DIRECTOR: Raymond Crowe,
M.D.
CO-DIRECTOR: Thomas Wassink, M.D.
Overall Aims
1. To search for vulnerability loci for schizophrenia.
2. To explore the genetic controls of neurodevelopment
as they relate to schizophrenia and other neurodevelopmental brain disorders.
3. To explore the relative roles of genetic and environmental
factors in the production of neurodevelopmental disorders.
4. To search for more primary phenotypes (endophenotypes,
i.e., personality traits, characteristic brain morphology, patterns of
cognitive dysfunction) that may be more closely linked to genotypes than
syndromal, criteria-based diagnostic entities.
Background and Rationale
Genetic studies of schizophrenia have, until recent years,
been primarily concerned with testing whether or not there are genetic
contributions to the illness. The conclusion, drawn from these various
family, twin, and adoption studies, is that there almost certainly are.
Though the exact mode of inheritance remains unclear, most authors report
an estimate of heritability of approximately 6070%.
In response to this, the focus of genetic research in schizophrenia has shifted from investigations measuring genetic effects to studies searching for specific disease vulnerability genes. Though great hope for finding "the gene" has been placed in these studies, their results to date have been disappointingly scant. A number of factors contribute to this frustration. Linkage analysis, the predominant method used to search for disease loci, makes specific assumptions about the mode of inheritance of the disease of interest. Schizophrenia, unfortunately, does not appear to conform to these assumptions. An approach to overcoming this hindrance, which we are actively pursuing, is to increase the sample size by forming large, multi-center collaborative initiatives. These will hopefully provide the requisite statistical power to detect genes of presumably small effect.
A complementary approach is to examine associations of specific candidate genes with illness. Association studies are not limited by the assumptions of linkage analysis and offer potential advantages when investigating diseases with complicated heritability. Like linkage analysis, however, association studies of schizophrenia have proven disappointing thus far, with few significant results and minimal replication. The paucity of findings here may be due to the tremendous number of genes involved in brain functioning, with the consequently high a priori probability of false positive associations. They may also be due, however, to a misdirected search. Almost all candidate gene studies of schizophrenia to date have examined neurotransmitter enzymes, receptors, or transporters. While these clearly are involved in the expression of schizophrenia and in its treatment, it is not clear that they play a causal role in the illness. Abnormalities in their levels or activity may rather be compensatory changes provoked by disruption of a more primary process.
Early brain development is increasingly implicated as the locus of this disruption. Studies from a variety of disciplines have provided a diverse body of evidence to support this hypothesis. The process of neurodevelopment involves many stages such as nerve cell replication, growth, differentiation, migration to the cerebral cortex, and the formation of synaptic connections between neurons. An error at any point in its programming could potentially give rise to the symptoms that comprise schizophrenia. Thus, it is essential that we deepen our understanding of neurodevelopment and its genetic controls, as these may be the most viable candidates for disease vulnerability loci.
The current aims of the Genetics and Epidemiology Research Unit reflect these trends, shifting from our previous emphasis on twin and pedigree studies to a focused search for schizophrenia vulnerability genes in the context of a broader exploration of neurodevelopment and neurodevelopmental psychiatric disorders. To pursue these aims, we have brought together an excellent group of investigators. The core consists of seasoned, well-funded researchers, such as Dr. Raymond Crowe, Dr. Michael Miller, and Dr. Joseph Piven, who, with their complementary interests and approaches, are surrounded by promising younger investigators, many of whom have obtained start-up funding of their own. Dr. Miller and Dr. Jeffrey Murray (serving as a consultant) are an especially important additon, as they bring an expertise in biochemical and molecular genetic methods that will strengthen all projects in the unit. There is a significant amount of interaction and sharing of information amongst these investigators, facilitating pursuit of a unified purpose.
This first aim, the search for vulnerability genes, is supported by Projects 1 and 2. In Project 1, Dr. Crowe continues his Genomewide Search for Genes for Schizophrenia that has been a part of the MH-CRC from its inception. In line with the need, as noted above, for multi-center initiatives, the MH-CRC has contributed its multiplex pedigrees to one such collaboration, and is participating in international efforts to replicate provisional linkage findings on chromosomes 3, 6, 8, and 22, with plans to pursue further investigations of regions on 2, 4, 9, and 10.
Project 2, headed by Dr. Wassink, a postdoctoral fellow in the CRC, bridges a number of the specific aims. Under this project, DNA will be gathered from all subjects who enter the CRC and will be placed in a repository for future use here as well as in collaborative studies. The immediate focus of his project is to analyze neurodevelopmental candidate genes for evidence of linkage disequilibrium with schizophrenia, with parental DNA being gathered for the control group. He plans first to examine genes for the nerve growth factors and their receptors, in which he will be aided by Dr. M. Miller, who has extensively investigated these genes in animal models, and Dr. Murray. His next focus, in line with our growing interest in cerebellar involvement in schizophrenia, will be genes involved in the patterning and differentiation of the midbrain/hindbrain region. A second intriguing aspect of this study is that all probands will undergo the full standard CRC workup. Thus, assuming schizophrenia is truly heterogeneous, we will be able to explore genetic effects within the patient group on parameters such as symptomatology, cognitive abilities, and brain morphology, that may be closer to the genotype than the ultimate diagnosis of schizophrenia.
Project 3, Family and Molecular Studies of Autism with Dr. Piven, represents the first link to a non-schizophrenic neurodevelopmental psychiatric disorder. Autism is a pervasive neurodevelopmental disorder, with brain morphological abnormalities overlapping those of schizophrenia. Autism also has a substantial genetic component: the relative risk to each unborn child in a sibship containing an autistic child is at least 100, with twin studies indicating that the increase in risk is due to genetic factors. Dr. Piven's study has two primary aims. The first, supporting Aim 4 of the Genetics Research Unit, is to explore the boundaries of a more primary phenotype underlying autism, a concept that is also relevant to our work in schizophrenia, by examining specific character traits of relatives from families with multiply affected sibs. This may identify independent aspects of personality which are highly heritable and which may, when combined with other genetic or environmental factors, produce the autistic phenotype. The second, as with Dr. Crowe's multiplex family study, is to carry out, as part of a multi-center collaborative effort, genomewide scans for vulnerability loci to autism. Lastly, the brain imaging obtained on Dr. Piven's subjects as part of his involvement in the Structural Imaging Research Unit will enable us, as with Dr. Wassink's study, to examine allelic effects on brain morphology within the subject group.
The remaining projects address more directly issues of genetic/environment interactions in the context of neurodevelopment. In Project 4, Dr. Miller brings his expertise in molecular methods and animal models to bear in examining the effects of an environmental agent on neurodevelopment. Using central nervous system (CNS) derived cell lines, he is investigating the effects of alcohol on nerve growth factors and on the intracellular mechanisms of neuronal proliferation. In rats, he is investigating the effects of alcohol on neuronal migration and survival. Complementing this molecular approach is Dr. Swayze's clinical investigation of fetal alcohol syndrome (FAS) (Project 5). This study was included in our previous submission, but was criticized for methodological reasons and because it appeared peripheral to the aims of the research unit. While these concerns had merit, the study has been productive (see Progress below) and, with the refocus of this unit, addresses more directly our specific aims and fits well with the other projects. As Dr. Swayze points out, twin case reports indicate a probable genetic predisposition to FAS. Of special interest to the CRC, the teratogenic effects of alcohol disrupt development of primarily midline brain structures, including the cerebellum. Linked to the work of Dr. Nopoulos below, FAS is associated with a high rate of brain septal and midline facial anomalies, with a positive association between the severity of facial dysmorphology and the likelihood of brain dysmorphology. To probe gene/environment interactions, Dr. Swayze is using the imaging capabilities of the CRC to examine the effect of maternal alcohol consumption on genetically determined patterns of brain morphology with the hope of identifying particular periods of vulnerability and resistance to environmental stress.
Projects 6 and 7, both with Dr. Nopoulos as the PI, explore midline structural brain abnormalities in two other syndromes, cleft lip and palate and childhood onset schizophrenia, both of which have complex patterns of inheritance with genetic/environment interactions. Facial and brain structures are of ectodermal origin, and their development is extensively intertwined. Subjects with schizophrenia have an increased incidence of dysmorphic facial features, and, as noted above, subjects with cranio-facial abnormalities have an increased incidence of structural brain abnormalities. In Project 6, Dr. Nopoulos, drawing on the resources of the CRC, assesses brain morphology and neurocognitive function in a group of subjects with cleft lip and/or palate (CLP). Thus, she examines midline development through a syndrome where, in at least a subset of cases, the etiology is primarily genetic, contrasted with Dr Swayze's work in FAS, where an environmental insult is the predominant etiological factor. Identifying relationships between cranio-facial and brain structural abnormalities and cognitive dysfunction in these populations may provide clues to the nature and sequelae of neurodevelopmental disruption in general. Also of note, most of the CLP subjects will have participated in Dr. Jeff Murray's genetic studies of this syndrome, and will therefore already have genotypes on a number of genes of interest, with more DNA available for examining additional candidate genes. In Project 7, Dr. Nopoulos examines the frequency and severity of brain septal abnormalities in a group of subjects with childhood onset schizophrenia ascertained by Judith Rapoport, M.D., and Jay Giedd, M.D., at the Child Psychiatry Branch of the NIMH. As these brain abnormalities most likely represent early disruptions of neurodevelopment, and childhood onset schizophrenia appears to be a more severe variant of the adult form of the illness, this data provides another integrated point of access into the neurodevelopmental origins of schizophrenia.
Thus, the Genetics/Developmental Neurobiology Research Unit incorporates a number of complementary research strategies in the search for vulnerability genes for schizophrenia and other developmental psychiatric disorders. We are using traditional linkage methods, incorporating both schizophrenic and autistic pedigrees into large, multi-center collaborative studies in order to increase our ability to detect disease genes of presumably small effect. We are using association studies with the rigorous haplotype relative risk method of case control to examine well-chosen candidate genes for their involvement in predisposing to illness. At a more fundamental level, based on the hypothesis that schizophrenia is primarily a developmental brain disorder, we are examining the interactions between genetic and environmental factors in an array of such disorders. With the CRC's imaging and cognitive assessment capabilities, we will be able to look for genetic effects across disease categories on pathophysiological correlates of illness, such as abnormal brain morphology and cognitive dysfunction. These "endophenotypes" may, in fact, be more closely linked to the disease genotype than our syndromally based diagnostic criteria, and they provide a potentially powerful window into the genetics of these disorders.
Progress
The focus of this research unit, as stated above, has shifted. In
1998, we recognize that the dichotomization of genetic and environmental
effects is arbitrary, and that their relationship is highly interactive.
Thus, the thrust of our efforts, rather than investigating estimates
and modes of heritability, is now directed towards identifying the
specific components of that heritability. That schizophrenia is, in
part, genetically determined is well established and, in spite of setbacks
to date, there is growing optimism about our ability to disentangle
complex patterns of transmission. While still valuing and carrying
forward our twin and epidemiological studies, the attention given to
focused molecular genetic investigations has increased, and they now
form part of a concerted effort to understand the molecular and systems
level mechanisms of both neurodevelopment and neurodevelopmental disorders.
Almost all of the projects in this unit are independently funded by
a wide range of both federal and private grants. Also, it has become
clear that large collaborative efforts are necessary to carry out linkage
analysis of complex diseases. Because of this, we have actively organized
and participated in such multi-center studies, as evidenced by the
linkage studies of Drs. Crowe and Piven.
Specific progress from those projects included in our previous submission is as follows:
An international collaboration, of which the pedigrees obtained in the Genomewide Search for Genes for Schizophrenia are a part, recently investigated two areas in the pseudoautosomal (PA) regions of the X chromosome using 92 pedigrees with a maternal pattern of inheritance. The results were equivocal in that neither support for linkage was found nor could linkage be definitively ruled out.
The group has also searched for linkage to regions of chromosomes 6p, 3, and 8p. Replications at a highly significant level of confidence were found for 6p and 8p, but not for 3.
Further, a genomewide scan from this collaboration obtained findings on regions of chromosomes 2, 4, 9, and 10 suggestive of linkage that will be followed with analyses of both flanking and higher density markers.
Two specific candidate genes were examined using the
candidate gene approach with the pedigrees from the Genomewide Search.
The genes examined included those coding for the principle brain synaptic
vesicular monoamine transporter (VMAT2) and the dopamine transporter,
both of which were excluded from complete linkage with schizophrenia
spectrum disorders.
An independent assessment of specific regions of chromosome
11q and the pseudoautosomal region of the sex chromosomes using only our
pedigrees found no evidence for linkage. A sib pair analysis using 37 sib
pairs concordant for schizophrenia found no significant difference in concordance
for sex between sibships with paternal versus maternal transmission
of schizophrenia.
A number of studies carried out by Roy et al (1994) compared familial cases from our pedigrees to "sporadic" cases on demographic, brain structure, symptom, and cognitive measures. The familial cases were found to have greater ventricular asymetry (left greater than right) and greater ventricular volume, while sporadic cases were more likely to have been born in the winter and to have had more severe psychotic symptoms.
Although the twin studies from the previous submission are not listed as projects in the current application, ascertainment and data collection continue. Data from six twin pairs from the Nagasaki study have been collected (2 concordant for schizophrenia, 2 concordant normal, and 2 discordant).
12 monozygotic normal twin pairs from the Iowa Twin Study have been enrolled. Interesting findings to date from this data include a moderate degree of consistency in size of cerebral structures, with intra-class r's for various brain regions ranging from 0.20 to 0.74
There was also a surprisingly large difference in full scale IQ (FSIQ) between twins (mean = 8.8, sd = 4.5)
A large proportion of twins had a PIQ/VIQ split greater than 10 (8 out of 24)
A surprisingly low intra-class r was found for measures such as FSIQ (r = 0.42, p = 0.10) when compared with presumably more environmentally determined measures such as level of education (r = 0.96, p = 0.0001).
Dr. Swayze's work continues to provide valuable information regarding the pathophysiology of FAS and, more generally, disrupted neurodevelopment. He has reported, in one of the largest high quality MRI studies of FAS, a high rate of midline structural brain anomalies including callosal agenesis, cavum septum pellucidum, and cavum vergae.
He has also reported a high rate of micrencephaly and a positive association between severity of facial dysmorphology and midline brain anomalies.
It will be interesting, therefore, to compare these results with those of Dr. Nopoulos as her work progresses. She has already shown, in preliminary data from her study of cavum septi pellucidi (CSP) in schizophrenia, an increased rate and severity of septal abnormalities in childhood onset subjects as compared with normal controls.
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