Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number

Mechanisms of mitochondrial DNA (mtDNA) maintenance have recently gained wide interest owing to their role in inherited diseases as well as in aging. Twinkle is a new mitochondrial 5'-3' DNA helicase, defects of which we have previously shown to underlie a mitochondrial disease, progressive external ophthalmoplegia with multiple mtDNA deletions. Mouse Twinkle is highly similar to the human counterpart, suggesting conserved function. Here, we have characterized the mouse Twinkle gene and expression profile and report that the expression patterns are not conserved between human and mouse, but are synchronized with the adjacent gene MrpL43, suggesting a shared promoter. To elucidate the in vivo role of Twinkle in mtDNA maintenance, we generated two transgenic mouse lines overexpressing wild-type Twinkle. We could demonstrate for the first time that increased expression of Twinkle in muscle and heart increases mtDNA copy number up to 3-fold higher than controls, more than any other factor reported to date. Additionally, we utilized cultured human cells and observed that reduced expression of Twinkle by RNA interference mediated a rapid drop in mtDNA copy number, further supporting the in vivo results. These data demonstrate that Twinkle helicase is essential for mtDNA maintenance, and that it may be a key regulator of mtDNA copy number in mammals.     

Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast

Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although the underlying mechanisms are unclear. In budding yeast, certain levels of reactive oxygen species (ROS) can promote recombination-mediated mtDNA replication, and mtDNA maintenance depends on the homologous DNA pairing protein Mhr1. Here, we show that the fusion of isolated yeast mitochondria, which can be monitored by the bimolecular fluorescence complementation-derived green fluorescent protein (GFP) fluorescence, increases the mtDNA copy number in a manner dependent on Mhr1. The fusion event, accompanied by the degradation of dissociated electron transport chain complex IV and transient reductions in the complex IV subunits by the inner membrane AAA proteases such as Yme1, increases ROS levels. Analysis of the initial stage of mitochondrial fusion in early log-phase cells produced similar results. Moreover, higher ROS levels in mitochondrial fusion-deficient mutant cells increased the amount of newly synthesized mtDNA, resulting in increases in the mtDNA copy number. In contrast, reducing ROS levels in yme1 null mutant cells significantly decreased the mtDNA copy number, leading to an increase in cells lacking mtDNA. Our results indicate that mitochondrial fusion induces mtDNA synthesis by facilitating ROS-triggered, recombination-mediated replication and thereby prevents the generation of mitochondria lacking DNA.     

Mitochondrial DNA copy number and cytogenetic damage among fuel filling station attendants

Fuel filling attendants are constantly exposed to the complex mixture of gasoline and all refinery environments are probably carcinogenic for humans. These workers are considered as an unorganized group in India and unaware of the risk. The present study was focused to monitor workplace pollutants (particulate matter size 10 [PM10 μm], total volatile organic compound [VOC], and carbon monoxide [CO]), benzene exposure (phenol), and to evaluate their genotoxicity effect with reference to relative mitochondrial DNA copy number (MtDNAcn), 8-OHdG (8-hydroxy-2′-deoxyguanosine), and micronuclei (MN) frequency (%) among fuel filling attendants. This study recorded 318 ± 134 and 1,050 ± 260 μg/m³ time-weighted average concentration of PM10 and CO, respectively. However, total VOC levels recorded were below the detectable level (BDL) to 290 ± 50 μg/m³. A total of 53 subjects (26 exposed and 27 control) participated in this study with similar sociodemographic information. It was noticed that fuel filling attendants were not using proper personal protective equipment (PPE) and are younger generation. The significantly (p = <.001) higher level of phenol, a metabolite of benzene, was detected in the exposed group. The significantly elevated level of urinary 8-OHdG (p = .01), MN frequency (p = .001), and relative MtDNAcn (p = .001) was observed in exposed group as compared to the control group. The study exemplify that workers were exposed to the benzene, workplace pollutant, and observed genotoxicity suggest malignancy risk. This study highlights the importance of biomonitoring in occupational settings to avoid malignancies. The possible engineering controls, frequent health check-ups, awareness about the risks, and PPE use can reduce health hazards.     

Recurrent copy number gain of transcription factor SOX2 and corresponding high protein expression in oral squamous cell carcinoma

Gene copy number aberrations are involved in oral squamous cell carcinoma (OSCC) development. To delineate candidate genes inside critical chromosomal regions, array‐CGH was applied to 40 OSCC specimens using a microarray covering the whole human genome with an average resolution of 1 Mb. Gene copy number gains were predominantly found at 1q23 (9 cases), 3q26 (11), 5p15 (13), 7p11 (7), 8q24 (17), 11q13 (15), 14q32 (8), 19p13 (8), 19q12 (7), 19q13 (8), and 20q13 (9), whereas gene copy number losses were detected at 3p21‐3p12 (15), 8p32 (11), 10p12 (8), and 18q21‐q23 (10). Subsequent mRNA expression analyses by quantitative real time polymerase chain reaction found high mRNA expression of candidate genes SOX2 in 3q26.33, FSLT3 in 19p13.3, and CCNE1 in 19q12. Tissue microarray (TMA) analyses in a representative OSCC collection found gene copy number gain for SOX2 in 52% (115/223) and for CCNE1 in 31% (72/233) of the tumors. Immunohistochemical analyses on TMA sections of the corresponding proteins detected high expression of SOX2 in 18.1% (49/271) and of CyclinE1 in 23.3% (64/275) of tumors analyzed. These findings indicate that SOX2 and CCNE1 might be activated via gene copy number gain and participate in oral carcinogenesis. The combination of array‐CGH with TMA analyses allows rapid pinpointing of novel promising candidate genes, which might be used as therapeutic stratification markers or target molecules for therapeutic interference. © 2009 Wiley‐Liss, Inc.     

Gene copy number regulates the production of the human chemokine CCL3-L1

Genetic variation in the chemokine system is likely to affect responses to infection, and influence the course of autoimmune and inflammatory disease. We and others have shown that the human beta-chemokine CCL3-L1, unlike its related non-allelic isoform CCL3, has high affinity for the chemokine receptors D6, CCR3 and CCR5. Moreover, CCL3-L1, but not CCL3, is susceptible to cleavage by CD26, creating a truncated -2 form with enhanced affinity for CCR1 and CCR5. Strong interaction with CCR5 means that CCL3-L1, and particularly its -2 variant, are by far the most potent natural HIV entry inhibitors described to date. Here, using real-time PCR we have shown that CCL3-L1 and a novel CCL4 isoform (termed CCL4-L1) can vary from 1-6 copies per diploid genome (pdg) in Caucasians and are occasionally completely absent. The other isoforms (CCL3 and CCL4) remain at two copies per dpg. Importantly, in a model system of pro-inflammatory chemokine production (LPS-activated monocytes)higher gene copy number correlates with an increased ratio of CCL3-L1 versus CCL3 mRNA, and enhanced chemokine production. Supernatants from samples with high copy number are able to more potently chemoattract CCR5-expressing cells, an effect blocked with anti-CCL3/CCL3-L1 antibodies. As a result of these studies, we hypothesize that genetic variation in CCL3-L1 gene copy number may affect the susceptibility to, or the progression or severity of, diseases in which this chemokine plays a role.     

Epidermal growth factor receptor expression and gene copy number in conventional hepatocellular carcinoma

Epidermal growth factor receptor (EGFR) is frequently overexpressed in hepatocellular carcinoma, but its relationship with EGFR gene copy number has not been studied. This study examined EGFR expression and gene copy number in hepatocellular carcinoma and evaluated their relationship to clinicopathologic features in 76 tumors. Moderate to strong expression of EGFR was observed by immunohistochemical analysis in 50 (66%) of 76 hepatocellular carcinomas. Fluorescence in situ hybridization (FISH) showed extra EGFR gene copies in 17 (45%) of 38 tumors. This was accompanied by gains of chromosome 7, indicating that this was the result of balanced polysomy rather than gene amplification. There was no correlation between EGFR expression by immunohistochemical analysis and gene copy number by FISH. EGFR expression showed borderline association with cirrhosis but not with other clinicopathologic parameters examined. EGFR overexpression is present in a majority of hepatocellular carcinomas, suggesting a role for EGFR antagonists in therapy. The increased expression does not correlate with an increase in the EGFR gene copy number.     

Epidermal growth factor receptor expression and gene copy number in fibrolamellar hepatocellular carcinoma

Increased expression of epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase, is associated with tumor progression in many carcinomas. Epidermal growth factor receptor inhibitors have shown promise in treating some of these tumors. Fibrolamellar hepatocellular carcinoma (FL-HCC) is an aggressive neoplasm that occurs in young patients with no history of cirrhosis. This study examines the expression and gene copy number of EGFR in FL-HCC. Formalin-fixed, paraffin-embedded FL-HCC (n = 13) sections were stained with a monoclonal antibody against EGFR. Fluorescence in situ hybridization analysis was performed using probes against EGFR gene and centromeric region of chromosome 7 (CEP 7). Epidermal growth factor receptor and CEP 7 signals were counted in 50 tumor nuclei per case as well as 300 normal hepatocyte nuclei. The EGFR to CEP 7 signal ratio was calculated for each case. Most (92%, 12/13) of FL-HCC showed strong and diffuse staining with anti-EGFR antibody. Fluorescence in situ hybridization was informative in 11 cases, 10 of which showed extra EGFR gene copy numbers (mean, 3.69; range, 3.13-5.0). Epidermal growth factor receptor was overexpressed in all these cases. The mean number of EGFR signals per cell in FL-HCC was double that of normal hepatocytes (3.69 versus 1.80); the mean EGFR/CEP 7 ratio in tumor cells was 1.05. In conclusion, EGFR is strongly overexpressed on the cell membrane in nearly all cases of FL-HCC. Similar gains of chromosome 7 are observed, indicating that the extra EGFR gene copies are due to polysomy rather than gene amplification. The strong expression of EGFR in FL-HCC tumors suggests that they may respond to treatment with EGFR antagonists.     

High-resolution copy number arrays in cancer and the problem of normal genome copy number variation

High-resolution techniques for analysis of genome copy number (CN) enable the analysis of complex cancer somatic genetics. However, the analysis of these data is difficult, and failure to consider a number of issues in depth may result in false leads or unnecessary rejection of true positives. First, segmental duplications may falsely generate CN breakpoints in aneuploid samples. Second, even when tumor data were each normalized to matching lymphocyte DNA, we still observed copy number polymorphisms masquerading as somatic alterations due to allelic imbalance. We investigated a number of different solutions and determined that evaluating matching normal DNA, or at least using locally derived normal baseline data, were preferable to relying on current online databases because of poor cross-platform compatibility and the likelihood of excluding genuine small somatic alterations.     

Mosaic copy number variation in schizophrenia

Recent reports suggest that somatic structural changes occur in the human genome, but how these genomic alterations might contribute to disease is unknown. Using samples collected as part of the International Schizophrenia Consortium (schizophrenia, n=3518; control, n=4238) recruited across multiple university research centers, we assessed single-nucleotide polymorphism genotyping arrays for evidence of chromosomal anomalies. Data from genotyping arrays on each individual were processed using Birdsuite and analyzed with PLINK. We validated potential chromosomal anomalies using custom nanostring probes and quantitative PCR. We estimate chromosomal alterations in the schizophrenia population to be 0.42%, which is not significantly different from controls (0.26%). We identified and validated a set of four extremely large (>10 Mb) chromosomal anomalies in subjects with schizophrenia, including a chromosome 8 trisomy and deletion of the q arm of chromosome 7. These data demonstrate that chromosomal anomalies are present at low frequency in blood cells of both control and schizophrenia subjects.