Maria Karayiorgou, M.D. - US grants

DESCRIPTION (Adapted from the Investigator's Abstract): The long-term objective of this proposal is the identification of genes contributing to schizophrenia susceptibility. The investigators propose to first identify the genomic regions which may harbor such genes by performing linkage studies in genetically isolated populations (populations that originated from a small number of founders and expanded in relative isolation). To this end, the investigators have collected families afflicted with schizophrenia from genetically isolated founder populations with accessible genealogy that differ in their demographic characteristics (sample size, growth rate) as well in their age of ancestry. Specifically, they have collected families from the island populations of Kosrae and Yap in Micronesia (genetic isolates of recent origin), and the Afrikaners in South Africa (genetic isolate of 'older' origin). All three sites offer excellent genealogical data that could significantly improve the ability to assess the evidence for increased allele or segment sharing among affected individuals by taking into account all known genealogical relationships. The investigators propose to perform a 10-cM genome-wide scan in the families they have collected and examine all 'candidate regions' identified through the 10-cM scan by denser marker coverage and construction of haplotypes. The identification of genes that increase susceptibility to schizophrenia is expected to have a major impact on the understanding of disease pathogenesis and, ultimately, lead to the development of new, less empirical therapies.

DESCRIPTION (provided by applicant): Hemizygous microdeletions of the 22q11.2 locus are among the most common chromosomal abnormalities. Individuals with the 22q11.2 microdeletion exhibit a spectrum of cognitive deficits. Notably, ~30% of children with the 22q11.2 microdeletion will develop schizophrenia or schizoaffective disorder in adolescence or early adulthood. The genetic basis of the cognitive deficits and psychiatric symptoms is under intense scrutiny. Our own efforts to identify schizophrenia-susceptibility genes from this locus were based on the assumption that variants in 22q11 genes may modulate disease risk in the general (karyotypically normal) population and used large family samples followed up by studies in appropriately designed mouse models. Our studies on the PRODH gene, in particular, along with independent confirmatory work by other labs have provided compelling evidence that deficiency in the levels of PRODH (and the ensuing increase in L-proline levels) is an important contributor to the psychotic and possibly cognitive symptoms associated with the 22q11 microdeletions. Here, we propose to analyze in depth the effects of two additional biological processes that we discovered to be affected as a result of the 22q11 microdeletions, which are likely to affect neuronal development, synaptic plasticity and protein abundance of several brain expressed genes. Specifically, we propose to utilize three reliable mouse models recently generated in our labs and a series of sophisticated morphological, electrophysiological and behavioral approaches to facilitate a better understanding in vivo of how these two important physiological processes impaired by the 22q11 microdeletions, affect the function of hippocampus and prefrontal cortex, two brain regions implicated in the pathogenesis of schizophrenia and especially the cognitive endophenotypes associated with this disorder. Our proposed analysis promises to provide valuable insights on the ways the 22q11 locus increases the risk of schizophrenia and related impaired cognitive endophenotypes. Our proposed research will also provide well-characterized animal models that can be used to test further hypotheses and facilitate future drug development efforts. Indeed, a major motivation for this proposal is to exploit existing genetic knowledge to identify new drug targets that will improve currently available treatments. Microdeletions of the 22q11.2 locus are the highest known genetic risk factor for development of schizophrenia and related cognitive impairments. Our genetic studies thus far have allowed us to uncover a handful of genes that are likely to account for the observed increased risk. Since schizophrenia and related cognitive disorders affect a substantial proportion of the population and cause severe, long-term disability, it is imperative to pursue genetic research that will shed light on the causes and will allow meaningful intervention and development of successful treatments. The funding requested here will allow us to study in detail, in vivo in a real organism, the 2 novel pathophysiological mechanisms that we have uncovered thus far and generate pre-clinical mouse models that can be used for appropriately targeted drug development.

DESCRIPTION (provided by applicant): Genetic dissection of multigenic disorders, such as schizophrenia, is truly challenging yet possible, particularly with the availability of the human genome sequence. Genomewide linkage scans in schizophrenia have suggested that susceptibility loci may be present on several chromosomes. It is possible that in founder populations there will be fewer susceptibility loci and alleles. In addition, founder populations often exhibit less environmental heterogeneity than do other populations and allow detailed genealogical research and reconstruction of extended multigenerational pedigrees. Availability of such large pedigrees should facilitate the detection of linkage. We have collected a large number of families with schizophrenia from the genetically isolated population of the Afrikaners from South Africa. We performed a 10-cM genomewide scan on 143 small families, 34 of which were informative for linkage. Using both nonparametric and parametric linkage analyses, we obtained evidence for a small number of disease loci on chromosomes 1, 9, and 13. The locus on chromosome 1 reached genomewide significance levels and represents a novel susceptibility locus for schizophrenia. In addition to providing evidence for linkage for chromosome 1, we also identified a proband with a uniparental disomy (UPD) of the entire chromosome 1. This is the first time a UPD has been described in a patient with schizophrenia, lending further support to involvement of chromosome 1 in schizophrenia susceptibility in the Afrikaners. We propose to fine-map the chromosome 1p locus identified through the 10-cM genomewide scan of the Afrikaner family samples using microsatellite markers for a 1-cM coverage and SNPs for a denser coverage. We also propose to use genotypic information from the proband with the UPD to identify and define an Afrikaner risk haplotype on chromosome 1. In addition, we propose to perform a 10-cM genomewide scan in a second set of multiply affected Afrikaner families that we have collected to identify additional loci or strengthen the evidence and narrow the regions of involvement for already implicated loci. Finally, we propose to collect neurocognitive data from multiply affected Afrikaner families to use as quantitative endophenotypes of schizophrenia in a QTL linkage analysis.

DESCRIPTION (provided by applicant): The biological mechanisms responsible for psychiatric disorders are largely unknown. Given the limited success of identifying significant individual risk conferring genetic variants, it is believed that discovery of responsible epistatic interactions among multiple genetic variants reflecting molecular elements in complex pathways will elucidate novel disease mechanisms. We will perform our research aimed at discovering such mechanisms through collaboration between our computational and genetic laboratories. We will develop systems-based computational and visual tools to discover such interactions and apply them to publically available as well as in-house genome-wide association data for two diseases: schizophrenia and bipolar disorder. We will validate the statistical significance of our results and replicate them in silico on independent data. Our computational methodology will be designed to analyze both single nucleotide polymorphisms (SNPs) as well as copy number variations (CNVs) using quantitative measures of the synergy inherent in pairs of genetic variants indicating possible joint involvement in pathways. We will biologically interpret the resulting computational outputs and attempt to genetically validate the identified interactions. If the resulting biological hypotheses involving two genes are deemed promising, we will test those using in vitro neurobiological experiments. If enough evidence is accrued to support the possibility of biological epistasis, we will eventually generate transgenic animal models for either one or both genes in an interacting pair using genetic or pharmacological approaches when feasible, designed to confirm a biological interaction and dissect the underlying mechanistic basis. The relevance of our proposed research to public health is evidenced by its potential to enhance our understanding of etiological mechanisms responsible for schizophrenia and bipolar disorder. In turn, these discoveries will be helpful for the development of highly needed diagnostic and therapeutic methods for these diseases.

DESCRIPTION (provided by applicant): Challenge Area: 14 --Stem Cells Challenge Topic: 14-MH-101*, "Developing iPS cells for mental disorders" The recent description of somatic cell reprogramming to an embryonic stem (ES) cell-like phenotype, termed induced pluripotent stem (iPS) cell technology, presents an exciting venue toward cell-based models of disease mutations. This technology affords for the first time the potential to analyze neuronal cells grown from individuals carrying a given genetic lesion and offers a unique way of direct assessment of pathology. Recent studies have established an important role for rare copy number variants (CNVs, genomic deletions and duplications) in the etiology of schizophrenia and autism. Rare, but recurrent CNVs affecting the CNTNAP2 gene, a member of the neurexin family, have been described in both disorders. Given the high penetrance and clearly delineated genomic structure, which offers clear clues regarding the underlying functional deficit, this CNV is ideal to model at the cellular level, as well as at the level of the organism. We propose to generate iPS cells and iPS cell- derived forebrain neurons from schizophrenic patients carrying variable size CNTNAP2 gene deletions and their unaffected relatives and undertake an initial characterization of their basic morphological and electrophysiological properties. A unique aspect of our proposal is that the proposed comparisons will use as a reference point data acquired both in vitro and in vivo from hippocampal and cortical neurons from an animal model genetically engineered to carry a Cntnap2 gene deletion. Cell lines generated from individuals with schizophrenia carrying specific well-defined structural mutations offer an unprecedented opportunity to recapitulate pathologic human neural tissue formation in vitro and provide a unique platform for studies aimed at both providing valuable insights into the disease mechanisms, and the potential discovery of new compounds to treat this devastating disorder. PUBLIC HEALTH RELEVANCE: There is considerable promise in generating induced pluripotent stem (iPS) cell lines from patients afflicted with central nervous system diseases, including psychiatric disorders. We propose to generate neurons from patients with schizophrenia carrying CNTNAP2 deletions, a genetic risk factor for the disease, study their properties and compare them to brain neurons of a knock/out mouse model of the same gene. This is an unprecedented opportunity for the field to combine data from human neurons carrying a bona fide genetic risk factor for schizophrenia and an established mouse model of the same genetic lesion.

Abstract Recent advances in the field of psychiatric genetics have brought about excitement and urgency to envision and design intelligently the next steps in order to inform the neurobiology of the psychiatric disorders and develop strategies for prevention, early intervention, or treatment. The primary goal of the meeting on the Genetic and Neural Complexity in Psychiatry is to facilitate a discussion on the future of research in Psychiatry as it is shaped by the recent advances in genetics and newly emerging concepts and approaches in neuroscience. This meeting will bring together experts and open a dialogue between geneticists and neuroscientists allowing direct translation and speedy application of recent findings. The first planned meeting in the proposed series of 3 bi-annual meetings, will focus on deep sequencing approaches, which will allow discovery of rare, highly penetrant mutations, as well as on approaches designed to understand their impact on gene function, including the generation of mouse models. It will also address emerging reprogramming technologies that allow development of cellular models from patients carrying specific rare mutations as well as the pros and cons of studying additional animal species with particular emphasis on non-human primates. Finally, it will highlight and discuss novel approaches to decipher circuitry and communication in the disease brain.

DESCRIPTION (provided by applicant): We propose to undertake a multi-pronged human genetic analysis on the Afrikaners, a European origin founder population from South Africa, to identify genetic risk factors for both familial and non-familial (sporadic) forms of schizophrenia. Our work during the previous cycle of funding for this grant has provided three important insights in the genetic and biological basis of sporadic and familial forms of the disease: First, that at least 10 percent of sporadic cases of schizophrenia in the Afrikaner population can be accounted for by rare de novo CNVs; second, that a substantial portion of familial cases of schizophrenia can be accounted for by rare inherited CNVs enriched in deletions and duplications of genic regions; and third that a portion of Afrikaner families with schizophrenia carry one or more disease risk genes located at chromosome 13q34. Our work during the last funding period of this award represents one of the first family-based studies to explore the important question of how CNVs contribute to the known inheritance patterns of schizophrenia and dictates the research direction of this project for the next funding cycle. Specifically we plan to (i) undertake a new, high- density genome-wide scan for CNVs using large cohorts of familial and sporadic schizophrenia, as well as control cohorts; (ii) pursue sequencing based approaches to determine whether the total number of potentially damaging mutations or the number of de novo mutations in genes affected by rare CNVs is significantly higher in the cases versus controls; (iii) undertake a comprehensive effort based on two complementary approaches to probe the genetic architecture of the 13q34 schizophrenia susceptibility locus with a final goal to identify one or more common or rare variants accounting for the linkage signal observed in this region. The advantage of doing this research in the unique Afrikaner population cannot be overemphasized. In addition to being an ethnically homogeneous population derived from a small number of founders, the family structure, the potential for genealogical research and the availability of detailed psychiatric records over several generations facilitates family-based studies of schizophrenia and high-confidence distinction of sporadic and familial cases. This project holds tremendous promise to shed light into the genetic etiologies of schizophrenia, especially in the context of newly established technologies that allow efficient screens of rare mutations. PUBLIC HEALTH RELEVANCE: Our recent work has provided important insights into the genetic and biological basis of both familial and non- familial forms of schizophrenia, providing evidence that rare genomic copy number variants (CNVs) play an important role in the genetic risk of both forms of the disease. We propose to pursue further work to characterize the full spectrum of CNVs in schizophrenia and inform the genetic architecture of the disease more fully. We also propose to move beyond the CNV data and characterize in a comprehensive manner (using next generation sequencing approaches) genes located in identified disease risk loci, for additional RARE mutations that affect the function or expression of these genes.

DESCRIPTION (provided by applicant): It is now evident that many cases of psychiatric and neurodevelopmental disorders, as well as disorders of cognitive function, are due to highly penetrant, rare genetic variants. 22q11.2 deletion is a prominent example of such a variant. Carriers of deletions in chromosome 22q11.2, which predominantly occur de novo, exhibit a spectrum of cognitive deficits and develop schizophrenia in adolescence or early adulthood at a rate of 25-30%. Recurrent 22q11.2 deletions account for as many as 1-2% of cases of sporadic schizophrenia in the general population. Because of its leading role in the genetic landscape of psychiatric disease and cognitive dysfunction, functional analysis of the 22q1.2 deletion holds great promise for providing the biological insights necessary for development of new treatments for these conditions. In this project, we propose to study the impact of 22q11.2 deletions on neuronal structure and function in exquisite depth. Our proposed research focuses on this highly significant problem using state-of-the-art techniques, reliable animal models, and patient- derived neurons, and is designed to improve our understanding of the chain of events leading from the mutation, through its effects on neural cells and circuits, to clinical phenotype and inform the development of new therapeutics. A major aspect of our effort will be to implement carefully controlled translational paradigms to test many of the alterations that we find in mouse models in neurons from patients. Along these lines, we propose to pursue detailed comparative studies between human and mouse, using cortical neurons derived from induced pluripotent stem cells (iPSCs) from humans carrying the 22q11.2 deletion. The strength of this approach is undeniable since it will allow in-depth analysis at the level of the individual neuron and synapse, which are otherwise inaccessible in patients.