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Environmental Factor, March 2013

Intramural papers of the month

By Monica Frazier, Mallikarjuna Metukuri, Ajeet Singh, and Darshini Trivedi

New mouse model advances study of mitochondrial diseases

Researchers at NIEHS have determined that POLG2, the accessory subunit of the DNA polymerase gamma complex, is necessary for mammalian embryogenesis and mitochondrial DNA (mtDNA) replication. The finding will help scientists better understand disorders caused by mutations in mtDNA or depletion of mtDNA, such as Alpers’ disease, an illness that causes dementia, seizures, and liver failure.

The DNA polymerase gamma complex is made up of a catalytic subunit and an accessory subunit. While approximately 200 pathogenic mutations in the catalytic subunit, which lead to mitochondrial diseases, have been described, the accessory subunit had not been well characterized. The mouse model developed in this study is the first mammalian model of the Polg2 gene, and allowed the scientists to study the accessory subunit.

The researchers monitored mice that were heterozygous for Polg2 for two years and found them to be no different than wild-type mice. However, they determined that a homozygous knockout of Polg2 was embryonic lethal. Further investigation into the knockout revealed a loss of mtDNA, structural defects of the mitochondria, and respiratory-chain failure seen via the lack of cytochrome c oxidase I activity.

These results will be critical to the future development of additional mitochondrial disease mouse models, and in determining the susceptibility of mitochondria to environmental agents. (MF)

CitationHumble MM, Young MJ, Foley JF, Pandiri AR, Travlos GS, Copeland WC. 2013. Polg2 is essential for mammalian embryogenesis and is required for mtDNA maintenance. Hum Mol Genet 22(5):1017-1025.

A new method to analyze case-sibling studies

NIEHS investigators have proposed an improved approach to testing genetic susceptibility factors and gene-exposure interactions using siblings. It treats case-sibling data as nuclear family data for which the parents are missing, and uses a statistical missing data method, the expectation-maximization algorithm, to increase statistical power, by exploiting the fact that the case and their control share parents. The method also permits the investigator to enroll cases who lack a sibling control, and controls whose case sibling is unavailable, such as in cases of death.

Nuclear family-based designs are commonly used in genetic epidemiology to study complex diseases that involve both genetic variants and environmental exposures. When parental information is unavailable, as with diseases occurring late in life, siblings can serve as controls. Such case-sibling studies are typically analyzed using conditional logistic regression (CLR), where each case is matched with at least one unaffected sibling as control.

The NIEHS authors compared the traditional CLR with the missing-parents approach and found that the latter improved statistical efficiency, when at least some of the cases had two or more control siblings available, particularly when testing for genetic effects. Furthermore, the missing-parents approach provided additional improvements in power, through inclusion of unmatched cases and controls. (DT)

CitationShi M, Umbach DM, Weinberg CR. 2012. Case-sibling studies that acknowledge unstudied parents and permit the inclusion of unmatched individuals. Int J Epidemiol; doi:10.1093/ije/dys212.

Novel loci for lung function identified by using genome-wide joint meta-analysis

NIEHS researchers, along with collaborators, identified novel genetic loci in the most comprehensive meta-analysis to date of gene-by-smoking interaction in relation to pulmonary function.

Meta-analysis generally refers to methods that contrast and combine results from different studies, to identify patterns among study results and provide sufficient sample size to detect moderate effects. The authors conducted genome-wide joint meta-analyses (JMA) of single nucleotide polymorphism (SNP) main effects and SNP-by-smoking interaction associations, using ever-smoking or pack-years, with cross-sectional pulmonary function measures in 50,047 study participants of European ancestry.

Three novel gene regions, DNER, HLA-DQB1/HLA-DQA2, and KCNJ2/SOX9, which were not previously related to pulmonary function, were identified from the JMA. The authors evaluated DNER, KCNJ2, and SOX9 and found them to be expressed in human lung tissue. DNER and SOX9 were also differentially expressed in human airway cells in smokers compared to non-smokers. The authors did not check for the two HLA genes, given the known expression of this class in a range of airway cell types.

By using JMA, the authors identified loci that would have remained unknown using standard genome-wide association study approaches. This study demonstrated the importance of applying JMA to determine novel genetic risk factors that might further uncover the mechanisms leading to reduced pulmonary function. (MM)

CitationHancock DB, Artigas MS, Gharib SA, Henry A, Manichaikul A, Ramasamy A, Loth DW, Imboden M, Koch B, McArdle WL, Smith AV, Smolonska J, Sood A, Tang W, Wilk JB, Zhai G, Zhao JH, Aschard H, Burkart KM, Curjuric I, Eijgelsheim M, Elliott P, Gu X, Harris TB, Janson C, Homuth G, Hysi PG, Liu JZ, Loehr LR, Lohman K, Loos RJ, Manning AK, Marciante KD, Obeidat M, Postma DS, Aldrich MC, Brusselle GG, Chen TH, Eiriksdottir G, Franceschini N, Heinrich J, Rotter JI, Wijmenga C, Williams OD, Bentley AR, Hofman A, Laurie CC, Lumley T, Morrison AC, Joubert BR, Rivadeneira F, Couper DJ, Kritchevsky SB, Liu Y, Wjst M, Wain LV, Vonk JM, Uitterlinden AG, Rochat T, Rich SS, Psaty BM, O'Connor GT, North KE, Mirel DB, Meibohm B, Launer LJ, Khaw KT, Hartikainen AL, Hammond CJ, Gläser S, Marchini J, Kraft P, Wareham NJ, Völzke H, Stricker BH, Spector TD, Probst-Hensch NM, Jarvis D, Jarvelin MR, Heckbert SR, Gudnason V, Boezen HM, Barr RG, Cassano PA, Strachan DP, Fornage M, Hall IP, Dupuis J, Tobin MD, London SJ. 2012. Genome-wide joint meta-analysis of SNP and SNP-by-smoking interaction identifies novel loci for pulmonary function. PLoS Genet 8(12):e1003098.

The effects of sulfite-induced lesions in DNA

In a new study, NIEHS researchers developed a mutagenesis reporter system in yeast that specifically detects mutations arising from damage within single-strand DNA (ssDNA). ssDNA arises transiently during DNA replication and repair, as well as transcription. Since repair opportunities are fewer for ssDNA, it is more at risk than the more prevalent double-strand DNA. This study helps researchers to understand the molecular mechanisms of ssDNA-specific damage, which has been implicated in causing mutation clusters and up to 40 percent of all mutations in some cancers.

To validate their approach, the authors first showed that the human APOBEC3G, an enzyme that acts preferentially on ssDNA, converted cytosines to uracils in ssDNA, resulting in clusters of multiple mutations on the same DNA strand. They then treated cells with sulfites, a common environmental agent found in the atmosphere and food supply. Sulfites similarly caused multiple mutations on the same DNA strand, by damaging cytosines in ssDNA. Neither APOBEC3G nor sulfites caused mutations in double-strand DNA controls. Additionally, the chemically-modified uracils formed by sulfites were resistant to repair initiated by an enzyme that removes uracil from DNA.

This study highlights the first in vivo system to identify the genomic risk from environmental agents that can cause ssDNA damage and how they might contribute to disease including cancer. (AS)

CitationChan K, Sterling JF, Roberts SA, Bhagwat AS, Resnick MA, Gordenin DA. 2012. Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent. PLoS Genet 8(12):e1003149. 

(Monica Frazier, Ph.D., is an Intramural Research Training Award (IRTA) fellow in the NIEHS Laboratory of Molecular Genetics. Mallikarjuna Metukuri, Ph.D., is a research fellow in the NIEHS Laboratory of Signal Transduction. Ajeet Singh, Ph.D., is a visiting fellow in the NIEHS Laboratory of Molecular Carcinogenesis. Darshini Trivedi, Ph.D., is an IRTA fellow in the NIEHS Laboratory of Toxicology and Pharmacology.)

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