How to find a disease signature in DNA

The German Center for Neurodegenerative Diseases or DZNE is part of the Helmholz Association and one of the German Centers for Health Research established to combat the most important common diseases. The DZNE investigates the causes of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease among others and develops novel strategies for prevention, treatment, and patient care. There are currently ten DZNE sites that each have their own research focus. The DZNE in Tübingen has a strong focus on genetics and functional studies for disease together with clinical medical professional.

Prof. Dr. Peter Heutink’s background is in genetics and over the years his research group has identified genes and risk factors for many diseases including Parkinson’s disease and Frontal temporal dementia. They continue to search for genetic risk factors for these diseases and to investigate the functional consequences of the mutations in post-mortem human brain samples and cell-based models. I discussed Prof. Dr. Peter Heutink’s research with him and a briefly edited version of our conversation follows.

Prof. Dr. Peter Heutink, can you tell me what exactly is genome variation?

Each human being is a unique individual. The blueprint for a human being is written in her or his genome. Together with influences from the environment, this blueprint determines the phenotype of an individual. There is not a single human genome but the individual genomes of the human population each contain millions of variations in its DNA. New DNA variants keep occurring through new mutations, which can be transmitted to children and in this way spread in the human population. Some of these variants are beneficial and because genetics and evolution are tightly bound to each other, they can increase the evolutionary fitness and can become frequent in our population. Some of them have minute effects or are neutral and some unfortunately are damaging and might even cause a disease. The last category usually remains rare because they reduce the evolutionary fitness.

Why do you study genome variation?

Neurodegenerative diseases are very common diseases of the elderly population and there is currently no effective therapy or cure available. Genetic mutations play an important role in the risk for developing neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Mutations that are biologically damaging can have such a strong effect that they cause a disease without other influencers. Although they are usually rare in the general population you find them in a few families where the disease follows a Mendelian inheritance pattern. Other mutations have a much smaller effect and by themselves are not strong enough to cause a disease and they can be quite frequent in the population because there is no evolutionary selection against them. However, if an individual carries several of these risk factors and is exposed to environmental risk factors as well, the combination could also lead to disease.

We try to identify genetic risk factors because they give us a unique handle to study the mechanisms that result in a disease. The mutations are the direct cause of the disease and we carry genetic mutations with us our whole life from the moment of conception, therefore they function at the very beginning of the disease process. The mutations are therefore an ideal starting point for research that aims to understand the mechanisms that lead to a disease and to start to work towards developing a therapy to fix or prevent the problem.

How exactly do you study genetic risk factors?

In genetics we look for genome variants that are shared between patients, but not shared with healthy individuals. There are many ways to look at DNA and to study variations in our genome but nowadays the most widely used techniques are DNA sequencing and genotyping.

The field of genetics has benefitted greatly from the technological developments that are the direct result of the Human Genome project. The goal of the Human Genome project was to sequence a complete human genome but the technology to do this efficiently did not exist at the start of the project in 1990. It took 10 years to complete the first draft of the human genome. Now, we can sequence a full genome in a matter of days. This allows us to compare the complete genomes of patients and healthy individuals and identify the differences. The costs for whole genome sequencing are still very high but there are good alternatives that work well for a fraction of the costs. Based on the variation that has been found in all the sequencing efforts on the human population we have collected 100 thousands variants that have been spotted on glass arrays that we can use to find regions of the genome shared much more frequently by patients then by healthy individuals. With these arrays we can now test thousands of individuals to search for genetic risk variants using what we call Genome Wide Association Studies (GWAS).

How is it possible to link genome mutations with a disease?

For Mendelian diseases, where a single mutation is enough to cause disease, we search for variants that are present in patients but not in healthy individuals. Often we have families available with multiple affected individuals and the variants should co-segregate with the disease. Once we have a list of these variants we use bioinformatics approaches to determine which of these variants are likely to have a damaging effect. Finally, we will test the identified variants in a cellular model system to confirm their damaging effect and study the mechanism by which they could cause the disease.

For weaker risk factors the situation is more complicated because a single risk factor is not sufficient to cause the disease and therefore also many healthy people in the population can carry some of the risk variants. We therefore have to use statistical methods and large numbers of patients and healthy individuals to find variants that are significantly more present in disease cases versus healthy controls. A second complication is that since whole genome sequencing on thousands of samples is still very, very expensive and we have to use genotyping arrays. With these arrays we can find a region where the risk variant is located using GWAS but we can’t detect the variant itself. In fact these regions often contain multiple genes and it remains difficult to prove which gene and risk variant is responsible for the increased risk. Our lab uses a series of approaches to resolve this problem. We can sequence the region identified by GWAS in our study samples and then use bioinformatics to identify those variants that are most likely to have a functional effect. Unfortunately, the knowledge of all the functions encoded in the human genome is still limited, especially for the noncoding part of the genome which is more the 98% of our genome. We work tirelessly to improve this by participating in international consortia such as the FANTOM consortium that has helped to find many new noncoding RNA genes, microRNAs and regulatory elements, such as enhancers, and we use this information to help find the most likely gene or variant that increases the risk for a given disease.

Although these approaches help they are still predictions and we need to obtain functional evidence from wet lab experiments. We cannot do this using classic cell biology anymore. With the speed by which we identify genetic risk factors, this would be completely impractical. We have identified hundreds of genes to investigate and therefore our lab has developed a fully automated cell culture system in which we can perform functional studies in high throughput. We can culture clonal cell lines such as neuroblastoma cells as well as patient derived induced Pluripotent Stem cells (iPSc) to study the effect of the identified mutations in detail by using, for example, microscopic assays but also transcriptome, epigenome and proteomic assays. It has taken time to develop this approach but last year we published our first systematic screen for new genes for Parkinson’s disease that was lead by a talented PhD student, Iris Jansen. We are using this approach now as a tool to investigate all the genes we identify with our genetic studies and in this way select the genes that are truely influencing disease risk.

What are the practical applications of these findings in clinics?

For genes associated with Mendelian forms of disease, a test can be developed that can be used to diagnose the disease.

But the main benefit of identifying disease genes is that they can serve as a starting point to study the molecular processes that lead to a disease.

The genes identified by GWAS can be used to study the underlying biology of a disease and help to find new targets to develop a therapy that can either slow down or stop the disease process.

Although the path from finding a genetic risk factor to developing a therapy is a long and slow process, I think we live in exciting times. Genetics has contributed a lot to our understanding of disease and has often guided researchers in developing model systems or choosing targets for therapy development. We keep finding new risk factors that sometimes confirm the mechanisms we work on, but also often show us new pathways that we did not consider before that have then opened up completely new lines of research towards new therapy. I am confident that in the coming years we will finally be able to develop effective therapeutic approaches that will benefit the patients and that genetics will play a crucial role in this.

Thank you Prof. Dr. Peter Heutink for taking the time to talk with the Neuromag about your research!

Anastasia Illarionova is a GTC master student in the DZNE in the lab of Prof. Dr. Peter Heutink.


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