Analysis of Shared Haplotypes amongst Palauans Maps Loci for Psychotic Disorders to 4q28 and 5q23-q31.

To localize genetic variation affecting risk for psychotic disorders in the population of Palau, we genotyped DNA samples from 203 Palauan individuals diagnosed with psychotic disorders, broadly defined, and 125 control subjects using a genome-wide single nucleotide polymorphism array. Palau has unique features advantageous for this study: due to its population history, Palauans are substantially interrelated; affected individuals often, but not always, cluster in families; and we have essentially complete ascertainment of affected individuals. To localize risk variants to genomic regions, we evaluated long-shared haplotypes, ≥10 Mb, identifying clusters of affected individuals who share such haplotypes. This extensive sharing, typically identical by descent, was significantly greater in cases than population controls, even after controlling for relatedness. Several regions of the genome exhibited substantial excess of shared haplotypes for affected individuals, including 3p21, 3p12, 4q28, and 5q23-q31. Two of these regions, 4q28 and 5q23-q31, showed significant linkage by traditional LOD score analysis and could harbor variants of more sizeable risk for psychosis or a multiplicity of risk variants. The pattern of haplotype sharing in 4q28 highlights PCDH10, encoding a cadherin-related neuronal receptor, as possibly involved in risk.

Characterization of a Novel Mutation in SLC1A1 Associated with Schizophrenia.

We have recently described a hemi-deletion on chromosome 9p24.2 at the SLC1A1 gene locus and its co-segregation with schizophrenia in an extended Palauan pedigree. This finding represents a point of convergence for several pathophysiological models of schizophrenia. The present report sought to characterize the biological consequences of this hemi-deletion. Dual luciferase assays demonstrated that the partially deleted allele (lacking exon 1 and the native promoter) can drive expression of a 5'-truncated SLC1A1 using sequence upstream of exon 2 as a surrogate promoter. However, confocal microscopy and electrophysiological recordings demonstrate that the 5'-truncated SLC1A1 lacks normal membrane localization and glutamate transport ability. To identify downstream consequences of the hemi-deletion, we first used a themed qRT-PCR array to compare expression of 84 GABA and glutamate genes in RNA from peripheral blood leukocytes in deletion carriers (n = 11) versus noncarriers (n = 8) as well as deletion carriers with psychosis (n = 5) versus those without (n = 3). Then, targeted RNA-Seq (TREx) was used to quantify expression of 375 genes associated with neuropsychiatric disorders in HEK293 cells subjected to either knockdown of SLC1A1 or overexpression of full-length or 5'-truncated SLC1A1. Expression changes of several genes strongly implicated in schizophrenia pathophysiology were detected (e.g. SLC1A2, SLC1A3, SLC1A6, SLC7A11, GRIN2A, GRIA1 and DLX1).

Characterizing runs of homozygosity and their impact on risk for psychosis in a population isolate.

An increased abundance of runs of homozygosity (ROH) has been associated with risk for various diseases, including schizophrenia. Here we investigate the characteristics of ROH in Palau, an Oceanic population, evaluating whether these characteristics are related to risk for psychotic disorders and the nature of this association. To accomplish these aims we evaluate a sample of 203 cases with schizophrenia and related psychotic disorders-representing almost complete ascertainment of affected individuals in the population-and contrast their ROH to that of 125 subjects chosen to function as controls. While Palauan diagnosed with psychotic disorders tend to have slightly more ROH regions than controls, the distinguishing features are that they have longer ROH regions, greater total length of ROH, and their ROH tends to co-occur more often at the same locus. The nature of the sample allows us to investigate whether rare, highly penetrant recessive variants generate such case-control differences in ROH. Neither rare, highly penetrant recessive variants nor individual common variants of large effect account for a substantial proportion of risk for psychosis in Palau. These results suggest a more nuanced model for risk is required to explain patterns of ROH for this population.

Deletion at the SLC1A1 glutamate transporter gene co-segregates with schizophrenia and bipolar schizoaffective disorder in a 5-generation family.

Growing evidence for genetic overlap between schizophrenia (SCZ) and bipolar disorder (BPD) suggests that causal variants of large effect on disease risk may cross traditional diagnostic boundaries. Extended multigenerational families with both SCZ and BPD cases can be a valuable resource for discovery of shared biological pathways because they can reveal the natural evolution of the underlying genetic disruptions and their phenotypic expression. We investigated a deletion at the SLC1A1 glutamate transporter gene originally identified as a copy number variant exclusively carried by members of a 5-generation Palauan family. Using an expanded sample of 21 family members, quantitative PCR confirmed the deletion in all seven individuals with psychosis, three "obligate-carrier" parents and one unaffected sibling, while four marry-in parents were non-carriers. Linkage analysis under an autosomal dominant model generated a LOD-score of 3.64, confirming co-segregation of the deletion with psychosis. For more precise localization, we determined the approximate deletion end points using alignment of next-generation sequencing data for one affected deletion-carrier and then designed PCR amplicons to span the entire deletion locus. These probes established that the deletion spans 84,298 bp, thus eliminating the entire promoter, the transcription start site, and the first 59 amino acids of the protein, including the first transmembrane Na(2+)/dicarboxylate symporter domain, one of the domains that perform the glutamate transport action. Discovery of this functionally relevant SLC1A1 mutation and its co-segregation with psychosis in an extended multigenerational pedigree provides further support for the important role played by glutamatergic transmission in the pathophysiology of psychotic disorders.

Copy number variants for schizophrenia and related psychotic disorders in Oceanic Palau: risk and transmission in extended pedigrees.

BACKGROUND: We report on copy number variants (CNVs) found in Palauan subjects ascertained for schizophrenia and related psychotic disorders in extended pedigrees in Palau. We compare CNVs found in this Oceanic population with those seen in other samples, typically of European ancestry. Assessing CNVs in Palauan extended pedigrees yields insight into the evolution of risk CNVs, such as how they arise, are transmitted, and are lost from populations by stochastic or selective processes, none of which are easily measured from case-control samples. METHODS: DNA samples from 197 subjects affected with schizophrenia and related psychotic disorders, 185 of their relatives, and 159 control subjects were successfully characterized for CNVs using Affymetrix Genomewide Human SNP Array 5.0. RESULTS: Copy number variants thought to be associated with risk for schizophrenia and related disorders also occur in affected individuals in Palau, specifically 15q11.2 and 1q21.1 deletions, partial duplication of IL1RAPL1 (Xp21.3), and chromosome X duplications (Klinefelter's syndrome). Partial duplication within A2BP1 appears to convey an eightfold increased risk in male subjects (95% confidence interval, .8-84.4) but not female subjects (odds ratio = .4, 95% confidence interval, .03-4.9). Affected-only linkage analysis using this variant yields a logarithm of the odds score of 3.5. CONCLUSIONS: This study reveals CNVs that confer risk to schizophrenia and related psychotic disorders in Palau, most of which have been previously observed in samples of European ancestry. Only a few of these CNVs show evidence that they have existed for many generations, consistent with risk variants diminishing reproductive success.

Familial transmission of schizophrenia in Palau: A 20-year genetic epidemiological study in three generations.

Our genetic epidemiological studies of schizophrenia and other psychotic disorders (SCZ) in the isolated population of Palau have been ongoing for 20 years. Results from the first decade showed that Palau has an elevated prevalence of SCZ and that cases cluster in extended multigenerational pedigrees interconnected via complex genetic relationships after centuries of endogamous, but not consanguineous, marriages. The aim of our second decade of research, which extended data collection into a third generation of young, high-risk (HR) Palauans, was to identify significant predictors of intergenerational transmission of illness. Our findings revealed that degree of familial loading and gender effects on reproductive fitness are important modifiers of risk for transmission of SCZ. Among 45 distinct multiplex families, we identified 10 high-density (HD) Palauan families, each with 7-29 SCZ cases, which contain half of Palau's 260 SCZ cases and 80% of the 113 SCZ cases with one or more affected first-degree relatives, indicating that familial loading is a major risk factor for SCZ in Palau. Cases that belong to multiply affected sibships are more common than cases with an affected parent. Furthermore, only 6/38 multiply affected sibships have an affected parent, strong evidence that many unaffected parents are obligate carriers of susceptibility genes. Although reproductive fitness is dramatically reduced in affected males, the 30% minority who do become fathers are twice as likely as affected mothers to transmit SCZ to an offspring. As they evolve, these HD families can help to elucidate the genetic mechanisms that predict intergenerational transmission of SCZ.

Early signs and symptoms of psychosis among Palauan adolescents.

AIM: This study was designed to identify early symptoms associated with the occurrence of psychosis during adolescence. METHOD: Participants were recruited in the Republic of Palau, an isolated island nation in Micronesia with a prevalence rate for schizophrenia of 1.99%. Diagnostic interviews were used to obtain reports of early and current symptoms from 112 genetically high-risk (GHR) and 208 genetically low-risk (GLR) adolescents (ages 16-23). Based on current psychotic symptoms, participants were sorted into three groups: non-clinical, at-risk/symptomatic risk and clinically symptomatic. RESULTS: Multivariate analysis of variance revealed several between-group differences on rates of early symptoms. Most notably, youth who were in the GHR-clinically symptomatic group reported significantly higher rates of early marijuana use than GLR-clinically symptomatic youth, who were significantly more likely to report early symptoms of depression and behaviour disorders. In addition, several gender based differences in the link between early symptoms and adolescent onset psychosis were noted. CONCLUSIONS: Findings are generally consistent with previous research on early indicators, though several unexpected findings suggest that results from this study may not be fully generalizable beyond this relatively isolated and culturally distinct Micronesian nation.

Adoption, family relations and psychotic symptoms among Palauan adolescents who are genetically at risk for developing schizophrenia.

PURPOSE: This paper focuses on the role of adoption and family relations as moderators of genetic risk for psychotic disorders. METHODS: Participants included 184 adolescents in the Republic of Palau identified to be at genetic risk for schizophrenia and other psychotic disorders. Palau is an island nation in Micronesia with a lifetime prevalence of 1.99% for schizophrenia and 2.67% for psychotic disorders more broadly defined. In Palauan culture, kinship adoption is a common cultural practice; 47 of the 184 participants had been adopted at an early age. The current study was designed to test the hypothesis that adoption would function as a protective factor among Palauan youth at genetic risk for the development of psychotic symptoms. Participants were evaluated for psychotic and other psychiatric symptoms using KSADS-PL. Concurrently, the Youth Self Report was used to assess the perceived quality of family relationships. RESULTS: Results indicated that adopted adolescents were more likely to develop psychotic symptoms than non-adopted adolescents. However, perceived family relations moderated the association between adoption status and psychotic symptoms, such that adopted adolescents with poorer family relations reported disproportionately higher rates of psychotic symptoms. Family relations also moderated the association between level of genetic risk and psychotic symptoms, independently of adoption status. CONCLUSION: Consistent with previous research, adolescents at high genetic risk who reported more positive family relations also reported fewer psychotic symptoms.

Genetic liability to schizophrenia in Oceanic Palau: a search in the affected and maternal generation.

While liability to schizophrenia (Scz) is due to genetic and environmental factors, specific factors are largely unknown. We postulate a two-hit model for Scz, in which initial liability is generated during fetal brain development: this "hit" is precipitated by environmental stressors biologically interacting with maternal genetic vulnerability to the stress. Additional liability to Scz is generated by individual genetic vulnerability. To evaluate these putative levels of vulnerability, we search in the genome of both affected individuals and their mothers for variation that differs, statistically, from that in the general population. For parental analyses, mothers were treated as "affected," rather than their offspring, and the fathers were treated as "controls". We used a sample from the Palauan population: 175 individuals diagnosed with Scz, broadly defined; 87 mothers and 45 fathers of affected individuals. Pedigree and diagnostic data were available on 2,953 living and deceased subjects. DNA from 553 individuals was genotyped for short tandem repeats (STR) spaced approximately every 10 cM across the genome. We tested for association between affection status and STR alleles; such an approach was reasonable, despite the widely spaced markers, because this population has far-ranging linkage disequilibrium (LD). Results for the truly affected individuals were modest, whereas results from the maternal generation were promising. For a recessive model and a test for excess allele matching across mothers, significant findings occurred for D20S481, D10S1221, D6S1021, D13S317, and D18S976. Regions in which at least two adjacent markers produced substantial association statistics include 2p12-11.2, 2q24.1-32.1, 6q12-14.1, 10q23.2-24.21, 12q23.2-24.21 and 17q23.2-23.3.

Recurrence risk to offspring in extended multiplex schizophrenia pedigrees from a Pacific Island isolate.

Genetic transmission plays a major role in the pathogenesis of schizophrenia. Family, twin, and adoption studies have consistently shown that risks in relatives are many times greater than the general population risk of approximately 1%. McGue, Gottesman, and Rao (1983; Am J Hum Genet 35:1161-1178) calculated risk estimates of 12.8% for offspring and 3.5% for nieces/nephews of schizophrenia patients based on a large data set of Western European families. The present study evaluated corresponding risk levels in Palau, an isolated population in Micronesia where the prevalence of narrowly (broadly) defined schizophrenia is 1.99% (2.67%) and cases cluster in extended pedigrees, 20 of which contain 80% of affected individuals. We hypothesized that offspring in these extended families would have a higher risk for schizophrenia than offspring in smaller schizophrenia pedigrees from more genetically heterogeneous populations. RDC diagnostic data based on complete ascertainment of cases and their families covering the past two generations were used to quantify empirical recurrence risks in the offspring and nieces/nephews of Palauan schizophrenia patients. Risks to 1st- and 2nd-degree offspring were approximately double the rates found in the smaller Western European families: 23.4% in the offspring of an affected parent, 6.4% in offspring with one affected aunt/uncle, and 15.0% in offspring with two or more affected aunts/uncles. Recurrence rates in offspring of an affected parent were 1.6 times higher in males (27.9%) than in females (17.7%). The high risk levels we found in Palauan offspring reflect the elevated population prevalence, strong familial aggregation, and multi-lineal transmission pattern of schizophrenia in Palau.

Linkage analysis of a completely ascertained sample of familial schizophrenics and bipolars from Palau, Micronesia.

We report on linkage analysis of a completely ascertained population of familial psychosis derived from the oceanic nation of Palau. Palau, an archipelago of islands in the Southern Pacific, currently has a population of approximately 23,000 individuals. The peoples of Palau populated these islands recently in human history, approximately 2,000 years ago. As both historical and genetic evidence suggest, the population is far more homogeneous than most other populations undergoing genetic studies, and should therefore prove quite useful for mapping genetic variants having a meaningful impact on susceptibility to psychotic disorders. Moreover, for our study, essentially all on-island schizophrenics (150) and individuals with other psychotic disorders (25) participated. By analysis of narrow (only schizophrenia) and broad (all psychosis) diagnostic schemes, two-point linkage analyses suggest that two regions of the genome harbor genetic variants affecting liability in most families, 3q28 (LOD = 3.03) and 17q32.2 (LOD = 2.80). Results from individual pedigrees also support 2q37.2, 2p14, and 17p13 as potentially harboring important genetic variants. Most of these regions have been implicated in other genetic studies of psychosis in populations physically quite distant from this Oceanic population, although some (e.g., 3q28) appear to be novel results for schizophrenia linkage analyses.