Condition of epic magnitude

Lately, Merogenomics has been focused on content related to the perturbation of menstrual cycles post COVID-19 vaccination or SARS-CoV-2 natural infection. Consequently what we are really discussing seems to be the interplay between the immune system and the hormonal balance that regulates the menstrual cycle (currently, it is not known what factors are exactly responsible for affecting the menstrual cycle).

This focus has brought to our attention a very important issue affecting an enormous number of women that appears to be linked to the immune system: endometriosis.

Endometriosis is the most frequently diagnosed “benign” chronic gynecological disorder; where a build-up of endometrial tissue outside the uterus (ectopic) is associated with chronic pelvic pain, painful menstruation and impaired fertility. These conditions often greatly deteriorate a woman’s quality of life, hence calling this condition “benign” for the many women afflicted with it does not seem proper.

However, it should be noted that the eutopic endometrium (residing in the correct place) is also recognized as distinctly different in women suffering from endometriosis when compared to healthy controls, and appears to play some role in the disease development.

The development of endometriosis is dependent on hormones - driven mainly by estrogen which is crucially involved in the thickening of the endometrium, and consequently endometriosis is found predominantly in women of reproductive age. Staggeringly, up to 50% of infertile women have endometriosis and it is estimated that it affects approximately 10% of all reproductive aged women, with one reported estimate claiming 175 million women worldwide are affected. Thus, it is a condition of overwhelming proportion and impact. But despite the increase in the studying of this condition, endometriosis still remains a fairly mysterious disease.

Both genetics and environmental factors seem to contribute to the disease. It is well known that daughters or sisters of patients with endometriosis are at increased risk of developing endometriosis. Based on twin studies, genetics are expected to account for approximately 50% of the endometriosis disease presentation, with environmental factors apparently playing an equal role, accounting for the other 50%. However, the genetic component, which has been investigated to a substantial degree, is what is referred to as polygenic or multifactorial in its inheritance. In other words, a combination of many small effect genetic alterations throughout the patient’s entire DNA that collectively contribute to the disease.

As indicated, the field of endometriosis genetics has been revealed to be complex with many candidate regions proposed to be contributing to the disease. These genetic regions play roles in: cellular proliferation, cellular differentiation, cellular migration, apoptosis, angiogenesis and inflammation. In other words, there appears to be many different ways to contribute to endometriosis from a genetic point of view, which at least to some degree could account for its frequency.

Due to the complexity, we will only focus on some of the findings - specifically those related the immune system.

Why the immune system?

Defective immune surveillance is an important part of endometriosis physiology. It is believed that the disease develops as a consequence of decreased clearance of the endometrial cells within the pelvic cavity due to the reduced immune surveillance to recognize and clear the cells.

It is currently suspected that cytotoxic killer immune cells such as natural killer (NK) cells or T-lymphocytes (T-cells) are supposed to be clearing these cells to prevent their implantation and growth outside the uterine cavity. In endometriosis, these cells are suppressed from performing their typical function possibly by the eutopic endometrium no less, hence why it was important to bring up this distinction in endometrium development.

Normally, NK cells are supposed to interact with intercellular adhesion molecule-1 (ICAM-1) at the surface of eutopic endometrium cells but instead during endometriosis the eutopic cells release soluble (not tethered to their cell surface) ICAMs that can bind to NK cells and prevent their direct interaction with the endometrial cells.

T-cells on the other hand might be prevented from proper function in a different manner by being induced to die by the eutopic endometrial cells as they exhibit the presence of specific receptors called FasL which when contacted by T-cells, signal these cells to undergo apoptosis (a specific type of cell death).

Other immune cells can also invade the area and promote inflammation through the release of their messenger molecules (cytokines).

Finally, the eutopic endometrium also itself undergoes reduced apoptosis as well, and collectively this appears to allow endometrial cells to then be able to produce a molecular footprint that helps these cells in: i) attaching to surface areas in other regions; ii) helping to disintegrate the extracellular matrix that would stop these cells from migrating to new tissue locations (using proteins called matrix metalloproteinases); or iii) promoting the supply of new blood flow (angiogenesis) through such molecules as vascular endothelial growth factor (VEGF), one of the most prominent contributors to the development of endometriosis.

These are just some examples of how complex and intertwined the disease state can be with the performance of the immune system and these specific examples were brought up to highlight how challenging it is to unravel endometriosis at the molecular level.

Inflammation and immunity-related genes in endometriosis

Let us jump now into the genetic contribution to the disease of endometriosis focusing specifically on the role of the immune system, however keeping in mind that numerous other genes outside that domain have been proposed as well.

Interleukin (IL)-16 is a pro-inflammatory cytokine, known to be associated with various ailments. The gene responsible for the production of this cytokine has mutations or variations that are different between women with endometriosis and healthy women. A genetic change means there is likely going to be some alteration taking place in the gene product or the protein that is being made based on the gene code template. Changes in the protein that can affect the natural biological function of that protein to some degree.

Also, there are different types of mutations or genetic variations. For example, a piece of genetic material can go missing, be duplicated, or change location. Genetic material is very malleable in such ways and so can produce a great number of possibilities as to how that genetic code gets arranged. But the most common variation is a mutation of a single nucleotide, or the basic unit of the genetic code (which is made up of a specific arrangement of four types of nucleotides). In other words, the most common genetic variation is when one nucleotide in the genetic code is changed for another nucleotide. Officially this is referred to as single nucleotide polymorphisms or SNPs for short.

Each of these SNPs has a specific identification number so that if you have your genome decoded (in other words, you have your entire DNA that you were born with decoded) you can investigate for the presence of specific SNPs of interest. Typically however, these polymorphisms are observed in specific ethnic groups being studied, and so might not extend to other ethnicities. Another important note is that these correlations between the disease state and specific types of SNPs (or any other type of genetic variation) might not actually be clinically valid as it may just be an observed coincidence, and confirming the clinical validation can be challenging. Nevertheless, it is these careful observations of what type of genetic variations a disease state appears to be associated with that will eventually allow us to build solid clinical predictability.

Moreover, these correlations are not just monitored for either the presence or absence of disease. Endometriosis is a complex condition with different stages of progression. Some of the genetic variations can also be liked to disease progression. For example, certain SNPs in the IL-16 gene have indeed been suggested to be linked to the disease’s progression.

These polymorphisms have also been observed in numerous other genes coding for immunological cytokines. Such SNPs associated with endometriosis were also observed in the IL1A gene, or the IL-10 gene, as an example.

The intracellular adhesion molecule-1 (ICAM-1) gene, the molecular product of which we introduced earlier, is one of the genes for helping establish immunocompetence (a properly functioning immune system).

ICAM-1 gene polymorphisms have been suggested to be associated with the risk of endometriosis but could also possibly work in an additive effect manner with other genes such as IL-6 (a gene encoding yet another immunological cytokine). This is what is referred to as the polygenic effect, or the simultaneous influence of many different genes at the same time - and at times, it is a simultaneous, additive effect of hundreds of different genes that work together to create a disease condition! And to make things more interesting is the fact that individually, such genetic variation might not have an effect but in the additive manner it does.

When quantity matters

Another way how genetics might be influencing disease development is not by having a change in the genetic code, but rather by how much a given gene is used. When a gene is used as a template to produce a protein, it is not used directly to produce the protein. Rather, it is used first to produce messenger RNA (mRNA) which is then used as a template to produce the protein. Any given gene is supposed to be used to a certain degree to produce some specific amount of mRNA which then results in some specific amount of protein. If the gene is used too much or too little to produce too much or too little mRNA, this will result in either too much or too little amount of protein and that can affect the proper biological function of such affected cells.

With regards to endometriosis, this exact effect was suggested for the production of cyclooxygenase (COX)-2 protein (this is referred to as expression). COX-2 is responsible for the production of prostaglandins that are involved in inflammation. Inhibition of COX-2 is done to provide relief from inflammation and pain. Higher levels of COX-2 protein in the ectopic endometrial tissue with corresponding higher levels of mRNA were seen in comparison to the eutopic endometrium (a 5-fold difference). Therefore, this abnormal production of COX-2 with concurrent abnormal production of prostaglandin could be contributing to the disease development and disease progression.

In addition, the expression of the COX-2 gene in endometriosis patients is also higher than in healthy women, while the same study also showed that there also was a polymorphism difference between women with endometriosis who remained fertile versus controls. That same polymorphism was also associated with more advanced stages of endometriosis in another study.

Another example of the quantity of a gene product affecting the disease state has also been observed in the already mentioned IL-10 gene. A SNP located on a promoter of IL-10 gene, or a section of the DNA code that regulates how regulatory proteins that are supposed to translate the DNA code into the production of mRNA template bind and initiate this process, results in decreased gene expression levels (meaning reduced production of corresponding IL-10 mRNA) in women with endometriosis. IL-10 cytokine is an anti-inflammatory cytokine, meaning that women with such a SNP in the IL-10 gene might be experiencing reduced ability to suppress inflammation which then could be a contributing factor to the development of endometriosis.

Differentiating specific conditions

Another example of polymorphism for a specific stage of endometriosis is a polymorphism in the protein tyrosine phosphatase non-receptor 22 (PTPN22) gene. The product of this gene, a protein called lymphoid-specific phosphatase, can potently inhibit T-cell activation. This gene PTPN22 polymorphism was notably observed in the advanced stages of endometriosis.

An interesting aspect has been noted with the genetic polymorphisms of matrix metalloproteinase (MMP)−12 and −13 (once again, working in unison in an additive effect) with regards to endometriosis. SNPs in these genes have been suggested to be linked with endometriosis penetration depth. SNPs in these two metalloproteinase genes were associated with shallow and not deep infiltrating endometriosis, and thus only promoted the development of initial ectopic implants, with other factors needed to promote deeper infiltration. Alternatively, the authors of that study also considered that this particular tandem polymorphism could have a preventive/protective role against more in-depth tissue penetration.

Let us look at two more examples examining specific endometriosis category types by genetics. In one study, a polymorphism in the Fc-receptor like-3 (FCRL3) gene was linked to an increased risk of infertility. This genetic association with infertility persisted irrespective of symptoms and endometriosis stage. This gene is involved in activating a crucial chain of molecular reactions that relay important cellular information to the outside world. It is called the NF-κβ/MAPK pathways and it is involved in the function and development and then subsequent function of the immune system, especially in inflammatory and acute responses.

Next were the genes called Caspase recruitment domain (CARD)10 and CARD11, where distinctive genetic alterations were previously detected in ovarian endometriosis. In addition, these genetic alterations were specific to the endometrial sample collected from patients. Such post-birth, tissue specific mutations (i.e. mutations that you were not born with) are referred to as somatic mutations. CARD11 is also involved in the activation of the NF-κβ signaling pathway.

The final point we want to bring up as an example of complex genetics that relates to the development of such complex conditions as endometriosis is that one can also have polymorphisms that can be protective against disease severity. One such example is a polymorphism in the Tyrosine kinase 2 gene (TYK2), the product of which can interact with other molecules that all together play an important role in autoimmune and inflammatory diseases. This TYK2 polymorphism was associated with protection against endometriosis-related infertility, especially in moderate/severe endometriosis. Interestingly this polymorphism was identified as a part of a greater genetic fragment called "CTATG" haplotype. Haplotypes are genetic fragments that are inherited from generation to generation. When sperm cells or eggs are produced, each parent’s genome is shuffled in the production of gametes’ DNA to ensure genetic randomness of the subsequent generation. When that genome is shuffled, it is often through defined size genomic fragments and these are referred to as haplotypes. These haplotypes, because of their specific size, often will carry many polymorphisms that help to identify them. That the TYK2 gene polymorphism described above was linked to numerous other polymorphisms that travelled together in the genetic fragment referred to as "CTATG" haplotype. And if you wonder about the name for this haplotype, it has to do with what nucleotide substitutions were found in this group of polymorphisms. Finally, this haplotype is quite rare.

Many genes make the molecular world go round – can we profit?

In our very last example, we will move away from the immune system and look at the Vascular endothelial growth factor (VEGF) gene that is important in angiogenesis (the development of a new blood supply network to a site). This is one of the most thoroughly studied genes with potential association for the development of endometriosis. What is worth mentioning here is that enough investigations have occurred so that a wide comparison can be done between different, independent investigations of the same polymorphisms. Such comparisons of numerous studies are referred to as meta-analysis and they are some of the most valuable and authoritative analyses available so whenever you see conclusions from a meta-analysis, you know it is a more powerful type of conclusion than any individual investigation can offer. With regards to the VEGF gene, what is interesting is that such a meta-analysis revealed the presence of both polymorphisms protective of endometriosis development (such a polymorphism conferred a reduced risk of developing endometriosis) as well as another different polymorphism that increased the risk of endometriosis. And with the VEGF gene, also several different haplotypes were uncovered that were associated with endometriosis using the meta-analysis investigation.

This exemplifies how genetics, in a myriad of fashions can intertwine their effects, sometimes in an additive fashion and sometimes in a contrasting manner towards the development of many diseases, including endometriosis.

While polygenic risk scores for endometriosis are not yet available for clinical use, they are still in development at the moment therefore it is not feasible to know how different polymorphisms translate to a concrete risk estimate but such a list of polymorphisms can commence informing women (with endometriosis and their available genetic code) to start investigating potential genetic causes behind their condition.

And in time, we do not doubt that there will be available a polygenic risk score test to help estimate the genetic predisposition for endometriosis. Such tests might not be empowered for clinical diagnosis or screening of the population for actual predisposition due to the complexity of involved genetics and the environmental influence, but it is expected that such genetic tests will aid with future risk stratification of both the disease development as well as the severity of the disease.

This article has been produced by Merogenomics Inc. and edited by Jason Chouinard, B.Sc. Reproduction and reuse of any portion of this content requires Merogenomics Inc. permission and source acknowledgment. It is your responsibility to obtain additional permissions from the third party owners that might be cited by Merogenomics Inc. Merogenomics Inc. disclaims any responsibility for any use you make of content owned by third parties without their permission.

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