Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in Myoclonus Epilepsy and Ragged-Red Fibers (MERRF) syndrome by a multiplex Molecular Beacon based real-time fluorescence PCR

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype–phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNALys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

Quantitative analysis of chromosomal CGH in human breast tumors associates copy number abnormalities with p53 status and patient survival

We present a general method for rigorously identifying correlations between variations in large-scale molecular profiles and outcomes and apply it to chromosomal comparative genomic hybridization data from a set of 52 breast tumors. We identify two loci where copy number abnormalities are correlated with poor survival outcome (gain at 8q24 and loss at 9q13). We also identify a relationship between abnormalities at two loci and the mutational status of p53. Gain at 8q24 and loss at 5q15-5q21 are linked with mutant p53. The 9q and 5q losses suggest the possibility of gene products involved in breast cancer progression. The analytical techniques are general and also are applicable to the analysis of array-based expression data.

High copy number I-Ab transgenes induce production of IgE through an interluekin 4-dependent mechanism.

To better understand the role of class II major histocompatibility complex molecules in both normal and autoimmune responses, we have produced a series of I-Ab transgenic mice. One of these transgenic constructs, designated NOD.PD, has the sequence of the NOD beta chain (Abeta(g7)) except at positions 56 and 57, where Pro-Asp replaces His-Ser. Several NOD.PD transgenic lines have been produced. One line of these mice carried a very high number of copies (>50) of the NOD.PD transgene. As has been described in other mice carrying high copy numbers of I-Ab transgenes, B-cell development was abnormal. The steady state numbers of mature B cells (IgM+/IgD(hi)) in the periphery were greatly reduced in transgenic mice compared to nontransgenic littermates. Surprisingly, rather than being accompanied by a generalized hypogammaglobulinemia, this B-cell deficiency was accompanied by elevated concentrations of IgG1 and IgE in the serum. Conversely, the levels of IgG2a were reduced in transgenic mice compared to nontransgenic littermates. Because this isotype pattern was characteristic of interleukin (IL)-4-induced class-switching, we then investigated the role of IL-4 in causing the observed phenotype. We crossed the high copy number transgenic mice with an IL-4-deficient strain of mice. As expected, the elevated levels of IgE in high copy number transgenic mice were eliminated when the IL-4 gene was inactivated. However, the reduction in the number of B cells was not ameliorated. These data indicate that the primary defect caused by the transgene was to reduce the number of B cells in these mice. This reduction was accompanied by a secondary increase in IL-4 production, which drove the remaining B cells toward the production of IgGl and IgE.

PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value.

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

Maternal Copy Number Variants (CNV) transmission to their Autism Spectrum Disorder (ASD) sons correlates with phenotypic traits

Background: Common neurodevelopmental disorder, global prevalence ~1 %; Persistent deficits in social communication and social interaction; restricted and repetitive behavior, interests, or activities; Highly heterogeneous clinical presentation; Male to female ratio ~4:1.

Genome-wide copy number variation (CNV) detection in Nelore cattle reveals highly frequent variants in genome regions harboring QTLs affecting production traits.

Background: Copy number variations (CNVs) have been shown to account for substantial portions of observed genomic variation and have been associated with qualitative and quantitative traits and the onset of disease in a number of species. Information from high-resolution studies to detect, characterize and estimate population-specific variant frequencies will facilitate the incorporation of CNVs in genomic studies to identify genes affecting traits of importance. Results: Genome-wide CNVs were detected in high-density single nucleotide polymorphism (SNP) genotyping data from 1,717 Nelore (Bos indicus) cattle, and in NGS data from eight key ancestral bulls. A total of 68,007 and 12,786 distinct CNVs were observed, respectively. Cross-comparisons of results obtained for the eight resequenced animals revealed that 92 % of the CNVs were observed in both datasets, while 62 % of all detected CNVs were observed to overlap with previously validated cattle copy number variant regions (CNVRs). Observed CNVs were used for obtaining breed-specific CNV frequencies and identification of CNVRs, which were subsequently used for gene annotation. A total of 688 of the detected CNVRs were observed to overlap with 286 non-redundant QTLs associated with important production traits in cattle. All of 34 CNVs previously reported to be associated with milk production traits in Holsteins were also observed in Nelore cattle. Comparisons of estimated frequencies of these CNVs in the two breeds revealed 14, 13, 6 and 14 regions in high (>20 %), low (<20 %) and divergent (NEL > HOL, NEL < HOL) frequencies, respectively. Conclusions: Obtained results significantly enriched the bovine CNV map and enabled the identification of variants that are potentially associated with traits under selection in Nelore cattle, particularly in genome regions harboring QTLs affecting production traits.

Small-plasmid-mediated antibiotic resistance is enhanced by increases in plasmid copy number and bacterial fitness

Plasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogen Haemophilus influenzae carrying the small plasmid pB1000 conferring resistance to β-lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise to H. influenzae populations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.

Genomic microarrays in chromosomal analysis of leukemia

Chromosomal alterations in leukemia have been shown to have prognostic and predictive significance and are also important minimal residual disease (MRD) markers in the follow-up of leukemia patients. Although specific oncogenes and tumor suppressors have been discovered in some of the chromosomal alterations, the role and target genes of many alterations in leukemia remain unknown. In addition, a number of leukemia patients have a normal karyotype by standard cytogenetics, but have variability in clinical course and are often molecularly heterogeneous. Cytogenetic methods traditionally used in leukemia analysis and diagnostics; G-banding, various fluorescence in situ hybridization (FISH) techniques, and chromosomal comparative genomic hybridization (cCGH), have enormously increased knowledge about the leukemia genome, but have limitations in resolution or in genomic coverage. In the last decade, the development of microarray comparative genomic hybridization (array-CGH, aCGH) for DNA copy number analysis and the SNP microarray (SNP-array) method for simultaneous copy number and loss of heterozygosity (LOH) analysis has enabled investigation of chromosomal and gene alterations genome-wide with high resolution and high throughput. In these studies, genetic alterations were analyzed in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). The aim was to screen and characterize genomic alterations that could play role in leukemia pathogenesis by using aCGH and SNP-arrays. One of the most important goals was to screen cryptic alterations in karyotypically normal leukemia patients. In addition, chromosomal changes were evaluated to narrow the target regions, to find new markers, and to obtain tumor suppressor and oncogene candidates. The work presented here shows the capability of aCGH to detect submicroscopic copy number alterations in leukemia, with information about breakpoints and genes involved in the alterations, and that genome-wide microarray analyses with aCGH and SNP-array are advantageous methods in the research and diagnosis of leukemia. The most important findings were the cryptic changes detected with aCGH in karyotypically normal AML and CLL, characterization of amplified genes in 11q marker chromosomes, detection of deletion-based mechanisms of MLL-ARHGEF12 fusion gene formation, and detection of LOH without copy number alteration in karyotypically normal AML. These alterations harbor candidate oncogenes and tumor suppressors for further studies.

The mitochondrial and autosomal mutation landscapes of prostate cancer

BACKGROUND: Prostate cancer (PCa) is the most common cancer in men. PCa is strongly age associated; low death rates in surveillance cohorts call into question the widespread use of surgery, which leads to overtreatment and a reduction in quality of life. There is a great need to increase the understanding of tumor characteristics in the context of disease progression.

OBJECTIVE: To perform the first multigenome investigation of PCa through analysis of both autosomal and mitochondrial DNA, and to integrate exome sequencing data, and RNA sequencing and copy-number alteration (CNA) data to investigate how various different tumor characteristics, commonly analyzed separately, are interconnected.

DESIGN, SETTING, AND PARTICIPANTS: Exome sequencing was applied to 64 tumor samples from 55 PCa patients with varying stage and grade. Integrated analysis was performed on a core set of 50 tumors from which exome sequencing, CNA, and RNA sequencing data were available.

OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Genes, mutated at a significantly higher rate relative to a genomic background, were identified. In addition, mitochondrial and autosomal mutation rates were correlated to CNAs and proliferation, assessed as a cell cycle gene expression signature.

RESULTS AND LIMITATIONS: Genes not previously reported to be significantly mutated in PCa, such as cell division cycle 27 homolog (Saccharomyces cerevisiae) (CDC27), myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3), lysine (K)-specific demethylase 6A (KDM6A), and kinesin family member 5A (KIF5A) were identified. The mutation rate in the mitochondrial genome was 55 times higher than that of the autosomes. Multilevel analysis demonstrated a tight correlation between high reactive-oxygen exposure, chromosomal damage, high proliferation, and in parallel, a transition from multiclonal indolent primary PCa to monoclonal aggressive disease. As we only performed targeted sequence analysis; copy-number neutral rearrangements recently described for PCa were not accounted for.

CONCLUSIONS: The mitochondrial genome displays an elevated mutation rate compared to the autosomal chromosomes. By integrated analysis, we demonstrated that different tumor characteristics are interconnected, providing an increased understanding of PCa etiology.

ATM down-regulation is associated with poor prognosis in sporadic breast carcinomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genomic screening of testicular germ cell tumors from monozygotic twins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Down-regulation of SLC8A1 as a putative apoptosis evasion mechanism by modulation of calcium levels in penile carcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)