A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system.The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.

BACKGROUND Prostate tumor-initiating cells (TICs) have intrinsic resistance to current therapies. TICs are commonly isolated by cell sorting or dye exclusion, however, isolating TICs from limited primary prostate cancer (PCa) tissues is inherently inefficient. We adapted the collagen adherence feature to develop a combined immunophenotypic and time-of-adherence assay to identify human prostate TICs. METHODS PCa cells from multiple cell lines and primary tissues were allowed to adhere to several matrix molecules, and fractions of adherent cells were examined for their TIC properties. RESULTS Collagen-I rapidly-adherent PCa cells have significantly higher clonogenic, migration, and invasion abilities, and initiated more tumor xenografts in mice when compared to slowly-adherent and no-adherent cells. To determine the relative frequency of TICs among PCa cell lines and primary PCa cells, we utilized zebrafish xenografts to define the tumor initiation potential of serial dilutions of rapidly-adherent α2β1hi/CD44hi cells compared to non-adherent cells with α2β1low/CD44low phenotype. Tumor initiation from rapidly-adherent α2β1hi/CD44hi TICs harboring the TMPRSS2:ERG fusion generated xenografts comprising of PCa cells expressing Erg, AMACR, and PSA. Moreover, PCa-cell dissemination was consistently observed in the immune-permissive zebrafish microenvironment from as-few-as 3 rapidly-adherent α2β1hi/CD44hi cells. In zebrafish xenografts, self-renewing prostate TICs comprise 0.02–0.9% of PC3 cells, 0.3–1.3% of DU145 cells, and 0.22–14.3% of primary prostate adenocarcinomas. CONCLUSION Zebrafish PCa xenografts were used to determine that the frequency of prostate TICs varies among PCa cell lines and primary PCa tissues. These data support a paradigm of utilizing zebrafish xenografts to evaluate novel therapies targeting tumor initiating cells in prostate cancer.

The synapsin 2 (Syn2) gene (3p25) is implicated in synaptogenesis, neurotransmitter release, and the localization of nitric oxide synthase to the proximity of its targets. In this study we investigated linkage and association between the Syn2 locus and schizophrenia. 37 pedigrees of Northern European ancestry from the NIMH Human Genetics Initiative collection were used. Four microsatellites and twenty SNPs were genotyped. Linkage (FASTLINK) and association (TRANSMIT, PDTPHASE) between markers and schizophrenia were evaluated. A maximum heterogeneity LOD of 1.93 was observed at marker D3S3434 with a recessive mode of inheritance. Significant results were obtained for association with schizophrenia using TRANSMIT (minimum nominal p=0.0000005) and PDTPHASE (minimum nominal p=0.014) using single marker analyses. Haplotype analysis using markers in introns 5 and 6 of Syn2 provided a single haplotype that is significantly associated with schizophrenia using TRANSMIT (nominal p<0.00000001) and PDTPHASE (nominal p=0.02). Simulation studies confirm the global significance of these results, but demonstrate that the small p-values generated by the bootstrap routine of TRANSMIT can be consistently anticonservative. Review of the literature suggests that Syn2 is likely to be involved in the etiology or pathogenesis of schizophrenia.

Background We conducted a phase II study in men with castration sensitive metastatic prostate cancer to test the hypothesis that AT-101, a small molecule Bcl-2 inhibitor, has clinical activity in patients initiating androgen deprivation therapy (ADT) for metastatic prostate cancer. Methods Patients with metastatic prostate cancer scheduled to start, or recently (within 6 weeks) initiated ADT, were enrolled. ADT with a luteinizing hormone-releasing hormone agonist and bicalutamide started 6 weeks prior to initiation of oral AT-101, 20 mg/day for 21 days of 28-day cycle. The primary endpoint of the study was percentage of patients with undetectable PSA (less than or equal to 0.2) after 7.5 months (1.5 months of ADT alone plus 6 months of combined ADT and AT-101). To assess for an association between CHD1 and drug sensitivity, FISH with confocal microscopy was assessed in a subgroup of patients. Results Fifty-five patients enrolled, median age 61 years, median PSA of 27.6 ng/dL, and 72% had a Gleason score > 8. Three patients had visceral metastases and the remaining patients had bone or nodal metastasis. An undetectable PSA was achieved in 31% of patients. Twelve patients experienced serious adverse events (SAEs), and seven were considered related to study therapy. The majority of related adverse events were gastrointestinal and nervous system disorders. CHD1 assessment was feasible with a non-significant association with therapeutic sensitivity in a small number of patients. Conclusion The combination of ADT and AT-101 did not meet the pre-specified level of activity for further development of this combination

Although the haplotype structure of the human genome has been studied in great detail, very little is known about the mechanisms underlying its formation. To investigate the role of meiotic recombination on haplotype block formation, single nucleotide polymorphisms were selected at a high density from a 2.5-Mb region of human chromosome 21. Direct analysis of meiotic recombination by high-throughput multiplex genotyping of 662 single sperm identifies 41 recombinants. The crossovers were nonrandomly distributed within 16 small areas. All, except one, of these crossovers fall in areas where the haplotype structure exhibits breakdown, displaying a strong statistically positive association between crossovers and haplotype block breaks. The data also indicate a particular clustered distribution of recombination hotspots within the region. This finding supports the hypothesis that meiotic recombination makes a primary contribution to haplotype block formation in the human genome.[Supplemental material is available online at www.genome.org and http://www2.umdnj.edu/lilabweb/Publications. htm.]It is known that in the human population, certain alleles of genetic markers within a short distance are in tight association (linkage disequilibrium or LD) and LD becomes weak or disappears when the markers are located farther apart (Ardlie et al. 2002). Chromosomal segments containing markers in LD are called haplotype blocks (Wall and Pritchard 2003). Haplotype blocks in the human genome were first described on a large scale for a 500-kb region of chromosome 5q31 (Daly et al. 2001) and the entire chromosome 21 (Patil et al. 2001), and subsequently in other regions of the genome (Gabriel et al. 2002;Twells et al. 2003;Olivier et al. 2004;Stenzel et al. 2004). Information on the haplotype structure of the human genome is of great interest because it can be used to significantly reduce the number of markers necessary for localizing genes responsible for complex diseases (Judson et al. 2002;Wang et al. 2002;Phillips et al. 2003). The progress of the Human HapMap Project has resulted in the comprehensive mapping of haplotype blocks across the entire human genome (The International HapMap Consortium 2003). However, very little is known about the mechanisms underlying the formation of haplotype blocks. It was noticed in the late 1990s that polymorphic frozen blocks (PFBs) were linked to form megabase haplotypes in the major histocompatibility complex (MHC) region. Regions between these PFBs appear to contain localized recombination "hotspots" (Gaudieri et al. 1997), indicating that in the human genome recombination hotspots may have a primary role in LD breakdown.There is a strong belief that meiotic recombination plays a primary role in shaping LD and therefore has a direct effect on the haplotype structure found in the human (Daly et al. 2001;Jeffreys et al. 2001Jeffreys et al. , 2005Cullen et al. 2002;Gabriel et al. 2002;Kauppi et al. 2003;Twells et al. 2003;Crawford et al. 2004;McVean et al. 2004). However, proving such a correlat...