Irina V. Tereshchenko

Association of synapsin 2 with schizophrenia in families of Northern European ancestry

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.

Enrichment of human prostate cancer cells with tumor initiating properties in mouse and zebrafish xenografts by differential adhesion

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.

AccuTyping: new algorithms for automated analysis of data from high-throughput genotyping with oligonucleotide microarrays

Microarray-based analysis of single nucleotide polymorphisms (SNPs) has many applications in large-scale genetic studies. To minimize the influence of experimental variation, microarray data usually need to be processed in different aspects including background subtraction, normalization and low-signal filtering before genotype determination. Although many algorithms are sophisticated for these purposes, biases are still present. In the present paper, new algorithms for SNP microarray data analysis and the software, AccuTyping, developed based on these algorithms are described. The algorithms take advantage of a large number of SNPs included in each assay, and the fact that the top and bottom 20% of SNPs can be safely treated as homozygous after sorting based on their ratios between the signal intensities. These SNPs are then used as controls for color channel normalization and background subtraction. Genotype calls are made based on the logarithms of signal intensity ratios using two cutoff values, which were determined after training the program with a dataset of ∼160 000 genotypes and validated by non-microarray methods. AccuTyping was used to determine >300 000 genotypes of DNA and sperm samples. The accuracy was shown to be >99%. AccuTyping can be downloaded from .

Ionizing radiation is a potent inducer of mitotic recombination in mouse embryonic stem cells

Maintenance of genomic integrity in embryonic cells is pivotal to proper embryogenesis, organogenesis and to the continuity of species. Cultured mouse embryonic stem cells (mESCs), a model for early embryonic cells, differ from cultured somatic cells in their capacity to remodel chromatin, in their repertoire of DNA repair enzymes, and in the regulation of cell cycle checkpoints. Using 129XC3HF1 mESCs heterozygous for Aprt, we characterized loss of Aprt heterozygosity after exposure to ionizing radiation. We report here that the frequency of loss of heterozygosity mutants in mESCs can be induced several hundred-fold by exposure to 5-10Gy of X-rays. This induction is 50-100-fold higher than the induction reported for mouse adult or embryonic fibroblasts. The primary mechanism underlying the elevated loss of heterozygosity after irradiation is mitotic recombination, with lesser contributions from deletions and gene conversions that span Aprt. Aprt point mutations and epigenetic inactivation are very rare in mESCs compared to fibroblasts. Mouse ESCs, therefore, are distinctive in their response to ionizing radiation and studies of differentiated cells may underestimate the mutagenic effects of ionizing radiation on ESC or other stem cells. Our findings are important to understanding the biological effects of ionizing radiation on early development and carcinogenesis.

ERG and CHD1 heterogeneity in prostate cancer: use of confocal microscopy in assessment of microscopic foci.

Biomarkers predicting tumor response are important to emerging targeted therapeutics. Complimentary methods to assess and understand genetic changes and heterogeneity within only few cancer cells in tissue will be a valuable addition for assessment of tumors such as prostate cancer that often have insufficient tumor for next generation sequencing in a single biopsy core.

A Phase II Study of AT-101 to Overcome Bcl-2–Mediated Resistance to Androgen Deprivation Therapy in Patients With Newly Diagnosed Castration-Sensitive Metastatic Prostate Cancer

UNLABELLED In a phase II multicenter study, men with castration sensitive metastatic prostate cancer were treated with AT-101, a small molecule Bcl-2 inhibitor, and androgen deprivation therapy. At the end of 7 cycles of therapy in 55 patients, an undetectable PSA was achieved in 31%. However, the combination did not meet the pre-specified level of activity for further development. 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. MATERIALS AND METHODS Patients with metastatic prostate cancer scheduled to start, or who had recently (within 6 weeks) initiated, ADT were enrolled. ADT with a luteinizing hormone-releasing hormone agonist and bicalutamide was started 6 weeks before initiation of oral AT-101, 20 mg/day for 21 days of a 28-day cycle. The primary endpoint of the study was the percentage of patients with an undetectable prostate-specific antigen (PSA) level (≤ 0.2 ng/mL) 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 chromodomain helicase DNA binding protein 1 (CHD1) and drug sensitivity, fluorescence in situ hybridization with confocal microscopy was assessed in a subgroup of patients. RESULTS A total of 55 patients were enrolled, with median age of 61 years and a median PSA level of 27.6 ng/dL. Of the 55 patients, 72% had a Gleason score ≥ 8. Three patients had visceral metastases, and the remaining patients had bone or nodal metastasis. An undetectable PSA level was achieved in 31% of the patients. Of the 31 patients, 12 experienced serious adverse events, 7 of which were considered related to study therapy. Most of the related adverse events were gastrointestinal and nervous system disorders. CHD1 assessment was feasible, with a nonsignificant association with therapeutic sensitivity in a small number of patients. CONCLUSION The combination of ADT and AT-101 did not meet the prespecified level of activity for further development of this combination.

Identification of possible genetic alterations in the breast cancer cell line MCF-7 using high-density SNP genotyping microarray

Context: Cancer cell lines are used extensively in various research. Knowledge of genetic alterations in these lines is important for understanding mechanisms underlying their biology. However, since paired normal tissues are usually unavailable for comparison, precisely determining genetic alterations in cancer cell lines is difficult. To address this issue, a highly efficient and reliable method is developed. Aims: Establishing a highly efficient and reliable experimental system for genetic profiling of cell lines. Materials and Methods: A widely used breast cancer cell line, MCF-7, was genetically profiled with 4,396 single nucleotide polymorphisms (SNPs) spanning 11 whole chromosomes and two other small regions using a newly developed high-throughput multiplex genotyping approach. Results: The fractions of homozygous SNPs in MCF-7 (13.3%) were significantly lower than those in the control cell line and in 24 normal human individuals (25.1% and 27.4%, respectively). Homozygous SNPs in MCF-7 were found in clusters. The sizes of these clusters were significantly larger than the expected based on random allelic combination. Fourteen such regions were found on chromosomes 1p, 1q, 2q, 6q, 13, 15q, 16q, 17q and 18p in MCF-7 and two in the small regions. Conclusions: These results are generally concordant with those obtained using different approaches but are better in defining their chromosomal positions. The used approach provides a reliable way to detecting possible genetic alterations in cancer cell lines without paired normal tissues.

Small scale genetic alterations contribute to increased mutability at the X-linked Hprt locus in vivo in Blm hypomorphic mice

BLM, the gene mutated in Bloom syndrome (BS), encodes an ATP-dependent RecQ DNA helicase that is involved in the resolution of Holliday junctions, in the suppression of crossovers and in the management of damaged replication forks. Cells from BS patients have a characteristically high level of sister chromatid exchanges (SCEs), and increased chromosomal aberrations. Fibroblasts and lymphocytes of BS patients also exhibit increased mutation frequency at the X-linked reporter gene HPRT, suggesting that BLM also plays a role in preventing small scale genomic rearrangements. However, the nature of such small scale alterations has not been well characterized. Here we report the characterization of Hprt mutations in vivo in Blm hypomorphic mice, Blm(tm1Ches)/Blm(tm3Brd). We found that the frequency of Hprt mutants was increased about 6-fold in the Blm(tm1Ches)/Blm(tm3Brd) mice when compared to Blm(tm3Brd) heterozygous mice or wildtype mice. Molecular characterization of Hprt gene in the mutant clones indicates that many of the mutations were caused by deletions that range from several base pairs to several thousand base pairs. While deletions in BLM-proficient somatic cells are often shown to be mediated by direct repeats, all three deletion junctions in Hprt of Blm(tm1Ches)/Blm(tm3Brd) mice were flanked by inverted repeats, suggesting that secondary structures formed during DNA replication, when resolved improperly, may lead to deletions. In addition, single base pair substitution and insertion/deletion were also detected in the mutant clones. Taken together, our results indicated that BLM function is important in preventing small scale genetic alterations. Thus, both large scale and small scale genetic alterations are elevated when BLM is reduced, which may contribute to loss of function of tumor suppressor genes and subsequent tumorigenesis.

Genetic Structures of Copy Number Variants Revealed by Genotyping Single Sperm

Background Copy number variants (CNVs) occupy a significant portion of the human genome and may have important roles in meiotic recombination, human genome evolution and gene expression. Many genetic diseases may be underlain by CNVs. However, because of the presence of their multiple copies, variability in copy numbers and the diploidy of the human genome, detailed genetic structure of CNVs cannot be readily studied by available techniques. Methodology/Principal Findings Single sperm samples were used as the primary subjects for the study so that CNV haplotypes in the sperm donors could be studied individually. Forty-eight CNVs characterized in a previous study were analyzed using a microarray-based high-throughput genotyping method after multiplex amplification. Seventeen single nucleotide polymorphisms (SNPs) were also included as controls. Two single-base variants, either allelic or paralogous, could be discriminated for all markers. Microarray data were used to resolve SNP alleles and CNV haplotypes, to quantitatively assess the numbers and compositions of the paralogous segments in each CNV haplotype. Conclusions/Significance This is the first study of the genetic structure of CNVs on a large scale. Resulting information may help understand evolution of the human genome, gain insight into many genetic processes, and discriminate between CNVs and SNPs. The highly sensitive high-throughput experimental system with haploid sperm samples as subjects may be used to facilitate detailed large-scale CNV analysis.

Abstract 2233: Rearrangement of ERG and CHD1 genes in prostate cancer as a marker of tumor heterogeneity

Efforts to develop targeted therapy based on genomic findings are complicated by the polygenic nature of drug resistance and genetic heterogeneity between separate biopsy cores. These findings have led to the conclusion that analysis of multiple biopsy cores and consideration of multiple targeted agents will be important. Because of our concern that these assessments may be missing an additional layer of heterogeneity within even a single biopsy core, which sequencing alone may not identify, we studied the genetic heterogeneity of cells in focal areas using TMA. Using confocal microscopy, we analyzed the most common and prostate cancer specific gene rearrangement (TMPRSS2-ERG fusion and CHD1 deletion) in neighboring single cells in a tumor focus without changing the stromal microenvironment. We evaluated 56 patients that underwent radical prostatectomy at CINJ with pathological assessment, grading, and ERG rearrangement status. Overall, ERG rearrangements occurred in 24/56 (42.9%) patients. The most frequent mechanism of rearrangements was deletion (58%), while a split event was confirmed in 25% of the ERG-rearranged cases. In 88% of cases the ERG rearrangement was associated with TMPRSS2-ERG gene fusion. A copy number gain of the non-rearranged ERG gene occurred in 10/56 (17.6%) cases. A heterogeneous mix of ERG deletions, translocations, and copy number changes occurred in 8/24 (33%) patients, with a combination of deletions and translocations alone occurring in 3/24 (12.5%) cases. 20/21 (95%) of analyzed cases with ERG-rearrangement were negative for CHD1 gene rearrangement (p Citation Format: Irina V. Tereshchenko, Hua Zhong, Marina Chekmareva, Urmila Santanam, Whitney Petrosky, Noriko Kane-Goldsmith, Jay A. Tischfield, Robert DiPaola. Rearrangement of ERG and CHD1 genes in prostate cancer as a marker of tumor heterogeneity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2233. doi:10.1158/1538-7445.AM2014-2233