Copy Number Distribution Research Articles

Counting Small RNA in Pathogenic Bacteria

Here, we present a modification to single-molecule fluorescence in situ hybridization that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (~200 nucleotide) nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 0 and 10 transcripts. The numbers of these RNA are both increased (by a factor of 2.5× for YSR35 and 3.5× for YSP8) upon a temperature shift from 25 to 37 °C, suggesting they play a role in pathogenesis. The copy number distribution of sRNAs from bacteria-to-bacteria are well-fit with a bursting model of gene transcription. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in cellular regulatory networks.

FGFR1 amplifications in squamous cell carcinomas of the lung: diagnostic and therapeutic implications.

Fibroblast growth factor receptor 1 (FGFR1) is a type 4 receptor tyrosine kinase. The receptor and its ligands play an important role in development and physiology. However, constitutive activation of FGFR1 by gene amplification, translocation or mutation is associated with various malignancies as, for example, breast cancer or myeloproliferative diseases. We have recently reported that FGFR1 amplification occurs in 20% of pulmonary squamous cell carcinomas, and preclinical tests have shown that these alterations are therapeutically tractable. These findings make FGFR1 amplification a potential biomarker for lung cancer treatment. Squamous cell carcinomas of the lung are characterized by an uneven FGFR1 gene copy number distribution. Therefore, fluorescence in situ hybridization assays need to address focality and heterogeneity of FGFR1 in these tumors. Here, we review our proposal for a reading and evaluation strategy. Furthermore, we highlight the emerging landscape of clinical trials with selective and unselective FGFR inhibitors and provide first response data from early clinical trials.

Comparison of class 2 transposable elements at superfamily resolution reveals conserved and distinct features in cereal grass genomes

BackgroundClass 2 transposable elements (TEs) are the predominant elements in and around plant genes where they generate significant allelic diversity. Using the complete sequences of four grasses, we have performed a novel comparative analysis of class 2 TEs. To ensure consistent comparative analyses, we re-annotated class 2 TEs in Brachypodium distachyon, Oryza sativa (rice), Sorghum bicolor and Zea mays and assigned them to one of the five cut-and-paste superfamilies found in plant genomes (Tc1/mariner, PIF/Harbinger, hAT, Mutator, CACTA). We have focused on noncoding elements because of their abundance, and compared superfamily copy number, size and genomic distribution as well as correlation with the level of nearby gene expression.ResultsOur comparison revealed both unique and conserved features. First, the average length or size distribution of elements in each superfamily is largely conserved, with the shortest always being Tc1/mariner elements, followed by PIF/Harbinger, hAT, Mutator and CACTA. This order also holds for the ratio of the copy numbers of noncoding to coding elements. Second, with the exception of CACTAs, noncoding TEs are enriched within and flanking genes, where they display conserved distribution patterns, having the highest peak in the promoter region. Finally, our analysis of microarray data revealed that genes associated with Tc1/mariner and PIF/Harbinger noncoding elements have significantly higher expression levels than genes without class 2 TEs. In contrast, genes with CACTA elements have significantly lower expression than genes without class 2 TEs.ConclusionsWe have achieved the most comprehensive annotation of class 2 TEs to date in these four grass genomes. Comparative analysis of this robust dataset led to the identification of several previously unknown features of each superfamily related to copy number, element size, genomic distribution and correlation with the expression levels of nearby genes. These results highlight the importance of distinguishing TE superfamilies when assessing their impact on gene and genome evolution.

A significant effect of the TSPY1 copy number on spermatogenesis efficiency and the phenotypic expression of the gr/gr deletion

AZFc deletions cause a significant phenotypic heterogeneity with respect to spermatogenesis; however, the reason for this is poorly understood. Recently, testis-specific protein Y-encoded 1 (TSPY1) copy number variation (CNV) was determined to be a potential genetic modifier of spermatogenesis. We performed a large-scale cohort study to investigate the effect of TSPY1 CNV on spermatogenesis and to elucidate the possible contribution of TSPY1 genetic variation to the phenotypic expression of AZFc deletions. Haplogrouping of the Y-chromosome and quantification of the TSPY1 copy number were performed in 2272 Han Chinese males with different spermatogenic statuses (704 males with the b2/b4 or gr/gr deletion and 1568 non-AZFc-deleted males). Our data revealed that the TSPY1 copy number distributions were significantly different among non-AZFc-deleted males with different spermatogenic phenotypes. Lower sperm production and an elevated risk of spermatogenic failure were observed in males with fewer than 21 TSPY1 copies and in those with more than 55 copies relative to men with 21-35 copies. Similar results were observed in males with the gr/gr deletion. These findings indicate that TSPY1 CNV affects an individual's susceptibility to spermatogenic failure by modulating the efficiency of spermatogenesis and strongly suggest that there is a significant quantity effect of the TSPY1 copy number on the phenotypic expression of the gr/gr deletion. To our knowledge, this CNV is the first independent genetic factor that has been clearly observed to influence the spermatogenic status of gr/gr deletion carriers. A combined genetic analysis of the TSPY1 copy number and the gr/gr deletion could inform the clinical counselling of infertile couples.

Transcription Stochasticity of Complex Gene Regulation Models

Transcription is regulated by a multitude of factors that concertedly induce genes to switch between activity states. Eukaryotic transcription involves a multitude of complexes that sequentially assemble on chromatin under the influence of transcription factors and the dynamic state of chromatin. Prokaryotic transcription depends on transcription factors, sigma-factors, and, in some cases, on DNA looping. We present a stochastic model of transcription that considers these complex regulatory mechanisms. We coarse-grain the molecular details in such a way that the model can describe a broad class of gene-regulation mechanisms. We solve this model analytically for various measures of stochastic transcription and compare alternative gene-regulation designs. We find that genes with complex multiprotein regulation can have peaked burst-size distributions in contrast to the geometric distributions found for simple models of transcription regulation. Burst-size distributions are, in addition, shaped by mRNA degradation during transcription bursts. We derive the stochastic properties of genes in the limit of deterministic switch times. These genes typically have reduced transcription noise. Severe timescale separation between gene regulation and transcription initiation enhances noise and leads to bimodal mRNA copy number distributions. In general, complex mechanisms for gene regulation lead to nonexponential waiting-time distributions for gene switching and transcription initiation, which typically reduce noise in mRNA copy numbers and burst size. Finally, we discuss that qualitatively different gene regulation models can often fit the same experimental data on single-cell mRNA abundance even though they have qualitatively different burst-size statistics and regulatory parameters.

Retained Heterodisomy Is Associated with High Gene Expression in Hyperhaploid Inflammatory Leiomyosarcoma

Inflammatory leiomyosarcoma (ILMS) is a soft tissue tumor that morphologically resembles conventional leiomyosarcoma (LMS) admixed with a prominent inflammatory infiltrate. Genetic data on ILMS are still limited but have suggested that this entity is characterized by hyperhaploidy (24–34 chromosomes). This low chromosome number is otherwise uncommon in neoplasia and has been found only in 0.2% to 0.3% of cytogenetically investigated tumors. Here, three ILMS were investigated using cytogenetic, single-nucleotide polymorphism (SNP) array, and global gene expression analyses. All cases displayed a hyperhaploid origin. Combined with previously reported cases, hyperhaploidy has been found in six of seven cytogenetically investigated ILMS. The copy number distribution of individual chromosomes is clearly nonrandom; the hyperhaploid clones of all six cases displayed disomy for chromosomes 5 and 20, and two copies of chromosomes 18, 21, and 22 were also common. All chromosomes identified as disomic showed a biparental origin by SNP array analysis; whether this is of pathogenetic importance is not known. Compared with conventional LMS, ILMS had a distinct gene expression signature. Furthermore, the number of chromosome copies correlated well with gene expression levels; disomic chromosomes showed higher gene expression levels than monosomic chromosomes, a finding that has not previously been reported for hyperhaploid tumors. Taken together, our findings suggest that disomy for some chromosomes, notably 5 and 20, as well as distorted gene expression achieved through massive loss of other chromosomes are essential features of ILMS.

Association analysis of the CCL3L1 copy number locus by paralogue ratio test in Norwegian rheumatoid arthritis patients and healthy controls

Genotyping of multiallelic copy number variants (CNVs) is technically difficult and can lead to inaccurate conclusions. This is reflected by inconsistent results published for the CNV C-C chemokine ligand 3-like 1 (CCL3L1) and its contribution to rheumatoid arthritis (RA) susceptibility. In order to draw robust conclusions about CCL3L1 involvement in RA, we have performed association analysis (CNVtools) using genotyping by the paralogue ratio test of a Norwegian RA case-control material (N=1877). We also analyzed the associations after stratification for anti-citrullinated peptide antibody (ACPA) status. Clear clusters representing specific copy number classes were evident, but significant differential bias was observed resulting in a systematic trend toward slightly higher apparent copy number for cases relative to controls. Controlling for bias revealed no significant differences in copy number distribution either between all patients and controls, or after ACPA stratification. Our results do not support involvement of the CCL3L1 CNV in RA susceptibility.

Population density approach for discrete mRNA distributions in generalized switching models for stochastic gene expression

We present a generalization of a population density approach for modeling and analysis of stochastic gene expression. In the model, the gene of interest fluctuates stochastically between an inactive state, in which transcription cannot occur, and an active state, in which discrete transcription events occur; and the individual mRNA molecules are degraded stochastically in an independent manner. This sort of model in simplest form with exponential dwell times has been used to explain experimental estimates of the discrete distribution of random mRNA copy number. In our generalization, the random dwell times in the inactive and active states, T_{0} and T_{1}, respectively, are independent random variables drawn from any specified distributions. Consequently, the probability per unit time of switching out of a state depends on the time since entering that state. Our method exploits a connection between the fully discrete random process and a related continuous process. We present numerical methods for computing steady-state mRNA distributions and an analytical derivation of the mRNA autocovariance function. We find that empirical estimates of the steady-state mRNA probability mass function from Monte Carlo simulations of laboratory data do not allow one to distinguish between underlying models with exponential and nonexponential dwell times in some relevant parameter regimes. However, in these parameter regimes and where the autocovariance function has negative lobes, the autocovariance function disambiguates the two types of models. Our results strongly suggest that temporal data beyond the autocovariance function is required in general to characterize gene switching.

Could basic research shed light on false positivity in photodynamic diagnosis?

Accumulating evidence has suggested that photodynamic diagnosis (PDD) using new optical imaging agents (5-aminolevlinic acid [5-ALA] or hexaminolevulinate [Hexvix]) has an advantage detecting the flat tumor of the bladder, especially carcinoma in situ, or tiny papillary tumors, which might be overlooked by conventional white-light cystoscopy.1 Recent randomized controlled trials have shown that PDD-assisted transurethral resection of bladder tumors (TURBT) yields better clinical outcomes in terms of progression-free survival than conventional TURBT.2 Most readers might have simple questions about PDD. How does is the red fluorescence imaging generated from cancer tissue? How is the optical imaging agent administered, and accumulated specifically, if true, in the tumor? Are there any adverse effects (AE) including photosensitivity? Intravesically or orally administered 5-ALA is uptaken to cells by active transport and incorporated into mitochondria. As ferrochelatase, a key enzyme of final process of heme synthesis, is inherently absent in tumor cells, an intermediary metabolite (protoporphirin IX [PPIX]) is specifically accumulated in tumor cells. Dysfunction of the transporter gene, which acts as a efflux pump of PPIX in tumor cells, might accelerate the specific accumulation of the agent.3 Regarding AE of 5-ALA, no severe photosensitivity, which is a common and severe AE of the conventional optical imaging photosensitizer, have been reported. As 5-ALA is not approved by Japan, a multi-institutional prospective phase I/II study is in progress as the investigator initiated clinical trial. One of the drawbacks of PDD, however, is a relatively higher false positive rate. Although red fluorescence is theoretically generated only from tumor cells, false positive fluorescence is often encountered in the clinical setting, leading to lowering specificity to 50–60% in the literature. Why does false red fluorescence take place? One of the possible reasons is an optical artifact as a result of tangential observation. Artifact is often observed at the bladder neck and prostatic urethra. Other possibilities have been reported to result from cystitis, squamous metaplasia, mild to moderate dysplasia, or hyperplasia. Can all false positivity be ignored as a non-cancerous phenomenon? It is well known that urothelial tumor is chronologically or spatially multifocal as a result of harboring either a field defect, or dissemination of the clonally-selected cancer cell. Non-random gene alterations have been reported to occur frequently at chromosomes 7, 9 and 17, and those alterations strongly affect patient outcome in bladder cancer. We hypothesized that false positive bladder mucosa might harbor premalignant lesion (Fig. 1).4 Lengauer et al. postulated the concept of chromosomal instability, which is evaluated by a higher aberrant fraction of the copy number, a non-modal fraction of the copy number distribution in each chromosome.5 The aberrant fraction of chromosome 9 by fluorescence in situ hybridization analysis was significantly higher in false positive fluorescent mucosa than that in the non-fluorescent counterpart. Array comparative genomic hybridization using established bladder cancer cell lines showed 9p24.1 to be a commonly deleted region for fluorescent positive cell lines.4 Hartmann et al. reported that the deletion of p53 was highly associated with chromosome 9 alterations in the flat urothelial lesions detected by fluorescent cystoscopy.6 Other investigators reported a close association between chromosome 9 alterations and fluorescent mucosa. A phase III randomized controlled trial proved that PDD-guided transurethral resection, with the resection of all visible fluorescent mucosae, yielded a statistically higher progression-free survival than did conventional transurethral resection.2 The study suggests that an incomplete resection, or ignoring the fluorescent mucosa with PDD except for optical artifacts, might thus leave a potential source for bladder tumor recurrence. Case presentation. 56 years, Male 2003/10/31 First presentation, Pathology: TCC, G2, pTa. 2006/4/26 Recurrence. (a) Representative case of positive of photodynamic diagnosis. Normal looking mucosa by white-light (conventional) cystoscopy. (b) Photodynamic diagnosis showing irregular red fluorescence, (c) Molecular cytogenetic by fluorescence in situ hybridization analysis showing a higher variant fraction of chromosome 9. Arrow: chromosome 9 monosomy. The pathological diagnosis was mild dysplasia. Could basic research shed light on false positivity of PDD? Yes, such false positive mucosa by PDD might be a potential reason for exploring the mechanism of oncogenesis in urothelial cancer. None declared.

Lack of support for association between the copy number variants in the FCGR locus and schizophrenia: A case control study

The missing heritability of polygenic schizophrenia after genome-wide association studies (GWAS) can be potentially accounted for by the fact that most dynamic multiallelic copy number variants (CNVs) overlap segmental duplications (SDs). The FCGR locus covers this category of complex CNVs and it has long been postulated to harbor variants conferring the risk of schizophrenia. However, such association remains unproven. We used a case-control design to investigate CNV-based association with the disease. Data were obtained from 598 unrelated schizophrenia patients and 959 normal controls of Han ancestry from Shanghai. A total of four copy number (CN) probes in the FCGR locus were detected using TaqMan® Copy Number Assay. SPSS version 16.0 was used for the statistical analyses. And the frequency distributions of target CN in FCGR locus were very similar between controls and cases, whereas the CNV frequency differed markedly among different target CN analyzed in the two cohorts. When compared with the predominant two copies per diploid genome, a distinct non-protein-coding CN deletion region containing regulatory sequences was detected by probe Hs04194069_cn. Taken together, we found no evidence of association of target CNVs in the FCGR locus with schizophrenia. However, our negative findings suggest that more detailed next generation sequencing-based association studies are needed to fully evaluate the contribution of this category of complex CNVs to the disease.

Stable multi-infection of splenocytes during SIV infection - the basis for continuous recombination

BackgroundRecombination is an important mechanism in the generation of genetic diversity of the human (HIV) and simian (SIV) immunodeficiency viruses. It requires the co-packaging of divergent RNA genomes into the same retroviral capsid and subsequent template switching during the reverse transcription reaction. By HIV-specific fluorescence in situ hybridization (FISH), we have previously shown that the splenocytes from 2 chronically infected patients with Castelman's disease were multi-infected and thus fulfill the in vivo requirements to generate genetic diversity by recombination. In order to analyze when multi-infection first occurs during a lentivirus infection and how the distribution of multi-infection evolves during the disease course, we now determined the SIV copy numbers from splenocytes of 11 SIVmac251-infected rhesus macaques cross-sectionally covering the time span of primary infection throughout to end-stage immunodeficiency.ResultsSIV multi-infection of single splenocytes was readily detected in all monkeys and all stages of the infection. Single-infected cells were more frequent than double- or triple- infected cells. There was no strong trend linking the copy number distribution to plasma viral load, disease stage, or CD4 cell counts.ConclusionsSIV multi-infection of single cells is already established during the primary infection phase thus enabling recombination to affect viral evolution in vivo throughout the disease course.

Evolution and function of the extended miR-2 microRNA family

MicroRNAs are essential post-transcriptional regulators. Many animal microRNAs are clustered in the genome, and it has been shown that clustered microRNAs may be transcribed as a single transcript. Polycistronic microRNAs are often members of the same family, suggesting a role of tandem duplication in the emergence of clusters. The mir-2 microRNA family is the largest in Drosophila melanogaster, with 8 members that are mostly clustered in the genome. Previous studies suggest that the copy number and genomic distribution of mir-2 family members has been subject to significant change during evolution. The effects of such changes on their function are still unknown. Here we study the evolution of function in the mir-2 family. Our analyses show that, in spite of the change in number and organization among invertebrates, most mir-2 loci produce very similar mature microRNA products. Multiple mature miR-2 sequences are predicted to target genes involved in neural development in Drosophila. These targeting properties are conserved in the distant species Caenorhabditis elegans. Duplication followed by functional diversification is frequent during protein-coding gene evolution. However, our results suggest that the production of microRNA clusters by gene duplication rarely involves functional changes. This pattern of functional redundancy among clustered paralogous microRNAs reflects birth-and-death evolutionary dynamics. However, we identified a small number of mir-2 sequences in Drosophila that may have undergone functional shifts associated with genomic rearrangements. Therefore, redundancy in microRNA families may facilitate the acquisition of novel functional features.

Transgene copy number distribution profiles in recombinant CHO cell lines revealed by single cell analyses

Clonally derived recombinant cell lines are highly desired to achieve consistent production of recombinant biotherapeutics. Despite repeated rounds of cloning by limiting dilution or single cell cloning, the resulting cell lines have often been observed to diverge, becoming a heterogeneous population and losing productivity over long-term sub-culturing. To understand the underlying molecular mechanisms, we developed quantitative polymerase chain reaction (qPCR) assays for the analysis of transgene copy number distribution in single recombinant cells isolated from Chinese hamster ovary (CHO) cell lines. Single cells were obtained by fluorescence activated cell sorting (FACS) technology and lysed directly in 96-well plates. qPCR assays were then applied to analyze the quantity and distribution of transgenes in those single cells. Results revealed multiple types of transgene copy number distribution profiles from those clonally derived CHO cell lines. The cell lines that maintained productivity over time displayed relatively constant and homogeneous transgene copy number distributions; while most of those cell lines exhibiting a loss of productivity over time showed varying degrees of transgene copy number heterogeneity and distribution drift with passaging. Some cell lines showed the existence of a significant portion of cells lacking the transgenes (referred to as negative cells in this study) and the percentage of those negative cells increased with subsequent generations. Criteria based on transgene copy number distribution profiles were developed to assess cell line suitability for clinical applications, which include (i) percentage of negative cells; (ii) standard deviation of qPCR threshold cycle (C(t) ) value, a measure of population heterogeneity; (iii) mean C(t) changes during aging, a measure of population drift. By implementing these criteria, undesirable cell lines were eliminated for further clinical and commercial applications.

Association of copy number variation in Fc gamma receptor IIIb gene with risk of Graves' ophthalmopathy

The functional polymorphism that explains the established association of Fc gamma receptor IIIb (FCGR3B) with Graves' disease (GD) and Graves' ophthalmopathy (GO) remains unidentified, but copy number variation (CNV) might be relevant. The aim of this study was to determine whether CNV for FCGR3B is associated with GD and GO. Genotype analysis involved 624 GD patients, including 397 without GO (GDnonGO) and 227 with GO (GDGO), and 227 healthy controls. The relative copy number (CN) ofFCGR3B was determined using a relative real-time quantitative polymerase chain reaction. Our findings indicated the distribution of the relative CN of FCGR3B significantly differed between the GDGO patients and the healthy controls (p = 0.02) but not between the total GD patients and the healthy control groups (p = 0.06). Individuals with less than 2 CN or more than 2 CN of FCGR3B were at significantly decreased risk of developing GDGO. In addition, GD patients with less than 2 copies of FCGR3B were at reduced risk for developing nodular hyperplasia and vitiligo, but at increased risk for myxedema. Our results suggest that CNV of FCGR3B is associated with the development or progression of GD in Taiwan Chinese population.

Measuring the Plasmid Copy Number of Single E. Coli by Digital PCR: Are Plasmids Partitioned in Clusters during Cell Division?

The presence of plasmids is important for the behavior of bacteria, as they often carry antibiotic resistance genes and genes involved in virulence of pathogenic strains. Furthermore plasmids are often responsible for horizontal gene transfer. In order to remain in the bacteria the plasmids have to provide complex regulation mechanisms and often inhere active partitioning during cell division. Fingerprints of the resulting plasmid stability can be found in the plasmid copy number distribution. However, these distributions have been rarely reported, since they have been experimentally difficult to obtain.We performed single cell measurements that allow to piece together the plasmid copy number distribution in a bacterial population. We use fluorescent activated cell sorting (FACS) to isolate single bacteria and subsequently utilize the single molecule resolution of digital PCR to determine the plasmid copy number of single cells.Interestingly, the measurement of the plasmid pZE which has a ColE1 like origin of replication showed a rather broad distribution. An unexpectedly high percentage of the cells had very low plasmid copy numbers, despite the high mean copy number of 120. This characteristic however disappeared for a mutant which additionally contains the active partitioning system parABC. The instability of the pZE copy number is unlikely to be the result of random plasmid partitioning; we explain the findings by clustered partitioning, i.e. clusters of plasmids are partitioned to the daughter cells rather than the individual plasmids. Results are high statistical fluctuations of the plasmid copy number. Other groups showed evidence for clustering of plasmids in E. Coli and also showed, that these clusters get dissolved if the plasmids contain parABC genes. Furthermore, theoretical models which employ clustered partitioning fits our data quantitatively.

Prognostic relevance of the expressions of CAV1 and TES genes on 7q31 in melanoma

The 7q31 locus contains several genes affected in cancer progression. Although evidences exist regarding its impact on tumorigenesis, the role of genetic alterations and the expressions of locus-related genes are still controversial. Our study aimed to define the 7q31 copy number alterations in primary melanomas, primary-metastatic tumor pairs and cell lines. Data were correlated with clinical-pathological parameters. Genetic data show that 7q31 copy number distribution was heterogeneous in both primary and metastatic tumors. Extra copies were highly accompanied by chromosome 7 polisomy, and significantly increased in primary lesions with poor prognosis. Additionally, we determined the mRNA and protein levels of the locus-related CAV1 and TES genes. TES mRNA level was associated with metastatic location. CAV1 mRNA and protein levels were significantly higher in thicker tumors, however, lack of protein was also observed in a subpopulation of thin lesions. Expressions of CAV1 and TES were not associated with 7q31 alterations. In conclusion, 7q31 amplification can predict unfavorable outcome. Alterations of TES mRNA level may predict the location of metastasis. CAV1 possibly affect the cancer cell invasion.

Association between copy number variation of complement component C4 and Graves' disease

BackgroundGene copy number of complement component C4, which varies among individuals, may determine the intrinsic strength of the classical complement pathway. Presuming a major role of complement as an effecter in peptide-mediated inflammation and phagocytosis, we hypothesized that C4 genetic diversity may partially explain the development of Graves' disease (GD) and the variation in its outcomes.MethodsA case-control study including 624 patients with GD and 160 healthy individuals were enrolled. CNV of C4 isotypes (C4A and C4B) genes were performed by quantitative real-time polymerase chain reaction analysis. Statistical comparison and identification of CNV of total C4, C4 isotypes (C4A and C4B) and C4 polymorphisms were estimated according to the occurrence of GD and its associated clinical features.ResultsIndividuals with 4, 2, and 2 copies of C4, C4A and C4B genes, especially those with A2B2 polymorphism may associate with the development of GD (p = 0.001, OR = 10.994, 95% CI: 6.277-19.255; p = 0.008, OR = 1.732, 95% CI: 1.190-2.520; p = 2.420 × 10-5, OR = 2.621, 95% CI: 1.791-3.835; and p = 1.395 × 10-4, OR = 2.671, 95% CI: 1.761-4.052, respectively). Although the distribution of copy number for total C4, C4 isotypes as well as C4 polymorphisms did not associate with the occurrence of goiter, nodular hyperplasia, GO and myxedema, <2 copies of C4A may associate with high risk toward vitiligo in patients with GD (p = 0.001, OR = 5.579, 95% CI: 1.659-18.763).ConclusionsThese results may be further estimated for its clinical application on GD and the vitiligo in patients with GD.

The landscape of inherited and de novo copy number variants in a plasmodium falciparum genetic cross

BackgroundCopy number is a major source of genome variation with important evolutionary implications. Consequently, it is essential to determine copy number variant (CNV) behavior, distributions and frequencies across genomes to understand their origins in both evolutionary and generational time frames. We use comparative genomic hybridization (CGH) microarray and the resolution provided by a segregating population of cloned progeny lines of the malaria parasite, Plasmodium falciparum, to identify and analyze the inheritance of 170 genome-wide CNVs.ResultsWe describe CNVs in progeny clones derived from both Mendelian (i.e. inherited) and non-Mendelian mechanisms. Forty-five CNVs were present in the parent lines and segregated in the progeny population. Furthermore, extensive variation that did not conform to strict Mendelian inheritance patterns was observed. 124 CNVs were called in one or more progeny but in neither parent: we observed CNVs in more than one progeny clone that were not identified in either parent, located more frequently in the telomeric-subtelomeric regions of chromosomes and singleton de novo CNVs distributed evenly throughout the genome. Linkage analysis of CNVs revealed dynamic copy number fluctuations and suggested mechanisms that could have generated them. Five of 12 previously identified expression quantitative trait loci (eQTL) hotspots coincide with CNVs, demonstrating the potential for broad influence of CNV on the transcriptional program and phenotypic variation.ConclusionsCNVs are a significant source of segregating and de novo genome variation involving hundreds of genes. Examination of progeny genome segments provides a framework to assess the extent and possible origins of CNVs. This segregating genetic system reveals the breadth, distribution and dynamics of CNVs in a surprisingly plastic parasite genome, providing a new perspective on the sources of diversity in parasite populations.

Ploidy in cyanobacteria

A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. Synechococcus PCC 7942 and Synechococcus WH7803 were found to contain 3-4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, Synechocystis PCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the 'Kazusa' strain more than 20 years ago. The results reveal that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.

Study on the copy numbers and mRNA expression levels of the programmed death-1 gene in chronic hepatitis B patients

To study the copy numbers and mRNA expression levels of the Programmed Death-1 gene in chronic hepatitis B patients and to analyze the differences of the copy numbers and mRNA expression levels of the gene in patients with different clinical outcomes. Real time PCR was adopted to detect the PD-1 gene copy numbers and their mRNA expressions in peripheral blood mononuclear cells (PBMCs) from 27 samples from healthy donors in Control group, 31 samples from chronic asymptomatic HBV carriers (ASC, n=31), 19 samples from chronic severe hepatitis B patients (CSH, n=19) and 29 samples from Primary hepatitis B Virus-related hepatocarcinoma (PHC, n=29). The differences and relationship of copy numbers and their mRNA expression levels among those groups were compared and analyzed by adopting Chi-square test and Rank sum test. PD-1 gene copy number deviated from 0 copy to 3 copies among all the 106 samples. In control group, ASC group, CSH group and PHC group, the percentages of cases of haploid (single) were 37.0%, 35.5%, 26.3% and 6.9%, respectively, the percentages of cases of diploid (double) were 55.5%, 58.0%, 63.2% and 82.8%, respectively, and the percentages of cases of triploid (triple) were 3.7%, 6.5%, 10.5% and 10.3%, respectively. The percentage of cases of polyploid (diploid and triploid) in control group, ASC group, CSH group and PHC group were 59.3%, 64.5%, 73.7% and 93.1%, respectively. The different distribution of PD-1 gene copy number of polyploid was significant in total samples (x2=9.583, P<0.05). Compared with Control Group and ASC group, the percentage of cases of polyploid in PHC group was lower with the x2 equals to 8.985 and 7.215 respectively and both with P less than 0.05. The difference between the two groups was statistically significant. The mean PD-1 gene copy numbers for these four groups were 1.59+/-0.63, 1.70+/-0.52, 1.84+/-0.60 and 2.00+/-0.37 while the median were 0.002 54, 0.002 72, 0.002 55 and 0.001 33 respectively. Except the control group, there was a uptrend in the other three groups while PD-1 gene mRNA expression presented a downtrend. The mean of PD-1 gene copy numbers of 2 and their mRNA expression levels were 19.59, 32.57 and 33.22 for PHC, CSH and ASC groups among which PHC group had the lowest value, there was significant differences found in the comparison with F=5.395 and P<0.05. PD-1 gene copy numbers and their mRNA expression levels were different in chronic HBV infected patients with different transformation. It is valuable to follow up the patients with more than 1 copy number of PD-1 gene in long term.