Human Cerebrovascular Malformations PDF

03.12.2021 RETTA L.2 HUMAN CEREBROVASCULAR MALFORMATIONS CEREBRAL CAVERNOUS MALFORMATION Last time we started to talk about CCMs disease. We said that CCM disease is...

03.12.2021 RETTA L.2 HUMAN CEREBROVASCULAR MALFORMATIONS CEREBRAL CAVERNOUS MALFORMATION Last time we started to talk about CCMs disease. We said that CCM disease is one of the cerebrovascular malformations, there are also other types of malformations including ArterioVenous malformation (AVM) that you can see here (fig.1). There is major difference between ArterioVenous malformations and cerebral cavernous malformations (CCMS). As you can see in the case of CCMs (fig.2) that capillaries are involved. They appear dilated and abnormally without normal shape, while in the case of ArterioVenous malformation we have a shunt without intervening capillaries. There are also another major differences called High-flow malformations, because the blood flow is very high while in CCM disease a low blood flow is present. This is also relevant from a diagnostic point of view because this can be diagnosed with cell angiogram while CCM is silent on angiography. So the only diagnosis fig.1 approaches that you can use for CCM are magnetic resonance images. Different forms of this disease exist: fig.2 one is sporadic and the other one is familiar, it can be inherited (the inherited form, represent the 20% of the total). Symptomatic disease, as said in the previous lectures, can occur at any age (between the third and fourth decade of life. Exceptions are present, in fact the 25% of cases occur during infancy or in childhood) and we already said that these malformations can affect different parts of central nervous system but in some cases it can occur even in the peripheral one (spinal cords etc.). There is as you can see here (fig.3) a huge difference between some Magnetic resonance methodologies, including some bit on the left as compared to the susceptibility (…?…). This is the same patient, as you can see here there is a big malformation that of course you can detect with this method that is more sensitive to (…?…). This technique is more sensitive in detecting these lesions and their identification also depends on diagnostic approach that has been used. Diagnosis in commonly made by MRI screening, even if detection is far more likely via gradient echo (GRE) or susceptibility-weighted imaging (SWI). These lesions can form “de novo” and they have a dynamic measure. Speaking about fig.3 clinical symptoms patients can may suffer from recurrent headaches, epileptic seizures, neurological deficits, intracerebral hemorrhages. Identification of biomarkers could be useful. There is some evidences about some potential biomarkers that could be very useful from diagnostic point of view because there is huge difference in the severity of this disease, even among the different members of the same family that carry the same causative mutation. It’s not enough, the mutation of one of the two genes to gives rise to 10 03.12.2021 RETTA L.2 the severe form of the disease. Additional events must occur and then biomarkers that can tell us about the risk of development of the severe form of the disease. Hence potential biomarkers can provide us some information about the disease. Among the tissues that are affected are mainly the Central nervous system, as you can see from the given analysis, (fig.4) there are normal vessels and capillaries that are supported by endothelial cells that surround the lumen of the capillary and also there are other cells including pericytes and astrocytes, involved in food processes. All together they form the so called blood-brain barrier (the barrier between the blood and brain). This is also called neurovascular unit (NVU). The neurovascular unit is composed by endothelial cells and supporter cells including fig.4 pericytes and astrocytes for food processing (all together they make the neurovascular unit). The basal membrane is also important and the extracellular matrix underline the endothelial cells also play a role in the formation of the blood brain barrier. In abnormal vessels there is a clear alteration , it can be seen that the lumen, for instance, is wider and bigger. There is also another alteration in the Neural Parenchyma and the astrocytes-food process do not reach the endothelial cells. The picture shows (fig.5) an electron microscopy imaging. On the left the fig.5 normal situation can be seen and again the lumen of the vessels, the endothelial cells and then pericytes that supports the endothelial cells can be detected. Besides supporting endothelial cells pericytes play also a trophic function: they provide some metabolites to the endothelial cells, so there is a communication between Pericytes and endothelial cells, not only from the structural point of view but also from functional point of view. This is not only a physical interaction but a matter of communication based on some molecules with a trophic function. On the right of the picture you can see the lesions, where the junction between endothelial cells are broken (or totally broken, this lead to hemorrhages). This can occur as consequence of the rapture of the intercellular junctions. This is an analysis that you can make at the immunohistochemistry level. The cause of this disease is a genetic one. CCMs is indeed a genetic diseases that is caused by a mutation in one of the three genes. CCM IS A GENETIC DISEASE In our DNA there are about 24,000 genes and approximatively 6000 genetic diseases are known and more than 1000 of them are hereditary. The development of therapeutic measures is mainly dependent on the understanding of the molecular mechanisms. The pathophysiological mechanism of genetic diseases are fundamental in order to develop new therapeutic approaches. We know that CCM is a genetic disease but how can we detect the molecular alteration? How is it possible to approach this issue? (Unintelligible discourse about the painting) An American group from Fox Chase cancer center identified the first CCM gene. This group didn’t know yet that the gene that they found was associated to CCM. They just identified it because they were interested in discovering a gene coding for a protein interacting with another protein of their interest called RAP1. RAP1 is a small monomeric GTPase. This group was interested in understanding the function of RAP1 because studies reported it as a tumor suppressor, by counteracting the tumors effect trough oncogenic defect of genetic defection in Ras. In order to understand more this potential tumor suppressor, this group tried to identify potential binding partners. This is an approach that researchers use often when they don’t know very much about a problem. If we find another protein that interact with the protein of our interest and we can identify some functions of this other protein, we can know some functional information about it. They applied a technique called hist to hybrid that can help in identifying binding partners of proteins. So they identified a new protein called KRIT1: stand for K- Ras revertant. It’s a protein able to revert the oncogenic phenotype induced by K Ras. So when you identify proteins you could also identify genes, actually using two hybrid techniques. Two years later different two groups from USA and France reported the same results, the gene that has been identified previously is 11  03.12.2021 RETTA L.2 the mutated gene in patients affected by CCM disease. This was the first report associating the gene and the CCM disease (genes are responsible for CCM disease). This was the first step, the identification of one gene, and it was done by comparing and studying people with inherited disease in the same family (familial type) we could identify mutated genes related to the diseases and we can study the segregation of the phenotype. People who belong from the family that don’t show the disease probably have not inherited the mutation. Few different groups obtain some fundamental results. It was the first milestone, the first step for treatment the disease, then after the identification of the gene people started to try to understand the molecular mechanism underlying the pathogenesis of the disease. For instance you can see 2 groups that characterize more than one gene encoding for rap1 protein, identifying, for instance, new exons that before were not detected, so they were able to show that the gene it was regarded previously was longer than previously believed. It was believed to be a gene encoding for a protein containing some amino acid, later on this 2 groups reported that the protein is 200 amino acid longer than previously reported. So they characterize the gene and protein of CCM encoded by this gene. Later on the same groups from France and USA, they identified the other 2 genes associated with CCM diseases, the CCM2 gene was identified in 2005. So other 2 gene were identified so this provide additional information fig.6 that characterize the molecular mechanisms underline the disease. Two of them are marked on chromosome 7. CCM1 marks on the long arm of Chromosome 7 and CCM2 marks on short arm of chromosome 7, while CCM3 is on the long arm of chromosome 3. So the three genes (fig.6) were identified, characterized and marked on the chromosomes. So step by step you can have a picture more and more clear. By analyzing the familiar form of CCM disease, researchers were able to show that 50% of patients were usually carriers of mutation on CCM1 gene. 20% of cases were associated to mutation on CCM2 gene. So theoretically the 30% of cases should be associated to the third gene, but they found out that only 10% of people were carriers of CCM3 mutation. So we obtain that 20% of people were not carrier of any of these three genes identified: this means that probably there is an additional gene that still needs to be identified. Regarding to this aspects some researchers think that maybe it’s not possible to identified all the mutation that affect this disease, because we have 20% of people that are not associated to mutation on these 3 genes. Maybe some mutations that affect these 3 genes, but are not detectable, can occur because an additional gene is involved. There are some aspects that need to be identified including the genetic cause of 20% of cases that are not associated to the mutation in the three known CCM genes. These are the 3 genes, once you identified the genes associated with the genetic disease then you can characterize the mutations that affect these genes. So different groups, by analyzing different families affected by this disease, didn't find mutation. More than 100 different mutations were identified on the CCM1 gene. As it can be seen this involves different mutations such as frameshift mutations, non-sense mutations, splice inside mutations and mis-sense mutation. A lots of frameshift mutation were identified but they are different because they are localized in different exons. KRIT1 has 16 coding exons on a total of 20 exons, only 16 of them are coding for amino acids. The first 4 exons are untranslated regions. They are regions present in mRNA but they are not coding for protein. (Question) There are exons where a lots of mutations happen. Different exons show different number and type of mutations. Genetic regions that are affected more frequently by mutations are called hotspots. For example exon 16 could be a hotspot because this can be affected more frequently by mutations. If a person carries mutation in the first 4 exons is not affected the function of protein. (Question) some regions can undergo more alterations, for instance oxidative damage that can give rise to mutations. There are other mutations that can affect the other two genes. CCM2 contains only 10 coding exons while CCM3 contain 10 exons but only 7 are coding ones. Also you can infer the size of protein, the size of protein is bigger in the case of CCM1 and smaller in the case of CCM2 and CCM3. CCM1 encodes for proteins with 736 amino acid while CCM2 and CCM3 have 444 and 212 amino acids. The fact that CCM1 is responsible 12 03.12.2021 RETTA L.2 for higher percentage of the patients (50%) it’s also due to its bigger size which is more subjected to mutations while the smaller one is responsible only for 10% of the disease. GENETIC ANALYSIS Once you identified the genetic cause of the disease you can make genetic diagnosis, up to this fig.7 identification it was possible to make only magnetic resonance diagnosis. After the identification of the genes, in addition to the clinical diagnosis, you can make genetic diagnosis and verify whether the patient is affected by sporadic or familiar form. In the case of the familiar form you can detect the causes of the mutation so you can detected the gene that is responsible for the disease. What could be the advantage of identifying mutation? If you find a relationship between the genetic alteration and the phenotype then this could be helpful in order to develop the therapeutic approaches. Basically the sporadic form is responsible for 80% of the cases. In the sporadic case the CCM lesions are usually single, while in the familiar form the lesions are usually multiple. What could be the reason of this difference? In the familiar forms patients carry the mutation in all cells because it’s a germ-line mutation (familiar could be transmitted). This can occur only if germ- line are also mutated. In the familiar form the germ-line mutations occur while in sporadic cases the cause of alteration is so called somatic mutation. What is the difference between somatic mutation and germ- line mutation? A somatic mutation affects the gene in somatic cells (only in few cells, the cells that give rise to the lesion), this like the mutation that occur in cancer. This is a mutation that affects few cells of the organism. Inherited genetic diseases are based on so called germ-line mutations, so mutations that occur in all the cells, including germ cells, because the disease can be transmitted. Familial cases represented just 20 percent of the cases and they are characterized by the presence of multiple lesions (fig.7). (Just a rant about his teaching method, attempted discussion). So the next step is what are the functions of these genes that are associated with the disease? We need to know their functions and also their dysfunctions. We need to know what they do normally and also what occurs when they are dysfunctional. Other groups tried to identify new functions. (Interlude about past students) These other groups identified new interactions between KRIT1 and other proteins (identification of binding partners) The first hypothesis was that KRIT1 contributed to the tumor suppressor functions of RAP1. Then they used the same screen with KRIT1 as bait to identify the function of KRIT1 and identify potential binding partners. Both groups identified the same protein that is called Icap1 (integrin cytoplasmic domain associated protein). So this is a protein that is associated to integrin, that mediates cell adhesion. These 2 groups identified Icap1 as a new partner of Krit1 and then what is the consequence of this discovery? The consequence of this discovery was that, because the functions of integrin and Icap1 were known, it provides insights about the function that Krit1 can play a role in regulating cell adhesion processes. It is called adhesin protein because it mediate cell adhesion. (…) Based on the information we can first of all present the structure of protein and indicate the binding site for the 2 binding interactors. Icap1 and rap 1, so this is krit1 and the structure of Krit1 was started fig.8 to be characterized. Icap1 is able to bind to so called N terminal region from the sequence of amino acids while Rap1 is able to bind to C terminal (fig.8). Since Krit1 interact with Icap1 and since Icap1 interact with Rap1 we know that the Icap1 regulate the integrin functions, you see this is a plasma membrane, this is an interface that attaches to the intercellular matrix and its mediate cell attachment to extracellular matrix. So we know RAP 1 and ICAP 1 play role in the functions mediated by this 2 proteins and this was the hypothesis. Krit1 when 13  03.12.2021 RETTA L.2 is present conjugate the …... Cell adhesions. when is absent this influence the function of the binding part and eventually influence cell adhesion. so CCM pathogenesis could be…..... and altered cell adhesion. altered cell adhesion.......... (About 30 minutes are missing because the teacher was discussing one-on-one with a student and half of the conversation was inaudible) 14