Explanation and illustration of the researcher Vittoria Cenni from the CNR of Bologna.

Scientific research on genetic diseases aims at identifying treatments to counter the onset and progression of the disease. Achieving this goal involves surpassing various levels of knowledge, listed below:

  1. The first level involves identifying the molecular mechanisms of the pathology responsible for altering the functioning of certain processes in the cell. These processes are key to life and their normal functioning.
  2. Once these alterations are identified, the second step is taken, in which pharmacological treatments are used to restore the correct functioning of the altered cell processes (identified in the first level). The validation of the efficacy of the drugs is conducted in the laboratory using cellular models (so-called in vitro experimentation), and subsequently using animal models (so-called in vivo experimentation). This phase can be particularly complex because the work done in the laboratory, under specific experimental conditions, implies the absence of alterations in some cellular functions that are present in other and different conditions. This can lead the researcher to misleading conclusions.
  3. When the results are unequivocal, clinical experimentation proper finally begins. The criteria that regulate the enrollment of patients, their number, and the methods of execution and administration of the drug are established by competent authorities, such as the AIFA (Italian Medicines Agency) or the FDA (Food and Drug Administration).

In addition to the activity of cellular and molecular biologists, preclinical studies, particularly those conducted in vitro, rely on the contribution of neurologists, surgeons, and bioinformaticians. In some cases, scientists expert in the field of medicine and nanotechnology are also involved.

Scientific research related to collagen VI deficiency diseases typically begins in the operating room, where a surgeon collects a very small tissue sample (usually skin, tendon, or muscle) from control donors (non-dystrophic patients hospitalized for various types of trauma due to road accidents, domestic incidents, or other causes) or from patients with mutations in the genes that produce collagen VI. It’s important to emphasize that the collection and use of biological material for research purposes are contingent on prior informed consent signed by the donor.

When a researcher wants to extend or include animal models in their study, a certain amount of tissue can be taken from wild-type animals (animals without any genetic mutations and thus considered healthy, used as control samples to be compared with animals that do not express collagen VI) or from animals that, through genetic engineering techniques, no longer produce collagen VI.

Mice are primarily used in these studies, but zebrafish (Danio rerio) are also utilized. The use of animal models is necessary when exploring the consequences of the complete absence of collagen VI on the functionality, morphology, and performance of the most affected organs, or when testing the efficacy of drugs and innovative treatments.

Tissue samples can be processed immediately for experimental evaluations, or stored in liquid nitrogen (-210°C) or formaldehyde for future evaluations. Alternatively, using standardized experimental protocols, the tissue fragments can be used to produce primary cell cultures. For example, muscle cultures will yield muscle cells (myoblasts) and muscle fibroblasts, skin will yield skin fibroblasts, and tendons will yield tenocytes and tendon fibroblasts. The purity of these cultures is usually confirmed through the presence of uniquely expressed proteins.

In the laboratory, cells grow and reproduce inside incubators at a constant temperature of 37°C, thanks to vitamin and growth factor-rich cocktails. The proliferative capacity of cultured cells is not infinite, so careful use is very important. To overcome this limitation, where possible, immortalized cell lines (which have characteristics similar to those of the tissues) can be used, available for purchase from certified companies.

To characterize the morphological and functional properties of the cells or tissues of patients, to understand the pathogenetic mechanisms caused by collagen VI deficiency in response to specific mechanical stimuli or pharmacological treatments, or to test the efficacy of specific drugs, cells or tissues can be subjected to a series of experimental investigations.

The most commonly used investigations include:

  • Scanning or Transmission Electron Microscopy (EM), which allows for the observation of nanometer-scale morphological alterations. To give an idea, the size of a tendon cell is around 100 micrometers, which corresponds to 100,000 nanometers.
  • Fluorescence and confocal microscopy (IF), which enable the evaluation of very small alterations and, in some cases, the creation of three-dimensional images of the observed structures. These investigations can use immunological reactions (biochemical reactions mediated by antibodies that help the researcher selectively identify certain molecules of interest) and fluorescent probes, which allow for the selective identification of certain proteins or organelles inside the cell, and possibly other molecules they bind to.
  • Biochemistry (WB), which allows for the analysis and quantification of alterations in terms of expression and activity of individual proteins, including collagen VI, or those involved in signaling mechanisms associated with cell proliferation, differentiation, or stemness.
  • RT-qPCR, which enables the assessment of the expression level of genes that encode key proteins or micro-RNAs, very small molecules capable of modulating the expression of other genes.
  • High-Throughput Analysis (HTS), extremely advanced bioinformatics techniques that allow for the simultaneous examination of a huge number of data obtained through genomics, transcriptomics, and proteomics approaches (the suffix “-omics” defines a large set of data related in this case to genes, transcripts (the RNA produced by genes), and proteins).

In summary, scientific research allows for the exploration and characterization of what regulates the onset and progression of diseases from a microscopic and molecular perspective and is the basis of clinical experimentation. It is extremely important to believe in and support research in every way, even when it does not seem to lead to sensational discoveries: an apparently insignificant evidence can represent the keystone to achieve great results.



Vittoria Cenni

Researcher at the Institute of Molecular Genetics of the CNR in Bologna

1 Tissue is a collection of highly specialized cells that ensure its maintenance and functions. Tissues also consist of an amorphous substance, called extracellular matrix, or ECM, which is produced and released by the tissue cells, and plays a structural role, as well as in protection and in the transmission of mechanical and biochemical signals between the cells themselves. In pathologies from collagen VI deficiency, which is a protein of the ECM, the tissues most affected are connective tissue, which includes the dermis, ligaments, and tendons, and muscle tissue.

2 Proliferation, differentiation, and stemness: Proliferation is the ability of cells to duplicate in response to specific proliferative stimuli that are activated during the growth or enhancement of the tissue to which they belong, or following a tissue trauma. Differentiation is the ability of cells to acquire well-defined functions. It is important to remember that differentiated cells lose the capacity to proliferate. Finally, stemness is the ability of some “dormant” cells to resume proliferating and differentiating, ensuring the restoration of heavily damaged tissue portions.