Genetics and animal model

Let us first re-introduce WAS and the gene associated with it: Wiskott-Aldrich syndrome (WAS) is characterized by severe immunodeficiency, thrombocytopenia (decreased platelet size and numbers), eczema, and lymphoreticular malignancies. Untreated patients die in the first decade of life. WAS patients lymphocytes have an altered cytoskeleton with a reduction in cell surface microvilli (microscopic cellular membrane protrusions increasing the cell surface that are involved in some functions like cellular adhesion or absorption) as well as defects in antigen receptor–induced signaling (reviewed by Remold-O’Donnell et al. 1996). 

Linkage studies were employed to map the WAS gene to Xp11.23. The gene subsequently was isolated by positional cloning and found to encode a cytoplasmic protein (WASP) of 502 amino acids. Consistent with the cell types affected in WAS, the gene (WASP) was found to be expressed specifically in cells of the lymphocytic and megakaryocytic lineages. 

Was gene is composed of 12 exons (part of the gene actually coding directly for a protein or part of a protein, selected during splicing as opposed to the introns which are cut out of the sequence before transcription) and mutations have been found on all the 12 exons in WAS patients. 

Usually, the mutations in the first 4 exons are associated with milder disease whereas the ones from exon 5-12 cause more severe disease.  Most patients with milder disease were noted to have missense mutations or occasionally splice site mutations.  The majority of patient with severe WAS had deletions, insertions, nonsense mutations or splice mutations.

splice site mutation= either an exon is skipped (not read ) or an intron is read whereas it should not have been this resulting in a defect in the protein

missense= a change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by a gene

nonsense:a change in one DNA base pair —>prematurely signals the cell to stop building a protein

insertion: changes the number of DNA bases in a gene by adding a piece of DNA

deletion: changes the number of DNA bases by removing a piece of DNA

Before the genetics ruling this disease was understood, milder forms of it were known as other diseases described by the only symptoms among the triad that was observed ; for instance:

X-linked thrombocytopenia (XLT)-> low concentration of platelets, or dysfunctional platelets which results in thrombocytopenia and therefore risks of hemorrhage .

x-linked neutropenia called XLN -> low concentration of neutrophils,majority of circulating white blood cells and serve as the primary defense which results in immunodeficiency. 

This small scheme shows where the Was protein is most expressed in a cell and we can see that it is in extracellular space which makes sense because it is mostly involved in the production of actin so as to build the cytoskeleton given its solid structure to the cell but mostly connecting it to the rest of the body by moving it (blood cell) ,transmitting chemical messages and binding to the other cells.

As said briefly before in the section Introduction to WAS, it is a genetic disease, X-linked recessive which means the big majority of the patients are male because they only have one copy of the X chromosome coming from their mother and if it is carrying the mutant allele, the individual is automatically affected by the syndrome.Here a scheme to summarize the X-linked recessive inheritance:

However, two types of female patients have been found :

One was a genuine homozygous female which is very rare because the father has to be affected by the syndrome and the mother has to be a carrier.It has been reported that she showed microthrombocytopenia and infections to the same degree as her hemizygous father and brother.

The other was a 14-month-old girl with a history of WAS in her family who presented with thrombocytopenia, small platelets, and immunologic dysfunction. Sequencing of the WASP gene showed that the patient was heterozygous for the splice site mutation previously found in one of her relatives with WAS. Sequencing of all WASP exons revealed no other mutation. Levels of WASP in blood mononuclear cells were 60% of normal.WASProtein expression levels in the patient’s PBMCs( peripheral blood mononuclear cells (= lymphocytes + natural killer …)) were lower than normal, but substantially higher than those in WAS patients.This means she had a milder form of the disease than the male patients presenting the same mutation.

The answer to how a heterozygous female patient could show symptoms is that she had a random pattern of X chromosome inactivation in her PBMCs.

X-inactivation (also called lyonization) is a process by which one of the copies of the X chromosome present in female mammals is inactivated. As  all female mammals have two X chromosomes, X-inactivation prevents them from having twice as many X chromosome gene products as males, who only possess a single copy of the X chromosome.This process is random (except for marsupials) and happens during the first stages of embryons. The inactivation of an X-chromosome is definitive for a cell and will be the same for all the cells coming by cellular division from that particular cell.This explains why the patient had the same X inactivated in all her PBMCs.However we can note that there is no X inactivation (it is reversed) in germinal cell so that each ovocytes contains an activated X.

To discover all that and in order to study even further the genetics and the physiology of the disease, researchers felt a need for a model animal.When they did that, the gene responsible for WAS had already been identified in human so, in the process of creating an animal model, their first step was to find a homolog gene in mice as it is a easy animal to study for both genetics and physiologically close to human mechanisms.

They chose to induce Was in mice by a disruption of the gene in exon 7 in RNA (messanger).This already shows a limit of the model as mutation on 12 exons can exist but this will be discussed later on.

They used restriction enzymes (for instance: EcoRV) specific to a unique site/sequence to cut the exon 7 from thymus cells (wild type mouse). Other enzymes were used to insert an antibiotic resistance gene called  neomycin-resistance gene that has too functions: disrupt and so induce the syndrome  and then a selection of the vector later.

A specific medium synthesized the piece of RNA into DNA. A plasmide is a circular DNA easily inserted. The exon 7 modified is put in a plasmide into a vector: a bacteria.This is not something that works 100% times so this is were the selection by antibiotics is used to select the bacteria in which the plasmid has indeed been inserted.Then they « infected » mouse cells with those bacteria so that the plasmide insert itself into the mouse DNA.

Here is a scheme (in French) that illustrates this technique of genetic engineering but for a human instead of a mouse and with the bacteria  E.coli.

 

 

Those cells are then injected in blastocytes (= first stage of the development of embryon) to create mice carriers.Those mice are then inbred together to obtain males that are automatically affected by the syndrome.

WAS-deficient mice are viable and fertile.

This was done in 1998.

In 2002, the model was ameliorated.

As the only curative treatment is bone marrow transplant ( much longer life expectancy if a matched donor is found) scientists needed to study this transplant in the case of WAS.

For the donor of bone marrow: they used B6 Cd45.1, Pep Boy mice because they are known to be very suitable and commonly used for transplant studies.

 

Then,for the WAS deficient mice: first choice of species was 129/SvEv-Wasptm1Sbs  because they are very suitable for studies in immunology and hematology but they didn’t survive the transplant because of a high susceptibility to Helicobacter a bacteria inducing lethal colitis for them.The solution was to inbred those one from 2 to 4 generations with B6, B6J, Black 6, C57 Black that are very common and resistant mice:

In this first study of 1998 ( http://www.cell.com/immunity/fulltext/S1074-7613(00)80590-7; see sources), researchers noticed that there was a marked absence of classic participants in the WAS triad (hematopoietic malignancies or eczema) in the young WASP-deficient analysed murine model. The development of these symptoms will surely be examined in older WASP-deficient models. Nevertheless, the majority of these WAS-deficient mice developed chronic colitis by 4 months of age. 

Colitis is a IBD (Inflammatory Bowel Disease) a lot like Crohn’s disease, apart from the fact that Crohn’s most commonly affects the end of the small bowel (the ileum) and the beginning of the colon, but it may affect any part of the gastrointestinal (GI) tract whereas Colitis affects the large intestine (colon). This disease, like said, is an IBD, it involves the  thickening of the innermost lining of the colon. 

Researchers found that the most severely involved 
colons were diffusely dilated with thickened walls and had
marked mucosal thickening due to crypt hyperplasia,
causing abscesses, and the presence of a mixed lymphocytic
and neutrophilic infiltrate (accumulation of lymphocytes and neutrophils (white blood cells)) within the lamina propria. In comparison to wild-type mice, it is to be marked that there is
a large increase in the number of CD4+ and CD8+ T cells in this tissue. 

This figure shows the difference between a same wild type (A and C) and a severely involved WASP-deficient murine model (B and D). Firstly, let us look at A and B. A hematoxylin and eosin staining of colonic tissue from a wild-type leads us to conclude that the mucosa from affected WASP-deficient colons is thickened with crypt hyperplasia and a lymphocytic and neutrophilic infiltrate in the lamina propria. Crypt abscesses are also present (arrow). Secondly, an immunohistochemical analysis of colonic tissue from C and D shows that CD4+ T cells are observed in the lymphocytic infiltrate in D. These cells are  largely absent from wild-type colons. 

Next semester we shall write about another study that we have considered, inspired by the research from the paper we treated this semester. We shall look more into the use of murine models, in our case, and their influence in finding cures and treatments options for WAS. To do this we will study genetic crosses between 129v/Ev mutant mice and C57/BL6J wild type mice largely used in genetics and the use of HSCT (Hematopoietic Stem Cell Transplantation) to treat WAS. 

Now, we are going to try to compare the murine model (the definitive one, ameliorated) with the reality of the disease in humans: 

In humans, the typical features of WAS are severe immunodeficiency, manifested as recurrent infections, plus eczema and thrombocytopenia. These features are usually associated with lymphopenia (decreased numbers of blood lymphocytes), lymphoreticular malignancies, defective T cell function, and poor immune responses to most protein antigens and all carbohydrate antigens. Young WASP-deficient mice share many common features with WASP-deficient humans. In particular, they show mild thrombocytopenia and lymphopenia as well as defective T cell activation. However, none of the WASP-deficient mice analyzed have developed eczema or hematopoietic malignancies. Likewise, their antibody responses to a TD antigen and a TI-II antigen appeared relatively normal. However, as the WASP-deficient animals have been maintained in a specific pathogen-free environment, it is not possible to fully assess their immunocompetence at this time; immunisations with other antigens or variations in the immunisation protocol may show defects. In addition, it is possible that other missing aspects of the human phenotype might appear in older mice. 

WASprotein

Actin cytoskeleton regulation is vital in the correct functioning of the immune system (cell migration, antigen uptake by white blood cells, T-cell activation, etc). WASp is a protein that plays a key role in the regulation of formation of actin filaments.

For the immune system to function correctly, all immune cells must work normally and in harmony. These cells require dynamic cytoskeletal rearrangements to allow effective cellular function. This is because these cells need to be mobile and of a specific shape in order for them to move from the blood stream into the infected or inflamed tissue (in a process named diapedesis). Several steps are involved in this process of tethering, rolling, arrest and extravasation. Once in the tissues, the actin cytoskeleton also plays a role in the uptake of debris or of microbes by a mechanism that is referred to as phagocytosis.  Finally the actin cytoskeleton also plays a role in the activation of T-cells when they encounter antigen presented on the surface of white blood cells (mostly dendritic cells or macrophages), an essential step in the development of the adaptive immune response. 

Migration is impaired in WAS and this will likely result in defective extravasation of monocytes and neutrophils (1). Inside the tissue, pathogens will be phagocytosed by neutrophils, macrophages and dendritic cells (2), which is reduced in WASp-deficient cells. Dendritic cells will migrate with the processed pathogen antigens to the draining lymph node (3), where they will present the antigens to lymphocytes (4). Both migration of dentritic cells and priming of T cells (by dendritic cells) is defective in WAS. Primed lymphocytes will proliferate (5), home to the inflamed tissue (6) and exert their effector function (7). 

WASp and so the actin cytoskeleton play main roles in an immune response, this explaining why people with WAS suffer from an auto immune disease. As we can assume, with faulty WASp comes impaired immue cells. An example of a WAS symptom is eczema. This symptom is due to an insufficient production of interleukins (4 and 13) which play a role in the differenciation of naive helper T-cells and Th2 cells. When the latter is produced in ample quantity, more interleukin 4 is produced and the loop is closed. When a patient suffers from WAS, WASp is erroneously produced and T-cell differenciation (and activation) is disrupted so interleukin 4 – 13 is insufficiently produced leading to eczema.

Below we shall explain briefly how WASp controls the formation of actin filaments.

WASp occurs in two states (in two shapes); an auto-inhibited and an active state.

Its activation depends on two associated components : a protein named CDC42 (a GTP-ase) and a lipid named PIP2. WASp is a multidomain protein (figure 5). It contains an N-terminal EVH1 domain, a C-terminal VCA domain and central B and GBD (GTP binding domain) domains. WASp possesses a CRIB (CDC42 and RAC interactive binding) with allows the binding of CDC42 and PIP2 to the protein in order to activate it. When WASp binds CDC42 bound to GTP, the protein “opens” up and exposes the VCA domain. The open conformation constitutes the “active” state of the protein. The VCA domain is made up of three parts: the verprolin homology motif (V), which binds actin monomers and delivers them to Arp2/3; the cofilin homology motif (C), which binds cofilin; and the acidic motif (A), which binds Arp2/3. When the Arp2/3 complex fixes onto the acidic motif, it activates, forming a stable site nucleus allowing actin monomers to bind to the complex (process referred to as nucleation) and subsequently to polymerize into a filament (figure 6 and 7). 

Nucleation followed by polymerization is not sufficient to form integrated actin networks, since these newly synthesized polymers would not be associated with pre-existing filaments. Arp2/3 operates by binding to already existing filaments and thus leads to the formation of a rather solid network of branched actin filaments. Actin capping proteins stabilize the newly formed filaments.

WASp’s control region (B and GDB domains) continuously tries to stop VCA domain activity. This region constantly binds CDC42 and PIP2 allowing WASp to stay activated and relieve the auto-inhibition of the protein. The N-terminal domain (WH1) may also be degraded so a second protein, WIP, prevents the WH1 domain from being damaged.

CDC42 and PIP2 localize the WASp-Arp2/3 complex to the plasma membrane. This membrane localisation plays is important for all actin-mediated activites described in above (scheme X). It is needed for migration and engulfment of microbes (phagocytosis) and it is needed for the membrane-located processes that involve the activation of the T-cells. 

The WH1-domain of WASp is mutated in patients with the Wiskott – Aldrich syndrome 

When a patient suffers from WAS, the N-terminal of WASp (WH1-domain) is mutated. The amino-acids ranging from 31 to 178 cause problems as they are substituted leading to a change in conformation (size of amino-acids are different) and physico-chemical properties (acidic/ basic/ hydrophilic/ hydrophobic properties). This means that it cannot bind to other substances (like CDC42 and PIP2 or WIP) making this protein useless, faulty or easily damaged.

Impaired WASp (or absence of CDC42 or PIP2) expression leads to a domino effect. It causes a locked conformation of the protein. If the protein is locked then the Arp 2/3 complex cannot bind to the protein and so the actin monomers cannot bind to Arp2/3. If one of these three components are missing then the actin cytoskeleton cannot be synthesised and the cell either dies or cannot move. WAS is a syndrome caused by a mutation on the WAS gene encoding the WASp protein. The protein is either not made or is faulty. Because this only touches the haematopoietic cells, blood cells are touched and so the immune system is weakened.

Introduction to WAS

 Wiskott – Aldrich syndrome is an X linked recessive illness that involves the host’s immune system. WAS is an immunodeficiency disorder, a disorder that compromises a person’s immune system, they have problems fighting against infections and other pathogens. Seeing as WAS is an X chromosome linked recessive illness, only males can be affected (with the possible exception that a female could be affected if both parents are carriers). The WAS gene situated on the X chromosome usually codes for WASp protein; a protein that is important in the fabrication of actin. Actin can be considered as the building blocks for the cytoskeleton located in the cell. The cytoskeleton allows the cell to move, change shape; it’s the structure that gives the cell its form. Individuals with WAS have a defect in the WAS and so in the WASp protein, leading to malfunctions in the production of actin. WASp has a big role in hematopoiesis; the process that creates of blood cells.

 The symptoms of WAS are qualified as a “triad”; they are three main symptoms that characterise the illness: microthrombocytopenia, immunodeficiency and eczema (inflammatory skin disorder characterised by abnormal patches of red, irritated skin). Other symptoms exist such as recurrent ear infections, bleeding, bruising etc. This triad was first observed in 1937 by Alfred Wiskott in three brothers that presented these symptoms and who died at an early age from intestinal bleeding and sepsis. They defined the illness as hereditary in the 1950’s. 

 

WAS affects 1 to 10 in every million new-born boys.
During the first year of life, bleeding (diarrhea, intracranial, at the umbilical stump or penis after circumcision) and eczema accompanied by recurrent infections are observed; petechia and purpura as well as oral mucosa are often present. From four to eight months, Otitis Media (ear infections), pneumonia, meningitis and sepsis are frequent
ly apparent. During this period fungal infections (ex: thrush) and chicken pox can be life threatening. 

Diagnosis can be decided by analysis of umbilical cord blood, at birth, with the observation of a low count of platelets that are significantly smaller. Diagnosis in children from two years up can be identified by a failure to produce antibodies to illnesses or vaccinations or by a skin test to asses T cell function. Thirty years ago, WAS was considered to be a fatal disorder with a life expectancy of only two to three years. Nowadays, the average life expectancy for boys is about 15 to 20 years without hematopoietic stem cell transplant. Engrafted children are expected to survive much longer.

These symptoms are explained by microthrombocytopenia: decrease in the number and size of platelets; the cells responsible for clotting and blood coagulation, and immunodeficiency. This is because of the defect in the making of actin; the cells are non-viable or too small if the amount and quality of the actin isn’t good enough. If somebody has WAS, they have platelet abnormalities, leading to bruising and problems with blood clotting or prolonged bleeding following a minor trauma. They also have defects with their lymphocytes causing a malfunctioning adaptive immune system. If someone’s immune system is weak or defective, they are exposed to a greater risk of infection and inflammatory disorders. 

Generally, when WAS appears to be fatal, death results from bleeding because of the abnormality and the reduced number of platelets but it can also result from infections due to the immunodeficiency, or from autoimmune disorders or cancer that tend to occur much more often than the usual frequency in the healthy population. At this point, we must say that WAS symptoms and therefore the patients’ quality of life worsen with time as it comes with a gradual decline in immunological functions.

Life expectancy and living conditions can be improved: firstly, antibiotics and immunoglobulin (antibodies) supplementation can be given to prevent infections, helping to support the immune system.

Some treatments are possible : Treatment options

Sources