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Research on rare genetic disorders can be utilised in understanding the mechanisms behind even more common diseases

The population in the north of Finland has a unique genetic background. This has enabled the study of rare diseases and the discovery of new genes, proteins and reaction paths.

Docent Reetta Hinttala from the University of Oulu and Johanna Uusimaa, Professor of Pediatric Neurology, have discovered mutations in the NHLRC2 gene as a cause of a severe childhood multiorgan disease. Currently, the function of NHLRC2 in cells is unknown, but it has been identified as crucial to embryonic development.

Reetta Hinttala studies hereditary neurological childhood diseases at the University of Oulu’s Faculty of Medicine. The children suffering from FINCA disease were found to have previously uncharacterised connective tissue formation in their lungs, degeneration of neuronal cells, and increased angiogenesis in the brain. The disease manifested at the age of two months, and progressed quickly.

“Since this was a new combination of symptoms, we suspected that it must indicate a new disease,” says Reetta Hinttala.

The patients’ exomes, i.e. genomic areas encoding proteins, were sequenced. Based on what we currently know, the majority of variants that cause diseases are within those areas that only cover around 1.5% of our genome.

“Exome sequencing revealed changes, variants, in the nucleotide order of the NHLRC2 gene. At that time, this gene or its variants had not been linked to any human disease or even described in scientific publications. So we started studying the protein encoded by the gene, particularly focusing on its function in cells.”

Help from the mouse model

In FINCA disease, mutations in the NHLRC2 protein cause severe tissue fibrosis and degeneration of nerve cells. Cell culture models can be used to study the role of the NHLRC2 gene in the emergence of nervous diseases. The image shows a cell culture model used to study the effects of the FINCA mutation on developing nerve cells in particular. The cell model consists of neural progenitor cells (NPC) isolated from fetal mouse brain. The effect of the mutation on nerve cells is studied by comparing nerve cells isolated from a FINCA mouse to those from a wild type mouse. These cells can also be modified by gene transfer. Some of the cells in the picture are colored green, because they express green fluorescent protein (GFP), that has entered them through gene transfer. The cell culture contains both transgenic and non-transgenic cells. The red color, on the other hand, indicates neuron-specific form of tubulin protein, which appears normally in all nerve cell cytoskeletons. DNA-binding dye has stained the nuclei of all the cells blue.

When you want to find out how a disease emerges and develops in humans, the only way of doing this is to study it in living organisms. Only then can we monitor what the protein that causes a disease is doing in the organism. In this particular case, we created a mouse model with the same mutation combination as the patients. A knock-out mouse, in which the NHLRC2 gene has been completely turned off, was obtained from the EMMA repository of the Infrafrontier infrastructure. EMMA (The European Mouse Mutant Archive) archives genetically modified mouse strains from around the world. In addition to this, Hinttala and her team used the CRISPR-Cas9 technology to create the identical point mutation that was observed in FINCA patients, to the mouse. By crossing the mouse carrying point mutation with the knock-out mouse, a model was created that has the same mutation combination as the FINCA patients.

“We are using our mouse model to determine the role of NHLRC2 in the development of the central nervous system in particular, through a project funded by the Academy of Finland. At tissue level, we have observed encouraging signs that the mouse model is exhibiting features similar to the FINCA disease, but it will still take some time to build a comprehensive picture of the phenotype.”

Cellular changes resulting from disturbances in the gene causing the disease were studied in the patients’ fibroblasts, using a transmission electron microscope, at Biocenter Oulu’s electron microscopy core facility. Fibroblasts are the main cell type present in connective tissue and they are specialized in secreting extracellular matrix proteins.

“The first indication that a mutation, or variant, is harmful is when the variant changes the protein’s amino acid code. In the patients of our study, mutations of the NHLRC2 gene indeed changed the code. Using cultured fibroblast from patients’ skin biopsies, we checked the expression of the NHLRC2 protein. Fibroblasts from healthy persons’ skin biopsies had a normal level of NHLRC2 protein whereas, owing to mutations, the protein had been almost completely eliminated in the FINCA patient derived cells. This would indicate that the mutations change the structure of the NHLRC2 protein so radically that the cell attempts to break up the harmful protein. This enabled us to ensure that these were not neutral variants.

As a result, the study discovered a gene that was necessary to normal embryonic development. The researchers drew the conclusion that the gene is vital during the first cell divisions in the mouse. Findings in FINCA patients also showed that the NHLRC2 protein plays a key role in maintaining a normal function of several organs in humans.

Cellular signalling paths provide valuable information

Researchers solved the crystal structure of NHLRC2 (Biterova et al. 2018).

The study of rare diseases may also lead to discovering the causes of more common diseases, specifically proteins with previously unknown mechanisms.

“By studying a rare disease, there is a high probability that we will discover reaction paths that play a role also in more common diseases.”

Cell functions are based on biochemical reactions. Reaction paths are either turned on or off, depending on the function of the cell. Cells change their behaviour on the basis of messages they receive from their environment. For example, when a hormone attaches to a receptor on the cell surface, the receptor activates a certain molecule inside the cell, which will carry the signal further. The signal often ends up in the nucleus and controls the reading of genes . There are large numbers of signalling paths. Cancer cells, for example, do not react to many of the messages intended for them. Instead, they strengthen signalling paths that cause the cell to divide, and therefore help the tumour to grow.

The NHLRC2 protein has an effect on many cell reaction paths and events. For the first time, researchers were able to show that a dysfunction of NHLRC2 leads to changes in the cytoskeleton and the formation of vesicles within cells. The cytoskeleton plays a major role in multiple cell functions, and without it the cells would not be able to survive.

According to Hinttala, the study of rare diseases can result in discoveries that are important to basic cellular biology. It is also valuable to learn how dysfunction of genes and proteins manifests in humans.

“With FINCA, a dysfunction of the NHLRC2 protein will result in serious tissue fibrosis and degeneration of neurons. More common diseases, such as liver cirrhosis and Alzheimer’s, have similar tissue manifestations.”

According to Hinttala, the study of rare diseases involves a high probability of discovering the reaction paths that also lead to more common diseases.”

“Severe childhood diseases are rarely caused by environmental factors, but the role played by such factors complicates the study of the adult-onset disease mechanisms . The diseases we are studying are most probably hereditary and primarily caused by a dysfunction of a single gene.”

Ari Turunen

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