AD-HIES: An Immune System Disorder

CHAPTER 1: Introduction

Description of the disorder

The autosomal dominant hyper-IgE syndrome (AD-HIES) an immune system disorder which brings about recurrent skin effects, a variety of lung infections and even affects the skeletal system leading to abnormalities in bones and teeth. Studies and research in this domain have identified over one hundred germline mutations in the signal transducer and activator of transcription 3 gene (STAT3) for people with the condition. An examination of these mutations involved in the condition by previous works shows that it results in a change of the single amino acids found within the STAT3 protein. Further, the mutations usually occur within the regions of the protein that are vital for its activation or the regions critical for its ability to successfully bind to DNA.

Epidemiology and frequency

Frequency

However, most of the times the disorders may be misdiagnosed or are usually unrecognized hence it is not easy to determine the real frequency in a population generally.

Mortality/Morbidity

There are more than 200 cases of the disease that have been described in medical studies. Even though it is a very rare condition

Sex

AD-HIES affects both males and females equally and within all races and ethnic groups.

Age

The condition may be present during infancy however; a diagnosis may not be easy till the adolescent age or sometimes adulthood.

Pathophysiology of STAT3 disease

Diagnosis

The AD-HIES is diagnosed by a comprehensive clinical evaluation of the patient history. Laboratory studies such as blood tests (levels of IgE and white blood cells), X-rays, MRI scans CT scans (lung infections and pneumatoceles) are then used after which a scoring system is used to help in diagnosis.

Symptoms

There are usually immunoglobulin IgE in the blood (thus the term hyper IgE). As a result of the abnormalities in the immune system, individuals with this health condition become highly susceptible to other bacterial and fungal infections which mainly affect the lungs and the skin. The key symptoms of AD-HIES include; dskin rash (eczema) beginning at birth or early infancy ages as well as Itchiness (pruritis). Babies are particularly vulnerable to bacterial infection, such as staphylococcal infections. They may result in boils and abscesses to form on the skin. These abscesses are referred to as “cold” abscesses because they don’t have the surrounding warmth as expected in such an infection.

Standard Therapies

The studies and researches on individuals who have various STAT3 mutations continues to lead the discovery of vital biological pathways that the protein is involved in and the ways in which they impact the normal functioning as well as health conditions (Darnell et al., 1994). As a result of the abnormalities in the immune system, individuals with this health .The significant role of the STAT3 protein in the development and maintenance of the bone tissue can be used to explain why the mutations in the gene result in the dental and skeletal abnormalities characteristic of the AD-HIES condition.

Location in the Pathway

The mutations in the STAT3 gene that result into the AD-HIES syndrome are as a result of the changes in the structure that in turn alter the function of the protein hence limiting its ability to normally regulate the activity of other genes as required. Since these changes lead to the impairment of the normal functioning of the protein encoded by the STAT3 gene, they are usually referred to as ‘loss-of-function mutations’. The inability of the STAT3 protein to transmit any signals leads to the disruption of the Th17 cells maturity. The Th17 cells are a subset of the immune system T-cells

Continue your exploration of Starbucks Strategic Management Overview with our related content.

Genetic Change

Residual function of the STAT3 exists due to the homo-dimerization of the wild type alleles within the protein which leave it functional by a proportion of between 20 to 30 percent. On the other hand, the gain of the function of STAT3 has been typically linked with neoplasms while the somatic mutations in the gene have been reported in a large population of LGL patients.

Disease and Associated Diseases

Autosomal recessive hyper IgE syndrome is another rare immunodeficiency disorder as a result of mutations in the ZNF341 gene. It also has the same characteristics with affected people being vulnerable to bacterial and lung infections.

Background

The signal transducer and activator of transcription 3 (STAT3) is one of the key transcription factors that acts as an arbitrator of various responses or reactions from the body cells to a range of cytokines, hormones as well as growth aspects. The STAT3 gene forms part of the broader family of genes referred to as the STAT genes (Zhong et al., 1994). Prior studies on human genetics assert that the STAT3 gene participates in a wide selection of cell physiological processes and additionally directs seemingly opposing reactions and responses.

The STAT3 genes are responsible for giving instructions for the development of proteins that form part of the vital pathways for chemical signaling in the body cells. The proteins encoded by the STAT3 gene are part of the signal transducer and activator of transcription protein family which usually respond to the hormones, cytokines, and growth factors when the kinases from the receptors phosphorylate them. The larger STAT family of proteins then respond by forming the heterodimers or homodimers which in turn translocate to the nucleus of the cell in which they essentially act as transcription activators. In other words, when the STAT family of proteins get activated by particular signals from the receptors, they then shift to the cell nucleus and then attach or bind themselves to certain areas of DNA. Some of the cytokines and growth factors that result in the activation this protein through phosphorylation as a response mechanism include; IFNs, EGF, IL5, IL6, HGF, LIF and BMP2. It is on the basis of this action of the STAT proteins that they are referred to as transcription factors (Acosta-Rodriguez et al., 2007). The STAT proteins mediate the actions of a wide range of genes in a response to a variety of stimuli in the cells. As such, the proteins play a key role in numerous functions of the cell, including cell growth, development and divisions as well as the overall movement of cells and their self-destruction, a process known as apoptosis. The GTPase Rac1 has been presented to control the binding of the STAT protein and regulation of its overall activity. Also, one of the specific inhibitors of the protein has been shown to be PIAS3, in accordance with existing body of knowledge. Apart from the Signal transducer and transcription activator 3 (STAT3), the STAT family also has six other transcription factors that make up the JAK-STAT chemical signaling cascade which transmit a wide array of regulatory factors that modulate the transcription of genes. The chromosomal region in which they are located is the 17q21 (Annunziato et al., 2012). In particular, the STAT3 may be activated by a range of ligands in response to massive signals including; IL-6, TNF-α, and VEGF.

Additionally, STAT3 is also significantly involved in a variety of other functions within the active tissues in which it is found all over the body. Some of these functions include the development of the immune system, the regulation of inflammation and the development and maintenance of the skeletal system. For the immune system, the protein transmits signals for the T cells and the B cells which control the response to any foreign invaders while for the skeletal system, the protein encoded by the STAT3 gene helps in the formation of specialized cells that develop or decompose bone tissue. For the inflammation response, when STAT3 gets activated by IL31 through the IL31RA, it then in turn regulates the differentiation between the naïve CD4(+) T cells turning them into the T-helper Th17 (Alberts et al., 2002). The Signal transducer and transcription activator specifically works in the arbitration of the cellular response to interleukins such as KITLG/SCF, LEP by recruiting other coactivators, e.g. NCOA1 or MED1 to move to the promoter region of the target gene. STAT3 then responds by binding to the interleukin-6 (IL-6)-responsive elements that have been previously identified in the promoter regions of the acute phase genes which code the protein (Yamaoka et al. 2004). Given the significant role of the protein coding gene in DNA binding transcription factor activity and sequence specific DNA binding alongside its association with the aforementioned health conditions, this current study resolves to further present an analysis of the gene. The underpinning objective is to specifically examine a particular region or section of interest in an attempt to determine whether or not a significant mutation site in the STAT3 gene is present within the patient sample DNA (De Beaucoudrey et al. 2008).

A DNA sequencing of some of its key regions through polymerase chain reaction (PCR) of previously designed primers and amplification from an unknown individual’s gene shall then be carried out. As such, this investigation shall include literature review subsection that will link the preexisting body of knowledge over the domain of human genes, specifically the STAT families (Chandesris et al. 2012). This will then be followed by a materials design and methods section that shall entail a comprehensive presentation of the techniques and methods used for the examination, from the point of the application of bioinformatics to obtain the genomic sequence in the chromosome, to the design and amplification of the primers of the key portions and finally to the laboratory PCR processes used to examine the primers. The discussion will then continue into a presentation and analysis of the obtained results in correspondence to the key portions role and association to disease and vital features of the gene such as SNPs. The current study shall then be ended with a conclusion and recommendations for future research based on the results.

Genomic Context of STAT3

This subsection presents an overview of the overall genomic context of the human STAT3 in terms of its genomic locations and genomic view. The cytogenetic location of the STAT3 gene on the chromosomes is given as; 17q21.2 which indicates that the gene is located along the long arm (q) of the chromosome 17 at the exact position 21.2 as illustrated in figure 1 below. As given by the Homo sapiens Annotation release 109, GRCh38.p12 the molecular location of the gene is given by; base pairs 42,313,324 to 42,388,503 on chromosome 17. The STAT3 gene within the context of the human genome is as illustrated by the information in figure 2 below while the whole genomic sequence of the STAT3 gene is as shown by figure 3. The materials and methods section will present further information on the sequence of the selected region of interest as per the primer 3 output results. Some of the main synonyms and symbols of the gene include; APRF; HIES; ADMIO; and, ADMIO1.

Genomic Locations for STAT3 Gene

chr17:42,313,324-42,388,568 (GRCh38/hg38)

Size: 75,245 bases

Orientation: Minus strand

chr17:40,465,342-40,540,586 (GRCh37/hg19)

Size: 75,245 bases

Orientation: Minus strand

STAT3 Features, Mutations and SNPs

The findings from previous studies and scholarly research further elucidate the understanding of the general biology of STAT3 as well as the clinical symptoms in individuals with various disease phenotypes (O’Shea et al., 2013). According to Yu et al. (2014) in their study, ‘Revisiting STAT3 signaling in cancer: new and unexpected biological functions,’ the autosomal dominant Hyper IgE syndrome (AD-HIES) is one of the conditions that arises as a result of the dominant and continued negative loss of the function mutations in the STAT3 gene. They presented that the condition is characterized by recurrent bacterial and fungal skin and lung infections which are linked with the development of cold abscess, the formation of chronic eczematoid rash, mucocutaneous candidiasis, and abnormalities in the primary structural connective tissue as well as the formation and development of arterial tortuosity or aneurysm. An important observation that the authors presented was that the loss of function of the STAT3 due to the mutations does not necessarily correspond to the complete absence of the protein or of its function, at least this has not been observed in humans to date (Holland et al., 2007).

CHAPTER 2: Materials and Methods

Data Collection/Mining

Bioinformatics

The data collection process basically involves the gene selection and the application of bioinformatics to data mine more information about the selected gene, in this case the STAT3. Further information about the gene such as the protein structure, its relation to any diseases and the products were obtained from credible online libraries which include the American National Center for Biotechnology Information (NCBI) and the Online Mendelian Inheritance in Man (OMIM). These online sources contain a wide range of information about disease genes. Therefore, the data collection began by searching for the chromosome from these sources, after which the specific gene, STAT3 is then selected. After choosing of the gene, the next step was then to identify the specific regions of interest. Again, the online libraries such as the NCBI and the European Bioinformatics Institute (EBI) provided useful information about the gene and its subsequent protein product including the sequence. Information about the amino acid sequence is significant for the identification of the domains or regions of interest. Such regions of interest depend on a number of functions, such as substrate and DNA-binding, membrane anchorage, interactions with other ligands. The selected regions of interest under investigation may also be sites for mutations that result in various health conditions and diseases. This selected segment for amplification ranges between 100 – 1000 bp.

Having selected the gene, finding about its functions, any possible involvement with disease and the characteristics of the STAT3 protein which it encodes, the next step was to determine the genomic sequence in the chromosome. Here, a significant source that has been used in the current study is the University of Santa Cruz (UCSC) Genome Bioinformatics site. The online library has a gene sorter link in which the name and symbol of the gene is inserted after which the genomic sequence can be downloaded. This is done by a clear distinction between the exons and the introns using upper case and lower case letters respectively.

Primer Design for PCR

The final process in the data collection entails the designing of the specific primers to be used in the laboratory polymerase chain reactions (PCR). The PCR primers are important for the amplification of the chromosomal sequence corresponding to the previously selected region of interest. To this end, another online source, a primer design website, known as the ‘Primer 3’ is used. The website gives several possible pairs of both forward and reverse primers. Also, the website gives the ability to specify the target area and the size, which in this case is an amplicon of between 100 – 1000 bp. This range gives better information about the sequence. The design website’s default settings are adjusted as required to get the needed primers. After designing the primers, by choosing the specific exons and introns corresponding to the region of interest, an in silico PCR is then run using the UCSC website and then the primers are ordered based on the results. The next step is then to work in the lab, setting up the PCR reactions and quantification of the DNA.

Aim and objectives

The main aim of the laboratory project is to determine location of the mutation in the STAT3 using a PCR approach in the laboratory with respect to the previously described disease. The specific objectives are:

Objectives

Primer selection and design

DNA amplification

Visualization through gel electrophoresis

DNA quantification and purification

DNA Sequencing

DNA Alignment

Materials:

Thermocycler

Agarose powder

Dehydrated Primers (forward and reverse) eurofins

Human DNA

Taq enzyme mix

TBE buffer

Gel Red

DNA step Ladder

Gel tray

Balance

Centrifuge

Electrophoresis chamber

Weighing boats

Microwave

Conical flask

Comb

BioDrop Spectrophotometer

Methods:

Primer Dilution

Primers are purchased when dehydrated and therefore are dissolved using specific volume of RNAse/DNAse free water. A total of 120 µl of the forward primers was used while 286µl for the reverse. The concentration of the stock solution was 100 µM. The stock solution was then diluted to ensure the concentration of primers was at 10 µM. This was done by using 180 µl of the RNAse/DNAse free water at a temperature of -20°C in a freezer.

Master mix

The above table illustrates the additions that are required to make this mix on the right, and the reaction tubes composition on the left. Each primer is then pipetted into the tube with the correct volume as given in the data sheet – of DNase- and RNase-free water. A dilution is then performed which produces 10 pmol/µl solution by adding of 20 µl of solution with 180 µl sterile water that is free of both DNase- and RNase.

PCR

A suitable quantity of the human X DNA solution (1 µl in this case) is then mixed into each of the 2PCR tubes and the same amount of sterile and deionized water is then added to the control experiment tube. The tubes are then softly mixed and centrifuged in a microfuge. The next step is then to insert the PCR tubes into a thermocycler. The figure below illustrates the specific PCR program that is used in the experiment.

The annealing temperature that is used is determined by;

X = Tm of your primers minus 5Tmto +5Tm selected Temperature (56°C, 56°C)

Making gel:

After amplification, the bands were then separated through gel electrophoresis. The gel is made through dissolving of the 1g agarose powder with 100ml of TBE to obtain a volume of 100ml. This is then microwaved, stirred, and then left to cool.

Setting the chamber, loading and Gel electrophoresis

While the flask of agarose cools, the gel casting tray is then set to ensure that the agarose does not drip from the tray. This is done by inserting the “comb”, and then sticking the ends of the casting tray with using an autoclave tape. When the agarose solution has cooled down, 5µl of GelRed reagent, which is used to stain the DNA, is then added. The liquid agarose is then transferred into the gel casting tray and waiting for some time, in this case, about 1-2hours for the gel to set. After this, the gel is then transferred in to the tank and submerged in a 0.5xTBE buffer. It is then loaded with 5µl DNA size markers as well as the control and experimental samples. Once best temperature had been established, and after repeating the PCR reaction to have enough DNA that will be sequenced, 5µl of the reaction was then consequently run on the gel. This was done at a constant 90V – ensuring that the sample slots are towards the cathode, such that the DNA then moved to the anode end.

Visualize UV

After the marker dye which had been previously mixed into the GreenTaq enzyme mix, had moved down up to two-thirds of the gel (in 40 minutes), the current was switched off and then the gel was removed from the electrophoresis tank. At this point, the DNA bands were then visualized and noted in gel documentation using a transilluminator.

PCR product purification

Below is an outline of the steps used to purify the PCR Primers;

The first step the addition of up to 5 volumes of Buffer PB to a volume of the PCR product and then mixing them in a tube.

A quick-column is then placed in the 2 ml collection tube.

The PCR/Buffer Mix from the first step is then added into the quick column and centrifuged for about a minute.

Any liquid in the collection tube is cast-off and then the quick column is placed into the tube.

About 750 µl of the Buffer PE is then added into the quick column and then centrifuged for a minute.

The quick column is then centrifuged again for 60 secs and placed into another centrifuge tube that is clean.

50 µl Buffer EB is then put into the quick column followed by a one minute centrifuge

DNA Quantification

For sequencing, the amplified DNA and primers were sent to MWG Eurofins. For each sequencing reaction, a 15 µl of amplified DNA at a concentration of 5µM for the forward sequencing and reverse sequencing reactions were used. The purified DNA was then quantified using a spectrophotometric analysis. This current study used the Biodrop, which basically works in the same way as a spectrophotometer but allows for the analysis of a smaller quantity of the sample. The process entailed placing a small quantity of the ‘blank’ in the machine and consequently pressing ‘control’ button. After this, the ‘blank’ solution was then wiped away gently but carefully with a small piece of tissue. The small quantity of the reaction mixture was then pipetted into the machine and then the ‘reaction’ button pressed and the result noted. The ‘blank’ or control was therefore the small quantity of the EB buffer that the DNA had been eluted in. A significant factor that was taken into consideration was to mix all the successfully amplified DNA together before removing it for absorbance measurement using the biodrop.. Once the process was done, the next step was to move to on to the sequencing part of the project.

DNA concentration

The biodrop will calculate the concentration of the DNA. The formula below is then used to calculate the amount of DNA: Nominal DNA concentration (µg/ml) = OD260 x 50 x dilution factor. Here the dilution factor is 0.

Using the PCR product as a template for further PCR experiments

If the bands are still very faint after the electrophoresis, it may indicate that the mean quantity of DNA that has been produced from the assay is not enough to be sequenced. Therefore, in such as case, a “re-PCR” is carried out to increase the amount of DNA. To do this, the same procedures that have been previously outlined are followed except: using a larger quantity of amplified DNA as model DNA and then correcting the total volume with the sterile water that is free of any DNase- and RNase-free. After re-PCR, the amplified DNA is then checked by conducting another gel electrophoresis examination, with the same procedure, as previously outlined.

Chapter 3: Results

Introduction

This section is a presentation of the all the results of the human gene project, from the data collection to the DNA amplification and quantification through polymerase chain reaction (PCR) to the sequencing results from MWG Microfins.

STAT3 Gene Structure Selected regions

Designing Primers

Primer3 Output

Using 1-based sequence positions

When the primers were designed, they had to be checked using In-Silico PCR to make sure they amplified the required sequence.

Polymerase Chain Reaction (PCR) results

The experiment amplified the selected regions of interest of the primers with an unknown human’s DNA. Both of the experiments exhibited normal PCR amplifications for all the exons and introns analyzed. The PCR products of the representative human X DNA and the amplified DNA from the primers are as shown below:

Gene Amplification

The images below represent the visualized agarose gel of the amplified STAT3 PCR products for unknown human DNA and amplified DNA from primers. All the exons are located in close proximity on the genomic DNA and were amplified in each of the PCR products. All amplified products showed the expected length and there were clear multiple bands that were observed at specific temperatures.

The first well represents the DNA step ladder from 100 bp to 1000 bp. There were a total of 7 wells, in which well number 1 did not have any band while wells 2, 3, 5, 6 and 7 showed very clear and bright bands. Consequently, the multiple primer dimers could be observed in wells 2, 3, 4, 5, as well as 7. In figure 7, which represents the amplification of the STAT3 gene, there were a total of 15 wells that were visualized. However, there were no visible bands in wells 1, 4, 10 and 13, while multiple bright bands were observed in wells 5, 6, 8, 9, 11 and 12 respectively. The primer dimers were clearly visualized in wells 2, 3, 5, 6, 8, 9, 11, 12, 14 and 15

There were a total of 15 wells that were visualized. However, there were no visible bands in wells 1, 4, 10 and 13, while multiple bright bands were observed in wells 5, 6, 8, 9, 11 and 12 respectively. The primer dimers were clearly visualized in wells 2, 3, 5, 6, 8, 9, 11, 12, 14 and 15. All in all, the agarose gel of the STAT3 PCR products for unknown human DNA and amplified DNA from primers showed bright bands for all the wells that were loaded with the samples and all the amplified products also exhibited the expected length

DNA Quantification Results

For sequencing, the purified amplified DNA from the PCR reaction was then quantified through spectrophotometric analysis using the Biodrop which basically works in the same mechanism as a spectrophotometer but allows for the analysis of just 2µl of the sample. The results are as indicated in the figure below:

BioDrop results: the concentration of 2 µl purified DNA

Sequencing and Alignment Results

The sequencing results from the forward and reverse primers are as shown below:

Forward Primer Sequencing

Reverse Primer Sequencing

Reverse Complement

Alignment

A comparison between the forward sequence and the reverse complement to identify presence of any overlapping sequences shows there are several matches but not a fully exact match. Hence the forward and reverse primer sequences above were then aligned using the Blast Alignment tool, to compares the two sequences.

Forward

The upper strand in the above figure, Query, represents the Forward primer sequence while the lower one, Sbjct, represents the Reverse primer sequence. The tool shows there is 98% homology between the sequences, with three gaps identified. The sequence of the important region of the amplified gene was then submitted again to BLAST, through a specialized search to help determine the significant features. The results indicated that the features included in that part of the sequence included signal transduction and activator of transcription isoforms 1 and 2. This means that amplification of the STAT3 was therefore successful.

Forward and reverse

Therefore, the Forward and Reverse Primers were then run again through the specialized web tool BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the existence of nucleotide polymorphisms. For both sequences, there were a total of 3 gaps; one deletion of C from the forward primer and two deletions of A and C from the reverse primers.

Chapter 4: Discussion

The project has achieved all its aims and objectives. The choice of the STAT3 gene for this current study presented an opportunity of learning and understanding the role of the gene, and its larger family in cell physiology as well as the effects of its mutations and single nucleotide polymorphisms (SNPs). The main aim of the study was to determine the location of STAT3 mutations by amplifying with an unknown human X’s DNA. The specific objectives to reach this aim included primer selection and design, followed by the amplification and visualization through gel electrophoresis, after which the PCR products were purified and quantified. The forward and reverse primers were sequenced and then aligned using the web based tool BLAST. In this case, as observed in the results, the promoter region and the significant exons were amplified. Usually this stage might be quite challenging, but however the researcher successfully conducted the operations and obtained well amplified STAT3 from the designed primers. Following this successful amplification and subsequent sequencing, the results showed that indeed the amplification had been specific with respect to the region featuring the potential signal transducer and activator transcription sub family sequence. Further, an analysis of the sequences showed that there were a total of three single nucleotide polymorphisms that fell inside the exon sequence and hence potential influencing on the expression of the protein. To this end, important information has been acquired about the STAT3 gene over the domains of; its binding activities, promoter regions, the expression etc. The cytokines activated by the STAT3 translocate to the nuclei of the cell after undergoing several translational modifications and dimerization, in which the protein then binds to the serum inducible element – bearing promoters to finally activate transcription (SIE, TTC(N3)GAA). The DNA binding domain which lies in the location between 320-494 can be mutated to lead to higher levels of IgE , inhibiting the binding activity of the serum inducible element therefore leading to a rare health condition known as hyper immunoglobulin E syndrome (HIES).

This current study has managed to isolate an interesting genomic fragment that contains a 5’ portion by using a polymerase chain reaction (PCR). Using the product of the PCR reaction as a probe, the genomic portion was isolated and the DNA quantified using a Biodrop. The identification and of new additions of function mutations of the Signal transducer and transcription, 3 gene alongside the studies on the individuals affected by the loss of function mutations can greatly help in expanding the overall understanding of the STAT3 function and pathophysiology as well as its significant role in the regulation of the immune system (Braunstein et al., 2003). Aside from the fact that the activation of STAT3 is a key event for the regulation of cell development, growth, migration, motility, and immune responses, its aberrant expression as a result of the constitutive activation has been linked with a variety of human malignancies such as the gastric, prostrate, breast, cervical, hepatocellular and nonsmall cell lung cancers. Further, recent studies have also reported that the single-nucleotide polymorphisms (SNPs) in STAT3 are significantly associated with susceptibility to other health conditions such as Crohn’s disease, Hyper-IgE syndrome (HIES), Shingles, Autoimmune lymphoproliferative syndrome and other autoimmune disorders (Yang et al., 2007). Studies and research in this domain have identified over one hundred germline mutations in the signal transducer and activator of transcription 3 gene (STAT3) for people with the Hyper-IgE syndrome (HIES) condition. An examination of these mutations involved in the condition by previous works shows that it results in a change of the single amino acids found within the STAT3 protein. Further, the mutations usually occur within the regions of the protein that are vital for its activation or the regions critical for its ability to successfully bind to DNA (Heimall et al., 2011).

The mutations in the STAT3 gene that result into the AD-HIES syndrome are as a result of the changes in the structure that in turn alter the function of the protein hence limiting its ability to normally regulate the activity of other genes as required (Woellner et al., 2010). Since these changes lead to the impairment of the normal functioning of the protein encoded by the STAT3 gene, they are usually referred to as ‘loss-of-function mutations’. The inability of the STAT3 protein to transmit any signals leads to the disruption of the Th17 cells maturity. The Th17 cells are a subset of the immune system T-cells (Zhang et al., 2005).. As a result of the abnormalities in the immune system, individuals with this health condition become highly susceptible to other bacterial and fungal infections which mainly affect the lungs and the skin (Anolik,et al., 2009). It is upon the foundation of the importance and action of the protein in DNA-binding and the relation to the listed diseases that this current study is grounded on. The main goal of this human genome project was to undertake a DNA sequencing of the selected key portions of the human STAT3 gene. As per the previously presented results, this study has managed to achieve this objective by amplifying a designed primer from an unknown individual’s DNA by applying the polymerase chain reactions processes. The PCR products, that is, the 15 µl of amplified DNA at a concentration of 5 ng/µl for the forward and reverse sequencing reactions and 15 µl of 10 pmol/µl of each primer were then sent for sequencing to MWG Eurofins. The images of the obtained sequencing results have all been presented in the chromatograms in the appendix.

To carry out the current study on DNA sequencing of the STAT3 gene, the first step was to use various data mining techniques and biotechnology information skills to first find out information about the STAT3 gene structure, as well as the structure of the protein which it encodes, the STAT3. The project then proceeded to design primers by using the services of Primer3 site. As such, the selected primers were: Forward:60.00C CAC TCC TCG CCT AGA GTT GG and reverse 59.8 0C ACC TTG GGA TTG TTG GTC AG. These temperature calculations were done with an assumption of 50 mM salt and 50 nM annealing oligo concentration. The sequencing and annealing positions of the two primers, forward and reverse at 241 base pairs were as follows:

chr17:42324961-42325201 241bp

CAC TCC TCG CCT AGA GTT GGA CCT TGG GAT TGT TGG TCA GCA CTC CTC GCC TAG AGT TGG cag cag gtg tgg ttt atg gca tgt cct ttc att ctg agc ccc gtg aga tgc ggg tga aga gat ttc caa ggc tgt gag agc ccc tct gcc tcc cca gct cag tcc cca ctc cct ccg cag acc cac tcc ttg cca gtt gtg gtg atc tcc aac atc tgt cag atg cca aat gcc tgg gcg tcc atc ctg tgg tac aac atg CTG ACC AAC AAT CCC AAG GT.

The above represents the genomic sequence in the chromosome 17, and not the sequence of cDNA. In the sequence of the STAT3, the exons can be distinguished from the introns by the type of capitalization, in which upper case letters represent the exons while the lower case letters represent the introns. The output from the Primer3 is as shown in the table in appendix. The included sequence size and the region size of the amplicon according to these results were at first 241 base pairs (bp).

Villarino et al. (2015) asserts the importance of STAT3 as a transcription factor that mediates the transmission of chemical signals to the cell nucleus after stimulation of cytokine and other growth factors. The authors further present that after the cytokine simulations, the cytokine receptors are phosphorylated by the Janus Kinase (JAK) which in turn result in the activation of the cytokine receptor allied kinases which then tyrosine-phosphorylate the STAT protein (Cimica et al., 2011). After this, two of the STAT molecules homo-dimerize or hetero-dimerize through their SH2 domain depending on the stimulation and then shift to the cell nucleus whereby they bind to certain DNA elements and then regulate the expression of the genes. In the scholarly article, the authors then describe the significance of STAT3; it promotes and controls the transcription of specific target genes that are important for cell proliferation, cell apoptosis, and cell differentiation. Villarino et al. (2015) further observe the importance of STAT3 in the modulation of both innate and adaptive responses. As such they list some of the cytokines involved which include; IFNs, IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, and IL-27 (Wormald, and Hilton, 2004).

Analysis of the PCR products

All the amplified products were analyzed through gel electrophoresis (Tris-HCl, boric acid and EDTA). This entailed using a specific volume of agarose solution for casting the gel as well as a suitable quantity of agarose powder. An addition of a suitable amount of 0.5x TBE was also added and mixed with the products. 5µl of GelRed reagent, was then added to stain the DNA . After this, it was then loaded with 5µl of 100 bp DNA size markers and an optimal temperature was then set. When the 5µl of the reaction was run on the gel at a constant 90V to 100V – ensuring that the sample slots are towards the cathode, the DNA then moved to the anode end. After which clear DNA bands could be observed as noted in the gel visualization results in figures 6 and 7 respectively. In figure 6, the first well represents the DNA step ladder from 100 bp to 1000 bp. There were a total of 7 wells, in which well number 1 did not have any band while wells 2, 3, 5, 6 and 7 showed very clear and bright bands. Consequently, the multiple primer dimers could be observed in wells 2, 3, 4, 5, as well as 7. In figure 7, which represents the amplification of the STAT3 gene, there were a total of 15 wells that were visualized. However, there were no visible bands in wells 1, 4, 10 and 13, while multiple bright bands were observed in wells 5, 6, 8, 9, 11 and 12 respectively. The primer dimers were clearly visualized in wells 2, 3, 5, 6, 8, 9, 11, 12, 14 and 15. All in all, the agarose gel of the STAT3 PCR products for unknown human DNA and amplified DNA from primers showed bright bands for all the wells that were loaded with the samples and all the amplified products also exhibited the expected length.

After the PCR reaction had amplified the DNA samples as shown by the multiple bright bands that were observed in the gel visualization figures 6 and 7 and the subsequent purification of the DNA, it was then quantified and then sent for sequencing. The quantification was done using a Biodrop machine that basically works as a spectrophotometer in spectrophotometric analysis. The formula used is as previously laid out in the methods section. By analyzing a smaller sample of 2µl of the purified products, the DNA that was sent for sequencing was at a concentration of 38.20 µg/ml. MWG Microfins then sequenced the samples, which include the forward and reverse DNA and Primers marked as SS D1 and SS D2 respectively.

STAT3 Gene Sequencing Key Selected Regions

The sequencing results from the PCR products that were sent for sequencing to MWG Microfins shows the arrangement of the introns for the regions of interest that were amplified at 100 bp. The sequences in the chromatograms are as indicated below. SSD1 F and SSD1 R represent the amplified DNA sequences while SSD2 F and SSD2 R represent the sequences of the forward and reverse primers respectively. The respective chromatograms for the amplified DNA and the primers, with visualizations of the nucleotides and the respective peaks were all presented in figures 9, 10, 11 and 12 in the appendix. These protein and gene sequences were further analysed by using various gene analysis software as laid in the methodology section. The gene analysis presented some of the novel features, especially in terms of SNPs. The results of the current study show that over half of the binding sites of the STAT3 are mapped to introns. A further analysis of the chromatogram sequences suggest that the SNPs that are associated with various autoimmune diseases are co-localized within these STAT3 binding sites. As such, some of the notable features of this gene include the DNA binding domain (Jiao et al. 2008) and the SH2 domain. Also, there is a major site of tyrosine phosphorylation located at Y705 as well as a major site of serine phosphorylation at the S727 site. Consequently, the tyrosine of the protein encoded by the gene activates the high affinity DNA binding at TTC NNN GAA. The stimulation of phosphorylation occurs due to various growth factors which include; LIF, OSM, IL-6, leptin, EGF, PDGF, and HGF. The C terminal of this domain acts a transcriptional activation domain to which the activity is enhanced through the serine phosphorylation at S727. The protein and nucleotide alignments below show the various deletions in these mutation sites that result in reduced function linked with the health conditions previously mentioned above.

Conclusion

This current study has managed to collect information about the STAT3 gene; design primers based on a selected genomic region and consequently conducted a PCR amplification assay to amplify the DNA. A successful sequencing of the purified PCR products was then carried out and the chromatograms presented in the results section. Consequently the study has managed to analyze the significant regions in the sequence in relation to the functions of STAT3 such as the binding activities and its association with various autoimmune diseases.

Future Work

This research has managed to sequence and analyze a small part of the STAT3 gene and presented that indeed there exist some significant features and mutations that are related to disease. Further structural and functional analysis of the gene and the binding sites, can help in the management of such health conditions.

Acosta-Rodriguez, E.V., Napolitani, G., Lanzavecchia, A., and Sallusto, F. (2007). Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat. Immunol. 8, 942–949.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002). Molecular Biology of the Cell.

Annunziato, F., Cosmi, L., Liotta, F., Maggi, E., and Romagnani, S. (2012). Defining the human T helper 17 cell phenotype. Trends Immunol. 33, 505–512.

Anolik, R., Elmariah, S., Lehrhoff, S., Votava, H.J., Martiniuk, F.T., and Levis, W. (2009). Hyperimmunoglobulin E syndrome with a novel STAT3 mutation. Dermatol. Online J. 15, 16.

Avery, D.T., Ma, C.S., Bryant, V.L., Santner-Nanan, B., Nanan, R., Wong, M.,Fulcher, D.A., Cook, M.C., and Tangye, S.G. (2008). STAT3 is required for IL21-induced secretion of IgE from human naive B cells. Blood 112, 1784–1793.

Avery, D.T., Deenick, E.K., Ma, C.S., Suryani, S., Simpson, N., Chew, G.Y.,Chan, T.D., Palendira, U., Bustamante, J., Boisson-Dupuis, S., et al. (2010). B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans. J. Exp. Med. 207, 155–171.

De Beaucoudrey, L., Puel, A., Filipe-Santos, O., Cobat, A., Ghandil, P., Chrabieh, M., Feinberg, J., von Bernuth, H., Samarina, A., Jannière, L., et al. (2008). Mutations in STAT3 and IL12RB1 impair the development of human IL17-producing T cells. J. Exp. Med. 205, 1543–1550.

Beitzke, M., Enzinger, C., Windpassinger, C., Pfeifer, D., Fazekas, F.,Woellner, C., Grimbacher, B., and Kroisel, P.M. (2011). Community acquired Staphylococcus aureus meningitis and cerebral abscesses in a patient with a hyper-IgE and a Dubowitz-like syndrome. J. Neurol. Sci. 309, 12–15..

Braunstein, J., Brutsaert, S., Olson, R., and Schindler, C. (2003). STATs dimerize in the absence of phosphorylation. J. Biol. Chem. 278, 34133–34140.

Buckley, R.H., and Becker, W.G. (1978). Abnormalities in the regulation of human IgE synthesis. Immunol. Rev. 41, 288–314.

Buckley, R.H., Wray, B.B., and Belmaker, E.Z. (1972). Extreme hyperimmunoglobulinemia E and undue susceptibility to infection. Pediatrics 49, 59–70.

Casanova, J.-L., Holland, S.M., and Notarangelo, L.D. (2012). Inborn errors of

human JAKs and STATs. Immunity 36, 515–528.

Chandesris, M.-O., Melki, I., Natividad, A., Puel, A., Fieschi, C., Yun, L., Thumerelle, C., Oksenhendler, E., Boutboul, D., Thomas, C., et al. (2012). Autosomal dominant STAT3 deficiency and hyper-IgE syndrome: molecular, cellular, and clinical features from a French national survey. Medicine (Baltimore) 91, e1–19.

Chen, Z., Laurence, A., Kanno, Y., Pacher-Zavisin, M., Zhu, B.-M., Tato, C., Yoshimura, A., Hennighausen, L., and O’Shea, J.J. (2006). Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells. Proc. Natl. Acad. Sci. U.S.A. 103, 8137–8142.

Fagerlund, R., Mélen, K., Kinnunen, L., and Julkunen, I. (2002). Arginine/lysine-rich nuclear localization signals mediate interactions between dimeric STATs and importin alpha 5. J. Biol. Chem. 277, 30072–30078.

Grimbacher, B., Schäffer, A.A., Holland, S.M., Davis, J., Gallin, J.I., Malech, H.L., Atkinson, T.P., Belohradsky, B.H., Buckley, R.H., Cossu, F., et al. (1999b). Genetic linkage of hyper-IgE syndrome to chromosome 4. Am. J. Hum. Genet. 65, 735–744.

Heimall, J., Davis, J., Shaw, P.A., Hsu, A.P., Gu, W., Welch, P., Holland, S.M., and Freeman, A.F. (2011). Paucity of genotype-phenotype correlations in STAT3 mutation positive Hyper IgE Syndrome (HIES). Clin. Immunol. 139, 75–84.

Holland, S.M., DeLeo, F.R., Elloumi, H.Z., Hsu, A.P., Uzel, G., Brodsky, N., Freeman, A.F., Demidowich, A., Davis, J., Turner, M.L., et al. (2007). STAT3 mutations in the hyper-IgE syndrome. N. Engl. J. Med. 357, 1608–1619.

Jiao, H., Tóth, B., Erdos, M., Fransson, I., Rákóczi, E., Balogh, I., Magyarics, Z., Dérfalvi, B., Csorba, G., Szaflarska, A., et al. (2008). Novel and recurrent STAT3 mutations in hyper-IgE syndrome patients from different ethnic groups. Mol. Immunol. 46, 202–206.

Kato, K., Nomoto, M., Izumi, H., Ise, T., Nakano, S., Niho, Y., and Kohno, K. (2000). Structure and functional analysis of the human STAT3 gene promoter: alteration of chromatin structure as a possible mechanism for the upregulation in cisplatin-resistant cells. Biochim. Biophys. Acta 1493, 91–100.

Kiu, H., and Nicholson, S.E. (2012). Biology and significance of the JAK/STAT signalling pathways. Growth Factors 30, 88–106.

Kofoed, E.M., Hwa, V., Little, B., Woods, K.A., Buckway, C.K., Tsubaki, J., Pratt, K.L., Bezrodnik, L., Jasper, H., Tepper, A., et al. (2003). Growth hormone insensitivity associated with a STAT5b mutation. N. Engl. J. Med. 349, 1139–1147.

Kovanen, P.E., and Leonard, W.J. (2004). Cytokines and immunodeficiency diseases: critical roles of the gamma(c)-dependent cytokines interleukins 2, 4,7, 9, 15, and 21, and their signaling pathways. Immunol. Rev. 202, 67–83.

Lim, M.S., and Elenitoba-Johnson, K.S.J. (2004). The molecular pathology of primary immunodeficiencies. J Mol Diagn 6, 59–83. Liu, J., Li, Q., Chen, T., Guo, X., Ge, J., and Yuan, L. (2011a). Destructive pulmonary staphylococcal infection in a boy with hyper-IgE syndrome: a novel mutation in the signal transducer and activator of transcription 3 (STAT3) gene (p.Y657S). Eur. J. Pediatr. 170, 661–666.

O’Brien, C.A., Gubrij, I., Lin, S.C., Saylors, R.L., and Manolagas, S.C. (1999). STAT3 activation in stromal/osteoblastic cells is required for induction of the receptor activator of NF-kappaB ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D3 or parathyroid hormone. J. Biol. Chem. 274, 19301–19308.

Renner, E.D., Rylaarsdam, S., Anover-Sombke, S., Rack, A.L., Reichenbach, J., Carey, J.C., Zhu, Q., Jansson, A.F., Barboza, J., Schimke, L.F., et al. (2008). Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome. J. Allergy Clin. Immunol. 122, 181–187.

Yamaoka, K., Saharinen, P., Pesu, M., Holt, V.E.T., 3rd, Silvennoinen, O., and

Yang, J., Liao, X., Agarwal, M.K., Barnes, L., Auron, P.E., and Stark, G.R. (2007). Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFkappaB. Genes Dev. 21, 1396–1408.

Zhang, Z., Welte, T., Troiano, N., Maher, S.E., Fu, X.-Y., and Bothwell, A.L.M. (2005). Osteoporosis with increased osteoclastogenesis in hematopoietic cellspecific STAT3-deficient mice. Biochem. Biophys. Res. Commun. 328, 800–807. 198

Zhong, Z., Wen, Z., and Darnell, J.E., Jr (1994). Stat3 and Stat4: members of the family of signal transducers and activators of transcription. Proc. Natl. Acad. Sci. U.S.A. 91, 4806–4810.