arranged sequentially on linear structures called chromosomes. Thus, a chro-

165

mosome is a large strand of DNA containing many individual genes. Humans have 23 pairs of chromosomes. One pair consists of sex chromosomes; the other 22 pairs are called autosomes. The location of a gene on a chromosome is called its locus. The different variants of a gene that may be found at that locus are called alleles. If the pair of alleles at a gene locus are identical to one another they are termed homozygotes; if the pair are not identical (i.e., they represent different variants of the gene) they are referred to as heterozygotes. Genetic transmission occurs because all individuals inherit one member of each chromosome pair paternally and one maternally. However, the inherited chromosomes are not identical to any of the original parental chromosomes. This is due to the phenomenon of ‘crossing over’ which occurs during meiosis. Meiosis is the process whereby gametes (sperm and egg) are created. Unlike other cells, gametes contain single chromosomes, not pairs. These single chromosomes are not simply one of the chromosomes from the original pair. This is the result of crossing over between the original pair during meiosis. Crossing over is represented schematically in Fig. 1. In the figure, the original pair of chromosomes is represented as one dark strand and one light strand. Three different genes are highlighted with upper and lower case letters signifying different alleles at the same locus. During meiosis these two chromosomes will literally cross over one another and exchange portions of their DNA. Fig. 1 demonstrates the result of a single cross over. In reality, multiple cross avers will occur, resulting in a new pair of

chromosomes where each member of the pair is a complicated combination of genes from the original pair. When meiosis is complete, one chromosome from each new pair will be found in each gamete. Thus, the chromosome transmitted from parent to child contains a mixture of genes from the parent’s original pair. Linkage occurs when two loci on the same original chromosome are so close to one another that crossing over rarely occurs between them. Closely linked genes usually remain together on the same chromosome in the same gamete, after meiosis has been completed. In Fig. 1, the A (or a) and the B (or 6) locus are close to one another but distant from the C (or c) locus. Crossing over is unlikely to occur between the A and B loci. Therefore, it is very likely that, after meiosis, the rearranged chromosomes will contain either alleles A and B or alleles a and b. It is highly unlikely that a child will receive a chromosome containing either pairs Ab or aB. In contrast, because the C locus is far from the A locus, it is more possible that these two loci will exhibit recombination. That is, one would expect crossing over to occur between the C and A loci resulting in new, recombinant chromosomes containing the pairs AC and aC. Two loci will recombine only if an odd number of crossovers occur between them. If two loci are very far apart, the probability of an odd number of crossovers is equal to that for an even number, so that the probability of recombination is 50%. Thus, genes on the same chromosome that are distant from one another are transmitted independently, as is the case for genes on different chromosomes.

I

Linkage methodology To demonstrate that a single gene is involved in the etiology of a disease, one can show that the putative disease locus is linked to a known genetic marker. To be useful for linkage analysis, a genetic marker must be a measurable human trait controlled by a single gene with a known, chromosomal location. In addition, the mode of inheritance of the marker must be known and the marker must be polymorphic (i.e., more than one version of the gene exists with a substantial frequency). Commonly used marker loci include blood groups, enzymes, proteins and systems that control immune response. Linkage analysis tests the cosegregation of disease and gene marker in each

A

Fig. 1. Schematic representation of single crossing over of original chromosome pair during meiosis in which DNA portions are exchanged, resulting in a new pair of chromosomes.

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individual meiosis in a family. Linkage between a disease locus and a marker locus is demonstrated by showing that the genes at the two loci are not transmitted independently. This is done by estimating the recombination fraction, the probability that the disease and marker genes will recombine during meiosis. Consider a hypothetical genetic marker locus such that individuals with genotypes Mm and mm are marker positive, whereas individuals with genotype MM are marker negative (i.e., individuals with at least one m gene exhibit the trait). Next consider a disease which is assumed to be autosomal dominant such that individuals with genotypes Dd and dd become ill whereas those with the genotype DD are healthy (i.e., individuals with at least one d become ill). To demonstrate linkage between the marker locus and the disease locus, the investigator collects a series of families where one parent is marker negative (MM) and healthy (DD) (i.e., homozygous for both traits or double homozygote) while the other parent is marker positive (Mm) and is afflicted with the disease (Dd) (i.e., heterozygous for both traits or double heterozygote). If the afflicted parent is known to have genotype MDlmd (i.e., M and D are on the same chromosome and m and d are on the other chromosome), then the expected distribution of children is given in Table 2. Haplotypes refer to the genetic contribution from one parent, which constitutes half of the child’s genotype. As column 1 indicates, the child always inherits an MD haplotype from the doubly homozygous parent. Four possible haplotypes can be inherited from the doubly heterozygous parent. TABLE

2

Distribution

ofchild

genotypes from MD/md

x MD/MD

mating

Marker status

Disease status

MD/MD

Negative

Well

MD/md

Positive

Ill

MDjMd

Negative

I11

Positive

Well

Child genotype

Probability genotype

of

MDlmD

i N.B.: 8= recombination

fraction

The first two child genotypes will occur when the marker and disease loci do not recombine and are therefore called nonrecombinants. The last two genotypes in the table are recombinants; their second haplotype is a recombination of the ill parents original haplotypes. Column two gives the probability of observing each genotype expressed in terms of the recombination fraction, 8. The recombination fraction ranges from 0 to 0.5. Low values indicate that the disease and marker loci are tightly linked together on the same chromosome. A value of 0.5 means that the two loci are either on different chromosomes or are very distant on the same chromosome. When the two loci are tightly linked (i.e., e=O) then the two recombinant genotypes should be very rare in matings of the type given in Table 2. The two nonrecombinant genotypes should be equally common. In this example, nonrecombinants can be distinguished from recombinants at the phenotypic level; nonrecombinants will be either marker negative and well or marker positive and ill, whereas recombinants will be either marker negative and ill or marker positive and well. Thus, in this example, linkage means that positive marker status and affliction with the disease will tend to travel together within a given family. In practice, the detection of linkage is more complicated than the preceding example because the genotypes and haplotypes at relevant loci may not be known with complete certainty. Thus, it is often not possible to determine from direct observation which children are recombinants and which are nonrecombinants. Fortunately, statistical techniques have been developed that allow one to estimate the recombination fraction in situations when genotypes and haplotypes are not well specified. The most common method of reporting linkage results is to compute an odds ratio. It compares the probability that linkage is present (0~0.5) with the probability of no linkage (8= 0.5). It has been customary for linkage results to be reported in terms of the logarithm to the base 10 of the odds ratio. The log of the odds ratio is called the lod score. Lod scores greater than 3 are considered to be evidence favoring linkage; lod scores less than -2 are considered to be evidence against linkage; lod scores greater than 2 but less than 3 are suggestive of linkage; lod scores between -2 and 2 are considered to be uninformative.

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Thus, a linkage analysis will support the hypothesis of linkage if the statistical computations indicate that the odds favoring linkage are 1000 to 1 (i.e., log (1000/l)= 3). Molecular genetic markers Although methods of linkage analysis have been available for some time, the ability to perform a linkage study has, until recently, been limited by the availability of genetic markers. Measurable traits, such as blood groups that are polymorphic enough to be suitable markers, are rare. That is, they cover only a very small proportion of the genome, making it, a priori, very unlikely that a known genetic trait could be mapped to a chromosomal locus. Fortunately, nature, in conjunction with the diligent and brilliant work of molecular geneticists, has provided a simple, but powerful, solution to this problem. During evolution, bacteria developed a mechanism to protect themselves from the invasion of foreign DNA. At the core of this mechanism are a group of enzymes called restriction endonucleases. These enzymes neutralize foreign DNA by literally cutting it into pieces. The locations of the cuts are determined by the sequence of nucleotides in the foreign DNA. For example, the restriction endonuclease called A/u1 will cut DNA between nucleotides containing guanine and cytosine wherever the nucleotide sequence AGCT (i.e., four nucleotides sequentially containing the bases adenine, guanine, cytosine and thymine) occurs. The enzyme EcoRI cleaves DNA between G and A within the nucleotide sequence GAATTC. Thus, if A/u1 is applied to the DNA sequence: TACGGCCAGCTCGAAGT

two fragments

are produced: TACGGCCAG

CTCGAAGT

The application of EcoRI would not cut this sequence because it does not contain the recognition site GAATTC. For a variety of reasons, a large proportion of human DNA is highly polymorphic as regards the lengths of fragments produced by restriction enzymes. That is, if one extracts the same chromosomal segment from different individuals, the length and number of fragments produced from this segment by one or more restriction enzymes

will vary substantially among individuals. Since restriction fragment lengths are highly polymorphic they are known as restriction fragment length polymorphisms or RFLPs. To date, over 3000 RFLPs have been catalogued and identified along the human genome. It is expected that most, if not all, genes will be within linkage distance of RFLP loci in the near future. Through the systematic testing of RFLPs along the human genome in families with inherited disorders such as Huntington’s disease, for example, it has been possible to isolate gene sites associated with transmission of the disorder. Linkage studies of schizophrenia While more comprehensive genome searches have yet to be completed in the linkage study approach to schizophrenia, a few preliminary and isolated analyses have been reported. Much interest in a specific region of the human genome has been generated by a recent intriguing report (Bassett et al., 1988) in which schizophrenia was found to consegregate with a partial trisomy of chromosome 5 (region 5ql l-13) in a family of Asian descent. Two members of the pedigree, a nephew and his maternal uncle, share a distinct chromosomal abnormality resulting in a narrowly defined schizophrenic syndrome, along with dysmorphic facial features and other structural anomalies. The strikingly similar phenotypic presentation of the two family members has led to speculation concerning the long arm region of chromosome 5 as a potential major gene site for the predisposition to schizophrenia. Following on the heels of this discovery, Sherrington et al. (1988) studied seven British and Icelandic families with multiple schizophrenic members in at least three generations. The total sample included 39 cases of schizophrenia, five cases of schizophrenia ‘spectrum’ (schizoid personality disorder), and another ten cases of ‘fringe’ phenotypes (i.e., alcoholism, phobias, major depressive disorder, etc.). Using two RFLP probes to track the inheritance pattern of schizophrenia in these families, Sherrington et al. demonstrated genetic linkage on the same region of the long arm of chromosome 5. Their results indicated the existence of a gene locus with a dominant schizophrenia-susceptibility allele. In addition, the inclusion of schizophrenia spectrum and ‘fringe’ phenotypes in the analysis improved their results,

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suggesting that these disorders may represent alternate manifestations of the same allele. A lod score of 3.22 was obtained when only cases of schizophrenia were included in the linkage analysis, while a lod score of 6.49 resulted when all cases were included. As outlined earlier, the magnitude of both of these scores provides strong evidence for linkage. While on the surface the linkage analysis provided by Sherrington et al. temptingly appears to resolve the etiology of schizophrenia, the results of two additional studies (Kennedy et al., 1988; St. Clair et al., 1989) contradict these findings. Using an even more detailed search of the same region of chromosome 5 with the aid of seven RFLP probes, Kennedy et al. (1988) found strong evidence against linkage of schizophrenia in a single large Swedish pedigree. Lod scores in this study were all less than - 2. Given these findings and the positive results of the Sherrington et al. study, Kennedy et al. suggest that strong evidence is provided for the genetic heterogeneity of schizophrenia. That is, there would appear to be different loci, leading to different neurophysiological abnormalities, resulting in a ‘final common pathway’ phenotype of schizophrenia in different families. St. Clair et al. (1989) examined 15 Scottish pedigrees utilizing methods quite similar to that of Sherrington et al., and also found no evidence for linkage regardless of how broadly the schizophrenic phenotype was defined. It should be noted, however, that the pedigrees examined by these investigators also demonstrated significant levels of affective illness. Eight of the 15 pedigrees also contained bipolar members, while four of the remaining seven pedigrees also contained major depressives. Given what appears to be a somewhat different pedigree composition between the studies of Sherrington et al. and St. Clair et al., the confounding issue of genetic heterogeneity remains intact. Clearly, further work is required in the study of the molecular genetics of schizophrenia. Not only are expanded linkage studies required in those families already examined but similar studies must be carried out in many more affected families. These studies must not only investigate linkage in chromosome 5, but also explore additional gene loci which potentially predispose to schizophrenia. However, these strategies alone may not be sufficient to resolve the linkage question.

Rather, the more complex issues of genetic and phenotypic heterogeneity will also need to be addressed.

GENETIC AND PHENOTYPIC HETEROGENEITY

The inconsistent and sometimes conflicting results of mathematical modeling and linkage studies have most often been interpreted to indicate that the schizophrenic disorders are genetically and phenotypically heterogeneous. Genetic heterogeneity means that more than one genotype can cause schizophrenia. In other words, there may be several different subtypes of schizophrenia, each having a different mode of genetic transmission. All of the analyses reviewed above assume that most cases of schizophrenia are transmitted through the same mechanism. The failure to recognize potential heterogeneity in the genetic mechanisms underlying schizophrenic disorders creates obvious fertile ground for conflicting findings. The hypothesis of genetic heterogeneity is appealing; it explains inconsistent results with a genetic hypothesis that has been substantiated in other areas of medical genetics. For example, Morton (1956) demonstrated that linkage between elliptocytosis and the Rhesus blood group could be conclusively demonstrated in some families and competently rejected in others. Ott (1985) discussed the case of Charcot-Marie-Tooth disease, a hereditary motor and sensory neuropathy. Although this disease appears clinically homogeneous, some families demonstrate linkage to the Duffy blood group while others do not. Glycogen storage diseases result in abnormal amounts of glycogen deposited in one or more organs. Ten different forms of the disease have been isolated, each corresponding to a different enzyme defect. Although the clinical manifestations of the ten subtypes are similar, each can be considered to be a different single major locus disorder (Vogel and Motulsky, 1979). The potential existence of more than one genotype which can cause schizophrenia requires research designs capable of isolating genetically homogeneous samples. However, homogeneity of clinical symptomatology will not guarantee genetic homogeneity. Creating samples that are homo-

169

geneous, based upon biological marker criteria, may be successful if these markers are under a substantial amount of genetic control and are known to be etiologically involved in the disorder. For example, the heterogeneity of the glycogen storage diseases is revealed if one studies the activity of relevant enzymes instead of the overt clinical manifestations. Propping and Fried1 (1988) provide an excellent review of recent attempts to begin exploring the genetic aspects of biochemical and pathophysiological mechanisms potentially related to schizophrenia. A more commonly used method to maximize genetic homogeneity is to study ill individuals from a single large pedigree. Although genetic homogeneity may be compromised by assortative mating, individuals from the same pedigree are more likely to have the same genetic disorder than individuals from different pedigrees. In contrast to genetic heterogeneity is phenotypic heterogeneity, which suggests that a given pathogenic genotype can be expressed as one of several phenotypes. It is not uncommon for a single gene to have different phenotypic effects in different individuals; this is known as pleiotropy. Thus mathematical modeling and linkage research must not only address clinical schizophrenia per se but will need to closely attend to the specification of phenotypes that may be genetically related to schizophrenia. For example, the results of Tsuang and Faraone (1984) suggest that the schizophrenialike psychoses are related to schizophrenia. A similar relation is suggested by Risch and Baron (1984) for schizotypal personality disorder. Ideally, phenotype specification should combine traditional diagnostic distinctions with the assessment of potential biological markers. Improved definition of alternate phenotypes (e.g., schizophrenia spectrum disorders) and, in particular, the mapping of relevant biochemical and physiological parameters will be of critical value. These latter measures offer the potential of increased penetrance and are likely to be more reliably assayed. If successfully mapped, the correlation of such measures with the clinical phenotype of schizophrenia will likely shed much light on the inheritance of schizophrenic disorders. The combination of genetic and phenotypic heterogeneity results in the situation diagrammed in Fig. 2. This diagram indicates that a pathogenic genotype can lead to a specific clinical phenotype

Population that carries the genetic vulnerability or diithesis

What

we now study

What

we want

to study

Fig. 2. Genetic and phenotypic heterogeneity model. (Reprinted with permission from Tsuang, M.T., Lyons, M.S. and Faraone, S.V. (1987) Problems of diagnoses in family studies. J. Psychiatr. Res. 21, 391-399.)

(e.g., schizophrenia), a spectrum disorder (e.g., schizotypal personality disorder), or no clinical symptomatology. This situation represents phenotypic heterogeneity. Furthermore, there are individuals called phenocopies who manifest the clinical phenotype but do not have the specific genotype. They may have another pathogenic genotype or they may be nonfamilial forms of the disorder; both of these situations correspond to genetic heterogeneity. Clearly, if one only studies the clinical phenotype, many genotypically abnormal individuals will be considered normal. This will make the mode of inheritance quite difficult to detect. As Fig. 2 suggests, progress in psychiatric genetics would greatly benefit from a more rigorous and comprehensive description of phenotypes related to pathogenic genotypes. Its importance lies in the ultimate development of more genetically homogeneous subtypes of schizophrenia.

FUTURE

DIRECTIONS

FOR

RESEARCH

Early family, twin, and adoption studies have provided a solid foundation for psychiatric ge-

170

netics and have paved the way for more sophisticated paradigms involving quantitative modeling and molecular genetics. As reviewed above, these technologies have as yet to provide unequivocal conclusions concerning the genetic transmission of schizophrenia. In part, the increased implementation of sound and rigorous family study methods will contribute significantly to the interpretability of results. Such improvements, as important as they may be, are not likely to eliminate significantly the level of inconsistency and conflict observed in current results, however. This is doubtless true due to potential complexities created by genetic and phenotypic heterogeneity. Psychiatric genetics has reached a point where the sophistication of available experimental tools, such as molecular genetic technologies and statistical procedures, has surpassed the ability to describe relevant phenotypes. It is unlikely that advances in statistical or linkage analyses per se will resolve the problems posed by genetic and phenotypic heterogeneity. Rather, what is required is the rigorous use of these technologies in populations which expand beyond the mere clinical phenotype (i.e., schizophrenic disorder) to spectrum phenotypes and biochemical correlates. The validation of spectrum disorder phenotypes is relatively straightforward. The basic experimental designs of psychiatric genetics, family, twin and adoption studies can indicate whether a specific disorder or set of clinical symptoms is more common among relatives of probands manifesting the clinical phenotype in comparison to relatives of normal control probands. The identification of symptom-free individuals who carry the pathogenic genotype is not an easy task. Ideally, biological genetic marker research will supply us with some biological abnormality that is expressed in symptom-free individuals who have the pathogenic genotype. Studies of discordant MZ twins would be most useful in this regard. Although the phenotypic heterogeneity of schizophrenia may eventually be resolved by careful neurodiagnostic and psychiatric assessments of patients and their relatives, the etiological or genetic heterogeneity of the disorder will remain. Is there a chromosome 5 variant of schizophrenia as the data of Sherrington et al. suggest? If so, what percent of cases are accounted for by this variant? To answer such questions in the presence of genetic

heterogeneity is likely to require large samples of schizophrenic families. Currently, two nationwide projects are attempting to recruit such samples. The first is an initiative from the National Institute of Mental Health (NIMH) that will pursue genetic linkage studies of schizophrenia, bipolar disorder and Alzheimer’s disease. For each disorder, NIMH is planning to recruit 200 families that manifest familial transmission. These families will be wellcharacterized psychiatrically, using structured psychiatric assessment instruments. In addition, blood samples will be collected from each family member for the establishment of permanent sources of DNA that will provide RFLP markers for linkage analyses. Another nationwide linkage study is a proposed Veterans Administration Cooperative Study that will use the resources of the VA hospital system to recruit the large sample of families needed for linkage research. In the past year, the Brockton VAMC has conducted a study to determine the feasibility of a major cooperative study investigating the molecular genetics of schizophrenia, bipolar disorder, and major depression. The principal aim of this study has been to determine the availability of multiply affected families and the degree of interest in collaboration among VA hospitals around the country. The feasibility study has demonstrated a strong foundation of support for this research throughout the national VA system. To date, a core group of VA hospitals from around the country, inspired by the potential of this proposal, have completed data collection for the feasibility study. Other sites participating in the feasibility study are in the final stages of data collection. A critical multidisciplinary mass of psychiatric epidemiological methods, statistical techniques and molecular genetic technologies is currently available to begin the final states of exploration of the genetics of schizophrenia. The availability of large samples of informative families is an essential, rate limiting factor in the process of elucidating the genotype-to-phenotype pathway. Although this process will require years of careful research by many individuals, it appears that the NIMH and VA systems among others, are capable of providing the resources and leadership needed for such an effort.

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