Association of CNVs with methylation variation.

Germline copy number variants (CNVs) and single-nucleotide polymorphisms (SNPs) form the basis of inter-individual genetic variation. Although the phenotypic effects of SNPs have been extensively investigated, the effects of CNVs is relatively less understood. To better characterize mechanisms by which CNVs affect cellular phenotype, we tested their association with variable CpG methylation in a genome-wide manner. Using paired CNV and methylation data from the 1000 genomes and HapMap projects, we identified genome-wide associations by methylation quantitative trait locus (mQTL) analysis. We found individual CNVs being associated with methylation of multiple CpGs and vice versa. CNV-associated methylation changes were correlated with gene expression. CNV-mQTLs were enriched for regulatory regions, transcription factor-binding sites (TFBSs), and were involved in long-range physical interactions with associated CpGs. Some CNV-mQTLs were associated with methylation of imprinted genes. Several CNV-mQTLs and/or associated genes were among those previously reported by genome-wide association studies (GWASs). We demonstrate that germline CNVs in the genome are associated with CpG methylation. Our findings suggest that structural variation together with methylation may affect cellular phenotype

Optimizing copy number variation analysis using genome-wide short sequence oligonucleotide arrays

The detection of copy number variants (CNV) by array-based platforms provides valuable insight into understanding human diversity. However, suboptimal study design and data processing negatively affect CNV assessment. We quantitatively evaluate their impact when short-sequence oligonucleotide arrays are applied (Affymetrix Genome-Wide Human SNP Array 6.0) by evaluating 42 HapMap samples for CNV detection. Several processing and segmentation strategies are implemented, and results are compared to CNV assessment obtained using an oligonucleotide array CGH platform designed to query CNVs at high resolution (Agilent). We quantitatively demonstrate that different reference models (e.g. single versus pooled sample reference) used to detect CNVs are a major source of inter-platform discrepancy (up to 30%) and that CNVs residing within segmental duplication regions (higher reference copy number) are significantly harder to detect (P < 0.0001). After adjusting Affymetrix data to mimic the Agilent experimental design (reference sample effect), we applied several common segmentation approaches and evaluated differential sensitivity and specificity for CNV detection, ranging 39–77% and 86–100% for non-segmental duplication regions, respectively, and 18–55% and 39–77% for segmental duplications. Our results are relevant to any array-based CNV study and provide guidelines to optimize performance based on study-specific objectives

Single chain human chorionic gonadotropin, hCGαβhCG\alpha \beta: Effects of mutations in the \alpha subunit on structure and bioactivity

The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the \alpha and $_\beta$ subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCG\alpha\beta) was used to investigate roles of critical residues of the \alpha subunit in hormone receptor interaction and biological activity. The \alpha subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type $_\beta$ subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCG\alpha P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCG \alpha T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the \alpha subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormonereceptor interactions

Single chain human chorionic gonadotropin, hCGαβ: effects of mutations in the α subunit on structure and bioactivity

The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the α and β subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGαβ) was used to investigate roles of critical residues of the α subunit in hormone receptor interaction and biological activity. The α subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type β subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGα P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGα T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the α subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions

Translational fusion of two beta-subunits of human chorionic gonadotropin results in production of a novel antagonist of

The strategy of translationally fusing the alpha-and beta-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active singlechain analog hCG alpha beta expressed using Pichia expression system. Using the same expression system, another analog, in which the alpha-subunit was replaced with the second beta-subunit, was expressed (hCG beta beta) and purified. hCG beta beta could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose- dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCG alpha beta and hCG beta beta using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCG beta beta interacts with two LH receptor molecules. These studies demonstrate that the presence of the second beta-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of alpha-subunit, the molecule is unable to elicit response. The strategy of fusing two beta-subunits of glycoprotein hormones can be used to produce antagonists of these hormones

Prenatal detection and characterization of a psu idic(8)(p23.3) which likely derived from nonallelic homologous recombination between two MYOM2-repeats

Mosaicism with an isodicentric 8 with a breakpoint at p23.3 [idic(8)(p23.3)] is very rare. We report the first prenatal case on a male fetus, in which obstetric ultrasound revealed multiple congenital anomalies at 28 weeks of gestation. Cytogenetic analysis of amniocytes showed mos 45,XY,-8,psu idic(8)(p23.3)[16]/46,XY,psu idic(8)(p23.3)[4], and that of cord blood lymphocytes revealed mos 46,XY, psu idic(8)(p23.3)[37]/45,XY,-8,psu idic(8)(p23.3)[13]. Fluorescence in situ hybridization studies revealed that the break-reunion occurred at the cytoband 8p23.3 within the physical position 2.08 Mb from the 8p telomere. Chromosomal microarray analyses further assigned the duplication/deletion breakpoint at 2.16 Mb (Agilent 244K) and at 2.19 Mb (Affymetrix SNP6.0). Analysis of microsatellite DNA indicated that the psu idic(8)(p23.3) was derived from the maternal chromosome 8. Together, these findings indicate that the fetus was nullisomic for ∼2.2 Mb from 8pter, trisomic for the rest of chromosome 8 in mosaic condition, and likely had breaks in MYOM2 repeats of the maternal chromosome 8

Expression of the Platelet-Derived Growth Factor Receptor in Prostate Cancer and Treatment Implications with Tyrosine Kinase Inhibitors

The platelet-derived growth factor receptor (PDGFR) is a receptor tyrosine kinase overexpressed in a subset of solid tumors and therefore is the target of drugs inhibiting this function such as imatinib mesylate (Gleevec). Thus far, drug therapy has played a limited role in the treatment of localized prostate cancer (PCa). This study characterizes PDGFR-β expression in a wide spectrum of PCa samples to provide empirical data as part of a rational treatment strategy. A survey of five published prostate expression array studies, including 100 clinically localized PCa, did not identify tumors with increased PDGFR-β expression level. Protein expression of PDGFR-β, as determined by immunohistochemistry, revealed 5% of clinically localized PCa and 16% of metastatic PCa cases to show moderate or strong expression. To develop a strategy to detect patients most likely to profit from Gleevec treatment, we analyzed cDNA expression array data from 10,000 transcripts for PDGFR-β expression and divided tumors in groups based on PDGFR-β expression level. Performing a supervised analysis to identify potential comarkers of PDGFR-β in PCa, we identified a set of genes whose expression was associated with PDGFR-β status including early growth response 1 (Egr1), an upstream effector of PDGF (4.2-fold upregulation), α-methylacyl-CoA racemase, as well as v-Maf and neuroblastoma suppressor of tumorigenicity (both with a 2.2-fold downregulation). Taken together, this study suggests that only a small subset of PCas may be amenable to tyrosine kinase inhibitors specific for PDGFR