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NHGRI s Irini Manoli Describes a New Array for Mitochondrial Research

Irini Manoli
Research Fellow, Human Biochemical Genetics Section,
National Human Genome Research Institute

Name: Irini Manoli

Title: Research Fellow, Human Biochemical Genetics Section, National Human Genome Research Institute

Professional Background: 2005 — present, fellow in biochemical genetics, Human Biochemical Genetics Section, National Human Genome Research Institute, NIH; 2002 — 2005, research fellow, Endocrine Section, National Center for Complementary and Alternative Medicine, NIH; 1999 — 2002, resident, pediatrics, Athens University Children's Hospital, Greece; 1997 — 1998, senior house officer, neonatology, John Radcliffe Hospital, Oxford, UK; 1996 — 1997, senior house officer, pediatrics, Basildon and Thurrock Hospital, Essex, UK.

Education: 2002 — MSc, Pediatric Endocrinology, University of Athens, Greece; 1994 — MD, University of Athens, Greece.

A paper published this month in The Pharmacogenomics Journal, [Alesci S, Manoli I, Michopoulos VJ, et al. Development of a human mitochondria-focused cDNA microarray (hMitChip) and validation in skeletal muscle cells: implications for pharmaco- and mitogenomics. Pharmacogenomics J. 2006 Mar 14; (e-pub ahead of print)], describes how researchers at the National Institutes of Health, the University of Athens, Greece, and George Washington University developed a human mitochondria-focused cDNA microarray, or hMitChip, to study the contribution of mitochondrial genomics to human physiology.

Specifically, a 501-gene array was designed using mitochondria-related nuclear genes, and was validated by detecting gene expression changes in the mitochondrial nuclear transcriptome in human skeletal cells, after being treated with dexamethasone, a synthetic glucocorticoid that is often used to treat patients with mitochondrial disorders. Because such a tool may have applicability in research done in common disease areas such as obesity, diabetes, and cancer, BioArray News spoke with paper co-author Irini Manoli this week to discuss hMitChip's utility as a discovery tool.

You have an extensive background in both neonatology and pediatrics. Are there any diseases or conditions especially relevant in pediatrics that inspired you to undertake this work?

I have been trained in pediatrics in both Greece and England, and I have a special interest in patients with metabolic disorders, particularly mitochondrial diseases in children. These disorders are quite rare, with an incidence of 10-15 in 100,000. They are severe and often lethal and they present as encephalomyopathies, but can also affect any other tissue in the body at any age, and have a particular mode of inheritance. Another characteristic is that they have a huge phenotypic variability, so the same mutation can cause different phenotypes and vice versa. Mitochondrial diseases are fascinating and very complex and we don't understand very much about them. So any tool that can help us understand the variant phenotypes and the underlying pathophysiology of these rare genetic diseases would be helpful.

I should also mention that mitochondrial dysfunction is increasingly recognized in more common conditions like obesity, diabetes, and neurodegenerative disorders, like Alzheimer's and Parkinson's disease, and cancer. So there is a very wide spectrum of diseases. in pediatrics, and more generally, that could be studied further with our chip.

When did you start working with this particular technology?

Three years ago when I came to NIH. It was developed before I came here by [co-authors and fellow NIH researchers] Salvatore Alesci and George Chrousos and [George Washington University researcher and co-author] Yan Su. Dr. Su printed the chips for us at Georgetown University at the time.

I worked at the National Center for Complementary and Alternative Medicine (NCCAM), where we were interested in using this technology to study for example the effects of dietary supplements that are advertised as anti-aging or energy boosting agents, effects that have obviously high relevance to mitochondrial function.

What was your role in the work that was done with this team?

I did the work with the dexamethasone-treatment of human skeletal myocyte cultures and I performed all the microarray experiments basically, although I wasn't involved in the design and the development of this version of the chip. This first pilot in vitro application helped us discover things that we wanted to improve, and we designed the next generation of the chip based on that, as is mentioned in the paper.

Which tools did you use to scan the chips and for the data analysis?

We used the ScanArray from PerkinElmer to scan the chips. We developed our own software (Excel Macro and File Maker Pro based) to evaluate individual hybridizations, make sure duplicate spots on the chip have good correlation, as well as a database to upload the data from all our experiments . Both were developed by Yan Su, who also did the statistical analysis.

What are the advantages of using something like this chip over a PCR test?

PCR is more focused on genotyping, and we don't have SNPs or mutations included in this chip, this is a cDNA microarray, so we look at gene expression levels. It could be compared to quantitative RT-PCR, and I say that because it is a small chip, it has only 500 genes. It is like a large multi- RT-PCR experiment, really. In one hybridization step you can compare expression levels of your two sets of samples. It's looking at genes at the RNA level than looking at mutations or alterations at the DNA level like with PCR techniques.

Would you characterize this as a discovery tool?

It's mainly focused for research purposes. It can be used on patient samples if one would like to study how mitochondrial genes are expressed in a particular disease or on different experimental models. For example, you can study trans-mitochondrial hybrids, a particular technique used for mitochondrial research and look into different mutations and their effect on mitochondria. So, yes, it's mainly research oriented.

For the paper you used dexamethasone to look at the effect it had on the nuclear mitochondrial transcriptome in human skeletal muscle cells. Why was this done to evaluate the validity of the chip?

We are very interested in pharmaco-mito-genomics because different medications can have effects on the mitochondria. But dex in particular is known to cause, after prolonged exposure and high dosage, a specific myopathy, called steroid myopathy — which presents itself with loss of skeletal mass and function.

Steroids are used for many anti-inflammatory purposes, for example in patients with rheumatic arthritis, asthma, or leukemia. and the mechanism underlying the steroid-induced myopathy is relatively unknown. On the other hand they are used sort of paradoxically in Duchenne muscular dystrophy, and some mitochondrial disorders. It's somehow controversial how they act there because you give glucocorticoids to improve the myopathy. There is also previous evidence from in vitro and clinical studies suggesting that on one hand short term exposure can lead to increased mitochondrial biogenesis and up-regulation of some genes but long-term exposure can cause myopathy and loss of muscle.

So we wanted to explore the mitochondrial involvement in steroid myopathy with this unique tool where you can study a great selection of nuclear genes encoding the different enzymes that could be involved in mitochondrial function. We believe this is a relevant clinical question to address. So we used human muscle cells,. and short versus longer periods of exposure to dex in our experiment.

What was the conclusion?

I would summarize it by saying that dex seems to have a bimodal effect. In the early phase you have induction of transcription of genes related to stress response like heat shock proteins, and polyamines, oxidative phosphorylation and protein processing. Then with the longer exposure we saw down-regulation of lipid metabolism-related genes, of GABPA, which is a transcription factor that is involved in mitochondrial gene regulation and induction of apoptosis. These findings are consistent with what was previously described, while we identified new targets of dex, like monoamine oxidase, an enzyme leading to increased hydrogen peroxide production and oxidative stress, that we studied more extensively.

How is the next generation of the chip different from the one you used for this experiment?

The primary alteration is that we made cDNA clones for the mitochondrial DNA-encoded genes, so that was an important addition We have also used data from recent proteomic studies and have included genes, for example, that are published in the Mitoproteome database, while we enriched our collection with genes in pathways of interest, like mitochondrial biogenesis. The new chip contains about 1,000 mitochondria-related genes.

How do you make something like this available to more researchers?

Since we are at the NIH, we only use it for our research and in collaboration with partners for now. People that were interested in using this technology would have to collaborate with us.

Are there some other questions that you would like to answer using the chip?

One can use it for other pharmaco- and nutri-genomic studies,. We are coming to an era where more effort is being put into the development of drugs that affect and improve mitochondrial function and biogenesis, as possible agents for insulin resistance, neurodegenerative diseases and cancer and we think our chip can be used in those kinds of studies.

We would also like to use the chip for studies on patients with mitochondrial disorders to try and understand genotype to phenotype correlation and mechanisms of disease in these patients.

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