High rate of disease-related copy number variations in childhood onset schizophrenia.

Copy number variants (CNVs) are risk factors in neurodevelopmental disorders, including autism, epilepsy, intellectual disability (ID) and schizophrenia. Childhood onset schizophrenia (COS), defined as onset before the age of 13 years, is a rare and severe form of the disorder, with more striking array of prepsychotic developmental disorders and abnormalities in brain development. Because of the well-known phenotypic variability associated with pathogenic CNVs, we conducted whole genome genotyping to detect CNVs and then focused on a group of 46 rare CNVs that had well-documented risk for adult onset schizophrenia (AOS), autism, epilepsy and/or ID. We evaluated 126 COS probands, 69 of which also had a healthy full sibling. When COS probands were compared with their matched related controls, significantly more affected individuals carried disease-related CNVs (P=0.017). Moreover, COS probands showed a higher rate than that found in AOS probands (P<0.0001). A total of 15 (11.9%) subjects exhibited at least one such CNV and four of these subjects (26.7%) had two. Five of 15 (4.0% of the sample) had a 2.5-3 Mb deletion mapping to 22q11.2, a rate higher than that reported for adult onset (0.3-1%) (P<0.001) or autism spectrum disorder and, indeed, the highest rate reported for any clinical population to date. For one COS subject, a duplication found at 22q13.3 had previously only been associated with autism, and for four patients CNVs at 8q11.2, 10q22.3, 16p11.2 and 17q21.3 had only previously been associated with ID. Taken together, these findings support the well-known pleiotropic effects of these CNVs suggesting shared abnormalities early in brain development. Clinically, broad CNV-based population screening is needed to assess their overall clinical burden.

Molecular genetic evidence for overlap between general cognitive ability and risk for schizophrenia: a report from the Cognitive Genomics consorTium (COGENT).

It has long been recognized that generalized deficits in cognitive ability represent a core component of schizophrenia (SCZ), evident before full illness onset and independent of medication. The possibility of genetic overlap between risk for SCZ and cognitive phenotypes has been suggested by the presence of cognitive deficits in first-degree relatives of patients with SCZ; however, until recently, molecular genetic approaches to test this overlap have been lacking. Within the last few years, large-scale genome-wide association studies (GWAS) of SCZ have demonstrated that a substantial proportion of the heritability of the disorder is explained by a polygenic component consisting of many common single-nucleotide polymorphisms (SNPs) of extremely small effect. Similar results have been reported in GWAS of general cognitive ability. The primary aim of the present study is to provide the first molecular genetic test of the classic endophenotype hypothesis, which states that alleles associated with reduced cognitive ability should also serve to increase risk for SCZ. We tested the endophenotype hypothesis by applying polygenic SNP scores derived from a large-scale cognitive GWAS meta-analysis (~5000 individuals from nine nonclinical cohorts comprising the Cognitive Genomics consorTium (COGENT)) to four SCZ case-control cohorts. As predicted, cases had significantly lower cognitive polygenic scores compared to controls. In parallel, polygenic risk scores for SCZ were associated with lower general cognitive ability. In addition, using our large cognitive meta-analytic data set, we identified nominally significant cognitive associations for several SNPs that have previously been robustly associated with SCZ susceptibility. Results provide molecular confirmation of the genetic overlap between SCZ and general cognitive ability, and may provide additional insight into pathophysiology of the disorder.

Experimental strategies for the genetic dissection of complex traits in animal models.

Current success in detecting complex trait loci in general, and quantitative trait loci (QTLs) using model organisms in particular, has attracted major biological and biomedical interest. The potential ability to identify genes and their function provides opportunities for new diagnostics and treatments of complex genetic diseases. Despite the success in gene mapping, however, cloning of complex trait loci or QTLs is not straightforward. A major obstacle lies in achieving fine mapping resolution for the detected loci. Compared to the rapid development of sophisticated statistical and molecular tools, development and analysis of experimental designs for various stages in QTL mapping experiments have barely been considered. In this study, novel and existing experimental strategies for QTL analysis are presented and evaluated.

Localization of genes controlling resistance to trypanosomiasis in mice.

Tsetse fly-transmitted trypanosomes (Trypanosoma spp.) cause "sleeping sickness' in man and have a serious impact on livestock-based agriculture in large areas of Africa. Multigene control of variation in susceptibility to trypanosomiasis is known to occur in mice, where the C57BI/6 (B6) strain is relatively resistant and the A/J (A) and Balb/c (B) strains are susceptible. Such resistance is also well described among several types of west African cattle. We report here the results of genome-wide scans for genes controlling this trait in the B6 mouse using crosses with two different susceptible strains. Regions on mouse chromosomes 5 and 17 were found to be important in determining resistance in both crosses while an additional region on chromosome 1 showed evidence of involvement in only one cross. We confirmed the size of the effect due to chromosome 17 in F3 intercross populations fixed for alternative parental chromosomes. The three loci are of large effect and account for most of the genetic variation in both F2 populations. We propose that they be designated Tir1, Tir2 and Tir3.

GWA study data mining and independent replication identify cardiomyopathy-associated 5 (CMYA5) as a risk gene for schizophrenia.

We conducted data-mining analyses using the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) and molecular genetics of schizophrenia genome-wide association study supported by the genetic association information network (MGS-GAIN) schizophrenia data sets and performed bioinformatic prioritization for all the markers with P-values ≤0.05 in both data sets. In this process, we found that in the CMYA5 gene, there were two non-synonymous markers, rs3828611 and rs10043986, showing nominal significance in both the CATIE and MGS-GAIN samples. In a combined analysis of both the CATIE and MGS-GAIN samples, rs4704591 was identified as the most significant marker in the gene. Linkage disequilibrium analyses indicated that these markers were in low LD (3 828 611-rs10043986, r(2)=0.008; rs10043986-rs4704591, r(2)=0.204). In addition, CMYA5 was reported to be physically interacting with the DTNBP1 gene, a promising candidate for schizophrenia, suggesting that CMYA5 may be involved in the same biological pathway and process. On the basis of this information, we performed replication studies for these three single-nucleotide polymorphisms. The rs3828611 was found to have conflicting results in our Irish samples and was dropped out without further investigation. The other two markers were verified in 23 other independent data sets. In a meta-analysis of all 23 replication samples (family samples, 912 families with 4160 subjects; case-control samples, 11 380 cases and 15 021 controls), we found that both markers are significantly associated with schizophrenia (rs10043986, odds ratio (OR)=1.11, 95% confidence interval (CI)=1.04-1.18, P=8.2 × 10(-4) and rs4704591, OR=1.07, 95% CI=1.03-1.11, P=3.0 × 10(-4)). The results were also significant for the 22 Caucasian replication samples (rs10043986, OR=1.11, 95% CI=1.03-1.17, P=0.0026 and rs4704591, OR=1.07, 95% CI=1.02-1.11, P=0.0015). Furthermore, haplotype conditioned analyses indicated that the association signals observed at these two markers are independent. On the basis of these results, we concluded that CMYA5 is associated with schizophrenia and further investigation of the gene is warranted.

Genetic architecture of prostate cancer in the Ashkenazi Jewish population.

BACKGROUND: Recently, numerous prostate cancer risk loci have been identified, some of which show association in specific populations. No study has yet investigated whether these single nucleotide polymorphisms (SNPs) are associated with prostate cancer in the Ashkenazi Jewish (AJ) population. METHODS: A total of 29 known prostate cancer risk SNPs were genotyped in 963 prostate cancer cases and 613 controls of AJ ancestry. These data were combined with data from 1241 additional Ashkenazi controls and tested for association with prostate cancer. Correction for multiple testing was performed using the false discovery rate procedure. RESULTS: Ten of twenty-three SNPs that passed quality control procedures were associated with prostate cancer risk at a false discovery rate of 5%. Of these, nine were originally discovered in studies of individuals of European ancestry. Based on power calculations, the number of significant associations observed is not surprising. CONCLUSION: We see no convincing evidence that the genetic architecture of prostate cancer in the AJ population is substantively different from that observed in other populations of European ancestry.

Analysis of 10 independent samples provides evidence for association between schizophrenia and a SNP flanking fibroblast growth factor receptor 2.

We and others have previously reported linkage to schizophrenia on chromosome 10q25-q26 but, to date, a susceptibility gene in the region has not been identified. We examined data from 3606 single-nucleotide polymorphisms (SNPs) mapping to 10q25-q26 that had been typed in a genome-wide association study (GWAS) of schizophrenia (479 UK cases/2937 controls). SNPs with P<0.01 (n=40) were genotyped in an additional 163 UK cases and those markers that remained nominally significant at P<0.01 (n=22) were genotyped in replication samples from Ireland, Germany and Bulgaria consisting of a total of 1664 cases with schizophrenia and 3541 controls. Only one SNP, rs17101921, was nominally significant after meta-analyses across the replication samples and this was genotyped in an additional six samples from the United States/Australia, Germany, China, Japan, Israel and Sweden (n=5142 cases/6561 controls). Across all replication samples, the allele at rs17101921 that was associated in the GWAS showed evidence for association independent of the original data (OR 1.17 (95% CI 1.06-1.29), P=0.0009). The SNP maps 85 kb from the nearest gene encoding fibroblast growth factor receptor 2 (FGFR2) making this a potential susceptibility gene for schizophrenia.

Association between the oxytocin receptor (OXTR) gene and autism: relationship to Vineland Adaptive Behavior Scales and cognition.

Evidence both from animal and human studies suggests that common polymorphisms in the oxytocin receptor (OXTR) gene are likely candidates to confer risk for autism spectrum disorders (ASD). In lower mammals, oxytocin is important in a wide range of social behaviors, and recent human studies have shown that administration of oxytocin modulates behavior in both clinical and non-clinical groups. Additionally, two linkage studies and two recent association investigations also underscore a possible role for the OXTR gene in predisposing to ASD. We undertook a comprehensive study of all 18 tagged SNPs across the entire OXTR gene region identified using HapMap data and the Haploview algorithm. Altogether 152 subjects diagnosed with ASDs (that is, DSM IV autistic disorder or pervasive developmental disorder--NOS) from 133 families were genotyped (parents and affected siblings). Both individual SNPs and haplotypes were tested for association using family-based association tests as provided in the UNPHASED set of programs. Significant association with single SNPs and haplotypes (global P-values <0.05, following permutation test adjustment) were observed with ASD. Association was also observed with IQ and the Vineland Adaptive Behavior Scales (VABS). In particular, a five-locus haplotype block (rs237897-rs13316193-rs237889-rs2254298-rs2268494) was significantly associated with ASD (nominal global P=0.000019; adjusted global P=0.009) and a single haplotype (carried by 7% of the population) within that block showed highly significant association (P=0.00005). This is the third association study, in a third ethnic group, showing that SNPs and haplotypes in the OXTR gene confer risk for ASD. The current investigation also shows association with IQ and total VABS scores (as well as the communication, daily living skills and socialization subdomains), suggesting that this gene shapes both cognition and daily living skills that may cross diagnostic boundaries.

Replication delay along FRA7H, a common fragile site on human chromosome 7, leads to chromosomal instability.

Common fragile sites are specific chromosomal loci that show gaps, breaks, or rearrangements in metaphase chromosomes under conditions that interfere with DNA replication. The mechanism underlying the chromosomal instability at fragile sites was hypothesized to associate with late replication time. Here, we aimed to investigate the replication pattern of the common fragile site FRA7H, encompassing 160 kb on the long arm of human chromosome 7. Using in situ hybridization on interphase nuclei, we revealed that the replication of this region is initiated relatively early, before 30% of S phase is completed. However, a high fraction ( approximately 35%) of S-phase nuclei showed allelic asynchrony, indicating that the replication of FRA7H is accomplished at different times in S phase. This allelic asynchrony is not the result of a specific replication time of each FRA7H allele. Analysis of the replication pattern of adjacent clones along FRA7H by using cell population and two-color fluorescent in situ hybridization analyses showed significant differences in the replication of adjacent clones, under normal growth condition and upon aphidicolin treatment. This pattern significantly differed from that of two nonfragile regions which showed a coordinated replication under both conditions. These results indicate that aphidicolin is enhancing an already existing difference in the replication time along the FRA7H region. Based on our replication analysis of FRA7H and on previous analysis of the common fragile site FRA3B, we suggest that delayed replication is underlying the fragility at aphidicolin-induced common fragile sites.

Advanced intercross lines, an experimental population for fine genetic mapping.

An advanced intercrossed line (AIL) is an experimental population that can provide more accurate estimates of quantitative trait loci (QTL) map location than conventional mapping populations. An AIL is produced by randomly and sequentially intercrossing a population that initially originated from a cross between two inbred lines or some variant thereof. This provides increasing probability of recombination between any two loci. Consequently, the genetic length of the entire genome is stretched, providing increased mapping resolution. In this way, for example, with the same population size and QTL effect, a 95% confidence interval of QTL map location of 20 cM in the F2 is reduced fivefold after eight additional random mating generations (F10). Simulation results showed that to obtain the anticipated reduction in the confidence interval, breeding population size of the AIL in all generations should comprise an effective number of > or = 100 individuals. It is proposed that AILs derived from crosses between known inbred lines may be a useful resource for fine genetic mapping.

Selective DNA pooling for determination of linkage between a molecular marker and a quantitative trait locus.

Selective genotyping is a method to reduce costs in marker-quantitative trait locus (QTL) linkage determination by genotyping only those individuals with extreme, and hence most informative, quantitative trait values. The DNA pooling strategy (termed: "selective DNA pooling") takes this one step further by pooling DNA from the selected individuals at each of the two phenotypic extremes, and basing the test for linkage on marker allele frequencies as estimated from the pooled samples only. This can reduce genotyping costs of marker-QTL linkage determination by up to two orders of magnitude. Theoretical analysis of selective DNA pooling shows that for experiments involving backcross, F2 and half-sib designs, the power of selective DNA pooling for detecting genes with large effect, can be the same as that obtained by individual selective genotyping. Power for detecting genes with small effect, however, was found to decrease strongly with increase in the technical error of estimating allele frequencies in the pooled samples. The effect of technical error, however, can be markedly reduced by replication of technical procedures. It is also shown that a proportion selected of 0.1 at each tail will be appropriate for a wide range of experimental conditions.

Detecting marker-QTL linkage and estimating QTL gene effect and map location using a saturated genetic map.

A simulation study was carried out on a backcross population in order to determine the effect of marker spacing, gene effect and population size on the power of marker-quantitative trait loci (QTL) linkage experiments and on the standard error of maximum likelihood estimates (MLE) of QTL gene effect and map location. Power of detecting a QTL was virtually the same for a marker spacing of 10 cM as for an infinite number of markers and was only slightly decreased for marker spacing of 20 or even 50 cM. The advantage of using interval mapping as compared to single-marker analysis was slight. "Resolving power" of a marker-QTL linkage experiment was defined as the 95% confidence interval for the QTL map location that would be obtained when scoring an infinite number of markers. It was found that reducing marker spacing below the resolving power did not add appreciably to narrowing the confidence interval. Thus, the 95% confidence interval with infinite markers sets the useful marker spacing for estimating QTL map location for a given population size and estimated gene effect.

Inheritance and mapping of Compact (Cmpt), a new mutation causing hypermuscularity in mice.

During selection for protein content in mice at the Technical University of Berlin, individuals showing high protein content and a compact exterior were noted. Animals showing this "Compact" phenotype were separated to form a new line. The present investigations were carried out on a Hungarian subpopulation of this line, selected for maximum expression of the Compact phenotype, and apparently at fixation for the relevant genes. Fertility and viability of the Compact subpopulation was normal. As compared to normal mice, carcass percentage values for male and female Compact mice were 9.4 and 6.8% greater, respectively; and the muscle:bone weight ratio in males was 1.61-fold greater. The Compact phenotype showed variable expressivity and was of intermediate dominance in males, but almost fully recessive in females. The hypothesis that a single gene is solely responsible for the Compact phenotype was rejected by maximum likelihood analysis. Linkage mapping using selective DNA pooling located a single locus (denoted Cmpt) strongly associated with the Compact phenotype on mouse chromosome 1. Fine mapping, using individual selective genotyping and haplotype analysis, located Cmpt to the region between D1Mit375 and D1Mit21, approximately one third of the way to D1Mit21.

Quantitative trait locus mapping in dairy cattle by means of selective milk DNA pooling using dinucleotide microsatellite markers: analysis of milk protein percentage.

"Selective DNA pooling" accomplishes quantitative trait locus (QTL) mapping through densitometric estimates of marker allele frequencies in pooled DNA samples of phenotypically extreme individuals. With poly(TG) microsatellites, such estimates are confounded by "shadow" ("stutter") bands. A correction procedure was developed on the basis of an observed linear regression between shadow band intensity and allele TG repeat number. Using this procedure, a selective DNA pooling study with respect to milk protein percentage was implemented in Israel-Holstein dairy cattle. Pools were prepared from milk samples of high and low daughters of each of seven sires and genotyped with respect to 11 markers. Highly significant associations with milk protein percentage were found for 5 of the markers; 4 of these markers confirmed previous reports. Selective DNA pooling accessed 80.6 and 48.3%, respectively, of the information that would have been available through individual selective genotyping or total population genotyping. In effect, the statistical power of 45,600 individual genotypings was obtained from 328 pool genotypings. This methodology can make genome-wide mapping of QTL accessible to moderately sized breeding organizations.

Deletion and insertion mutations in short tandem repeats in the coding regions of human genes.

In vitro studies in bacterial, yeast and eukaryotic systems have demonstrated the existence of deletion and insertion 'hot-spots' involving repetitive sequences. Slipped-strand mispairing (SSM) has been suggested to be the mechanism involved. Progress in human molecular genetics has allowed the identification of many mutations causing diseases. Analysis of sequences involved in these mutations provides an opportunity to investigate the contribution of short tandem repeats to the naturally occurring mutations in coding regions of human genes. We have analyzed the sequences surrounding 625 disease-causing mutations in the coding regions of three genes: the cystic fibrosis transmembrane conductance regulator, beta globin and factor IX. Altogether, 134 (21%) insertion and deletion mutations of 4 base pairs or less were identified. In 47% of these mutations, the deletions and insertions occurred within a unit repeated tandemly 2- to 7-fold. These were classified as SSM mutations. The proportion of SSM mutations was significantly higher than expected by chance. The estimated net proportion of deletion and insertion mutations attributed to SSM was 27%. These results indicate that very short repetitive sequences contribute significantly to the generation of deletion and insertion mutations in human genes, and to the evolution of diversity of their coding regions.

Difference in frequencies of the cystic fibrosis alleles, delta F508 and W1282X, between carriers and patients.

One major mutation, delta F508, causing cystic fibrosis (CF) is found in most populations around the world. Among CF patients of Jewish Ashkenazi origin two major mutations, W1282X and delta F508 were found. We compared the relative frequencies of the two major mutations found in this patient population to their relative frequencies in the healthy population. The studied patient population included the entire CF Jewish Ashkenazi patient population in Israel (238 chromosomes), and a small group of Jewish Ashkenazi patients in the USA (57 chromosomes). Among these, 79 (27%) chromosomes carried the delta F508 mutation, and 151 (51%) the W1282X mutation. In addition, we have analyzed the results of screening 1,946 unrelated healthy Jewish Ashkenazi individuals for the delta F508 and the W1282X mutations. Surprisingly, an almost equal number of carriers of the delta F508 (35) and W1282X (36) was found. The difference between the relative proportions of the mutations in the two groups is statistically significant (p = 0.025). A striking manifestation of this difference is revealed in the analysis of patients' genotypes. There were 36 patients homozygous for W1282X, while only 7 patients were homozygous for delta F508, although the number of delta F508 carriers in the general Jewish Ashkenazi population is almost equal to the number of W1282X carriers. This difference in allele frequencies found between healthy carriers and CF patients in the Jewish Ashkenazi population might not be unique to this ethnic group nor to the CF disease. The results indicate that the common practice of inferring general population epidemiologic parameters directly from patients information is liable to introduce biases.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic autoinhibition during rest and sodium-pump inhibition in isolated rat portal vein preparation.

In the presence of cocaine and corticosterone low-frequency (2 Hz) nerve stimulation evoked release of [3H]noradrenaline measured from isolated rat portal vein preparation. In normal Krebs solution exogenously applied l-noradrenaline (3 X 10(-8)-10(-6) M) significantly reduced the nerve-evoked [3H]noradrenaline release. The IC50 value of L-noradrenaline proved to be 1.8 X 10(-7) M. Yohimbine (3 X 10(-7) M) maximally blocked the alpha 2-adrenoceptors and enhanced nerve-evoked [3H]noradrenaline release. In the presence of 5.9 mM external K+, ouabain up to 10(-4) M did not affect either the resting or the stimulation-evoked release of radioactivity from tissues. In the absence of external K+ both the resting and the nerve-evoked release of [3H]noradrenaline increased markedly. When K+ was readmitted to preparations which had been kept in K+-free solution both the resting and the stimulation-evoked [3H]noradrenaline release were greatly reduced temporarily. In K+-free solution L-noradrenaline (10(-6) M) and yohimbine (3 X 10(-7) M) failed to significantly alter the nerve-evoked release. However, 3 X 10(-6) M yohimbine in K+-free solution significantly increased the stimulation-evoked release of [3H]noradrenaline. It is concluded that presynaptic alpha 2-adrenoceptor-mediated "negative feed-back" is present in rat portal vein preparations which can be inhibited by the preferential alpha 2-adrenoceptor blocker, yohimbine. However, if the Na+-pump is inhibited (which by itself enhanced the transmitter release), presynaptic autoinhibition is more pronounced, since a high concentration of yohimbine is required to block it.

Genetic control of resistance to trypanosomiasis.

To map the genetic sources of trypanotolerance in mice, a linkage analysis of survival following trypanosome challenge was performed by selective genotyping in a large F2 population produced by crossing the resistant C57BL/6 and susceptible BALB/c inbred mouse lines. We report evidence of a chromosomal region of large effect, possibly comprising more than one resistance locus, on Chromosome 17; and of further loci on Chromosomes 1 and 5. Together, these genes can account for all of the difference between the mean parental phenotypes.