NC DNA DAY Genes and Diseases Cystic Fibrosis Presenter Notes

Transcription

1 NC DNA DAY Genes and Diseases Cystic Fibrosis Presenter Notes Title slide Hello! Today we are going to learn about how your physical characteristics are inherited through genetic material and how mutations in this genetic material can sometimes lead to disease. We will be focusing on the genetic disease, Cystic fibrosis, which is caused by a mutation in DNA We will lead you through an activity that puts you in the shoes of a clinician, where you will determine which patient of four possible patients has a cystic Fibrosis. And we will talk about how scientists and doctors are working together to come up with unique and novel treatments for cystic fibrosis patients. What is DNA? So What IS DNA? DNA is the most basic molecular building block of you! Your DNA dictates your physical appearance but it also plays a role in determining your susceptibility to disease and even your personality! As you know, YOU are a unique individual, and part of what makes you unique is your distinct variation of DNA. What is DNA Day? DNA Day commemorates two major events in the history of our understanding about DNA. In April 1953, James Watson and Francis Crick determined the structure of DNA. 50 years later, in April 2003, the Human Genome Project determined the entire sequence of human DNA. Research scientists are using the knowledge and technology generated by this project to further understand how your DNA sequence can contribute to disease. Variations in the DNA of different individuals can cause visible changes in individuals First of all, lets take a minute to look around us. Just in this room, we can see many examples of genetic variation. Some genetic traits, such as the color of your hair or the color of your skin are controlled by multiple genes A gene is a segment of DNA that codes for a particular trait

2 Others traits, such as the ability to roll your tongue or inability, are controlled by only one gene. How are Genetic Traits Inherited? These traits, the characteristics that help define who we are, can be inherited. This is a photo of Amelia s family. (Amelia is one of the creators of this lesson.) All of our traits, from the color of our hair, to the color of our eyes, to the shape of our nose and chin are a combination of our parents traits. How are Genetic Traits Inherited? Our traits are inherited through our genes, remember, a gene is a segment of DNA that codes for a particular trait. Genes are carried on chromosomes, which are condensed and highly organized structures made of DNA We all have 23 pairs of chromosomes, as shown here. We inherit one chromosome from each parent. For every gene, we inherit one copy from our mother, and one from our father. This means that every person has two copies of each gene- one of each chromosome in the pair. For example on one pair of my 23 chromosomes I have one copy of the gene for tongue rolling from my mother and one copy of the gene for tongue rolling from my father. Different versions of genes are alleles For each gene, you might have 2 different alleles for example one allele for brown eyes and one for blue. OR you might have 2 of the same allele for example, two alleles for blue eyes. Your combinations of alleles determine what traits you will have, such as Can you roll your tongue? The ability to roll your tongue. Who can roll their tongue? (Raise hand) If you can, you carry a dominant allele, either one copy or two.

3 Who cannot? (Raise hand) You carry two copies of a recessive allele (point to blue alleles). When we saw an allele is dominant this just means that if you inherit both the dominant and recessive allele, the dominant one will be expressed. The only way that a recessive allele can be expressed is if you carry two copies. It s important to point out here that if a trait is dominant, it is NOT necessarily more common! For example, the trait of having six fingers on one hand, which is called Polydactyly, is a dominant trait. Does anybody know anyone with 6 fingers? Probably not, it is extremely rare! Dominant does not mean that it is more common. Some diseases are caused by genetic factors that can be inherited So far we have looked at the inheritance of traits that do not cause a disease. Now, we will shift gears and talk about how the inheritance of some genetic factors can lead to disease. In order to understand genetic diseases however, I first want to review how information from DNA is translated into the proteins that make up our bodies and how this process can sometimes go wrong which in some cases can lead to disease. Genes contain instructions to make proteins. So far we have learned about genes. On a molecular level, a gene is a segment of DNA. That piece of DNA tells your cells how to make a certain protein or series of proteins. In order to make that protein, the information on the DNA, shown here in dark blue, must first be copied into RNA shown here in light blue. This process is called transcription. That RNA copy then can be used by machinery in your cells to make proteins, shown in green. This process is called translation. Proteins are long intricately folded chains of amino acids. Amino acids are the most basic molecular building blocks of proteins. Proteins work together to form the functional machinery that makes up a cell. How does an Altered Gene Result in an Altered Protein? Let s look more closely at how a change in DNA can result in the wrong protein. We ll use this sentence - SAM AND TOM ATE THE HAM - as an example of a DNA message. Much like the alphabet, Information in DNA is stored in as a series of 4 distinct molecules called bases. Each base has been assigned a letter, A C T or G.

4 This series DNA bases is read in three- letter groups. Each three- letter group stands for a specific amino acid, similar to one word in a sentence. As we learned, these amino acids are strung together to make a protein, which is a complete functional unit, much like a completed sentence. What happens If we change a single letter in our sentence SAM AND TOM ATE THE HAM, For example the A in HAM to an I This substitution changes the meaning of the sentence. In this case the sentence no longer makes sense. Similarly, if we change a letter in the DNA sequence the central A to a C one of the amino acids in the protein will be different. This new protein may not work properly. A change in the DNA is called a mutation. This is just one example of a mutation - there are many other ways that an altered gene can result in an altered protein. There are many types of mutations. Variations in the DNA of different individuals can cause physical changes in those individuals or even cause disease. An example of a disease caused by a genetic mutation is Cystic Fibrosis. Cystic Fibrosis 1 in 30,000 Americans have it and there are 2,500 new cases per year Can lead to organ inflammation Infertility Most notably the inability to clear the thick mucus in the airway Diseases are mostly defined by the symptoms a patient can experience. Initially, antibiotics became the common treatment for CF, but because this didn t lead to people being cured, scientists began thinking about how CF is a genetic disease. To know the cause of CF, we first need to see what healthy lungs look like. The lungs are lined with specialized cells Here is a depiction of what the lungs look like and here is a close up view. At first glance, some people might think that the lungs are just big hollow bags. In reality they are more like sponges, which increases the area inside the lungs that is available for blood to meet with the air. Your lungs need to be clean in order to function properly. The air you breathe is not always clean. For this reason, your lungs have a cleaning system. Dust, dirt, and germs are trapped

5 in the mucus that lines your nose, trachea and lungs. The glands that make the mucus are called mucus glands. The dirty mucus is then pushed out of your lungs and into your throat by tiny hairs called cilia. This system is called the mucus escalator. Without it, we would have no way to get rid of germs from our lungs and we would be sick all the time!! These special cells clear dust and excess mucus Think of this ball as mucus and the guy inside is dirt. Without the hands in the crowd, that ball would fall and not move. The inability to move the dirt or other particulates causes breathing problems. So what helps the hands move the dirt?...right, it s the mucus layer. Cystic Fibrosis Transmembrane Regulator (CFTR) Protein helps clear the mucus Let s remember how proteins are made, you have a gene in the DNA that is (have them answer transcribed) and then RNA is (have them answer translated) into protein. Remember, these genes in our chromosomes are inherited. Now that we have our protein, let s see how it is incorporated into the cell membrane. The CFTR in lung cells [Point everything out] These are lung epithelia cells (can anyone tell me what epithelial means, cells that line surfaces in the body). These are the Cilia (green), and here are the cell s nuclei (blue). Here is CFTR present on the surface of the cells below the cilia. Slide definition: Epithelial Cell Cells that form the covering or lining of all internal and external body surfaces Mutations in the CFTR gene blocks CFTR protein function In a common mutation in the DNA of the CFTR gene, the amino acid sequence is changed in the protein and the protein does not fold properly. This mutated protein gets degraded before it ever reaches the plasma membrane. As you saw before the normal mucus layer is thin and can move well to move dirt out. The CF mucus is thick and a lot harder to move. This makes it difficult to breathe. Now what is the relationship between the mucus consistency and CFTR? Cystic Fibrosis airways are dehydrated and cannot clear mucus In Cystic Fibrosis, the outside of the lung cells (airway) is dehydrated. Instead of being able to move freely like in the healthy airway, the cilia in a CF airway are trapped in thick mucus (think GAK!) and cannot move. The thick mucus gets stuck in the lungs and becomes a breeding ground for all sorts of infectious bacteria. How does water move in and out of cells? Answer: osmosis/water follows concentration gradient of solute Turns out, the CF airway mucus has a lack of water above the cilia. It is the CFTR protein that helps regulate the balance of water in the mucosal layer.

6 Cystic Fibrosis airways accumulate bacteria and inflammatory cells So what happens with this thick mucus layer? Turns out dirt is just the beginning of your problems, you also have to worry about bacteria such as PA. PA is an opportunistic pathogen, which means it infects people who are immunocompromised or already sick. Although the genetic disease is bad, it is the PA infection which is the ultimate demise of the CF patients. You also get buildup immune cells in the lung. Slide definitions: Opportunistic pathogen An infection that does not cause disease in a host with a healthy immune system, but does in a host who s immune system is compromised. Another way to think of it is: A pathogen that causes disease when there s an opportunity. Immunocompromised A deficient immune system How is CF inherited? Now that we know that mutations in the CFTR gene cause CF, let s get a better idea of how this disease is inherited. Pedigree chart Geneticists use a type of chart called a pedigree chart to understand how diseases are inherited in families. Symbols are used to represent people and lines are used to represent genetic relationships. The open circles in this chart represent non diseased females and open squares represent non diseased males. The line connecting the male and female in generation I mean they had 4 children, which are shown in generation II. In sum, this pedigree chart shows a man and woman had 4 children and passed on no disease traits. Pedigree 2 This pedigree chart, on the other hand, represents a typical method of cystic fibrosis inheritance. The same family structure shown is the same as in the slide before except this time the mother and father each have 1 mutant CFTR allele and one normal CFTR allele. The mother and father do not have cystic fibrosis because the disease requires 2 mutant alleles but they are considered carriers of the disease allele. Because CF is a recessive disease, you need 2 mutant copies of the CFTR gene to have the disease. Now take a look at their 4 children. Notice that 1 child has CF (black square), 2 children are carriers of the mutant allele (half open/half black), and 1 child has 2 normal alleles (open circle). This is an example of what the 4 children may inherit in a family based on their parent s genotypes. Since mom and dad each can donate a mutant or a normal CFTR allele, their children each have a 25% chance of inheriting both mutant alleles (diseased), 50% chance of inheriting 1 mutant and 1 normal allele (carrier), and 25%

7 chance of inheriting 2 normal alleles. In this example, the first son inherited both mutant copies and therefore has CF while 2 of his siblings are carriers and 1 sibling has 2 normal copies. Draw a punnett square on the board and use it to explain the pedigree 25% affected (ff) 50% carrier (Ff) 25% normal (FF) ****Note that the pedigree does not always work out statistically like a punnet square. Each child is a separate and independent event. Slide CF risk: In this example, how many children are likely to inherit CF? Teacher: give students some time to try and come up with the answer and then draw the punnett square on the board and explain: In this case, the dad has CF and will pass on only mutant CFTR alleles and the mom is a carrier so she will pass on either a normal or mutant allele so there is a greater chance that the children will inherit a mutant allele. 50% CF (ff) 50% carrier (Ff) Now it s your turn Now it s your turn! Classroom activity Pedigree Activity 1 This activity uses paper bags and fuzzy balls to simulate random genetic inheritance. Display Classroom pedigree slide on board/overhead/computer screen. You will need to add details about the genotypes that are chosen from the balls in the bags to the pedigree. You can either draw the pedigree on the board and fill it in, or you could use the annotating tool in your power point presentation (lower left corner) to draw the letters for the genotypes.

8 F f F f F F F f Spouse Spouse Pre activity- label 5 bags: Person A, Person B, Person 1, Person 2 and Spouse with Ff genotype Person A bag: Place one ball of each color Person B bag: Place one ball of each color Spouse bag: Place one ball of each color Person 1: Will be based on random drawing by class Person 2: Will be based on random drawing by class Students choose Assign one color ball to dominant allele and a different color to recessive allele. 1) From Person A bag, have a student choose a ball to represent the allele passed on by Person A to Person1. Fill in the blank for this allele for Person 1. Return the puff ball to Person A s bag.

9 2) From Person B bag, have a student choose a ball to represent the allele passed on by Person B to Person 1. Fill in the blank for this other allele for Person 1. Return the puff ball to Person B s bag. 3) Repeat process for person 2 and 3. 4) Now add puff balls to Person 1 s bag and puff balls to Person 2 s bag, based on the genotype chosen in steps ) For third generation have a student come up and pick one ball from person 1 to represent the allele passed on by Person 1 to child 4. Repeat for child 5. Spouse of Person 1 can only pass on one type of allele (F). 6) For child 6 (and 7and 8) have a student pick one ball from Person 2 and one ball from Spouse to represent the alleles passed on by Person 2 to child 6. Repeat for children 7 and 8. Student Questions. 1) How many of the third generation (grand children) have CF? Depends on pedigree 2) If a person is a carrier will they show symptoms of the disease? No. 3) If you were to do this activity again, would you get the same pedigree? Not necessarily! Each allele that is passed on to the next generation is a random, independent event. Just because a punnet square gives you the possibilities of offspring doesn t necessarily mean those will be the ratios of the genotypes of the actual offspring.

10 Pedigree Activity 2 Hand out the student pedigree sheet. Ask students to complete pedigree, both shading in boxes where appropriate and adding the genotypes. Known: The grandparents are alive and healthy. Person 3 has CF Person 5 has CF Person 6 and Person 8 are not carriers Let the students see if they can fill in the genotypes Here is the logic: If Person 5 has CF, then the circle should be shaded in and her genotype is ff. Therefore, Person 1 is a Carrier, genotype of Ff and should be half shaded. If Person 3 has CF, but the grandparents were alive and healthy, then they must be carriers. Both are genotype Ff and should be half shaded.

11 If Persons 6 and 8 are not carriers, then they are FF. Therefore, Person 2 is either FF or Ff. Person 2 could be either genotype and could have offspring that are all FF. Student Questions What genotypes must the grand parents have? Ff and Ff How many grandchildren (third generation) have CF? 1. How many carriers in this family? A,B,1, and carrier are all definitely carriers. Person 2 may also be a carrier. What genotype does Person 2 have? Do you know for certain? Person 2 may be Ff or FF. Just because she didn t pass on a recessive allele to her offspring does not mean that she doesn t have one. She has a 50% chance of passing on the recessive allele to each child, and each inheritance event is independent of the others. Answers:

12 Sequence DNA and look for mutations in the cftr gene How will we determine if Person 2 has a mutation one of their alleles for CFTR? We could sequence the DNA of the cftr gene and look for mutations that are known to cause cystic fibrosis There are about 900 mutations in the cftr gene that lead to cystic fibrosis, so we have lots of mutations to look for! We have sent samples from Person2 to the lab and have had their DNA sequenced. Remember each person has 2 copies of every gene, or 2 alleles. You can see the DNA sequences on the handout. HANDOUT 2. How will we determine if Person 2 has a mutation in CFTR? In order to determine if mutations in the cftr gene for Person2 results in mutations in the CFTR protein, we need to remember how proteins are made. DNA is transcribed to RNA which is translated to protein. If there is a mutation in the DNA, there could possibly be a mutation in the protein. On your handout, you can see the partial DNA sequences for the two copies of the CFTR gene from Person 2. We have to do two things to check if there are mutations in the patient s CFTR proteins. First, we have to transcribe the DNA to RNA. Second, we have to translate the RNA sequences to protein sequences. Using the instructions on your worksheet, determine the protein sequences for each patient s CFTR protein. PAUSE FOR STUDENTS TO COMPLETE THE WORKSHEET Which patient has the defective CFTR protein? Here are the results from the worksheet. Yours should look like this. You now have the partial CFTR protein sequence for each allele of Person 2. Comparing the protein sequence to the normal CFTR protein sequence, does Person 2 carry a mutated protein sequence? Based on DNA sequencing, we have found that allele 2 has two mutations in the cftr gene. One mutation does not cause the amino acid to change. This is called a silent mutation. The other mutation caused an amino acid to change and this is called a point mutation. This leads to a defective CFTR protein. Patient 2 therefore is a carrier of CF. How do you treat CFTR? We just found out that patient 2 is a carrier of a defective CFTR protein. If both parents are carriers, there is the potential that each could pass on a recessive mutated allele to their children. How do you treat cystic fibrosis?

13 Current cystic fibrosis treatments There are currently three therapies that are used together to treat cystic fibrosis 1. Bronchodilators à these are oral, inhaled, or injectable drugs that open up (dilate) the airways of the lungs 2. Airway clearance techniques à once the airways are opened up, you need to clear the mucus out since your lungs are not doing this on their own. By simple coughing, you can clear some mucus. There are also assist devices, such as those shown here. These devices contain small metal balls inside of them. When you blow into them, the metal ball vibrates, which causes vibrations through your mouth, trachea, and into your lungs. These vibrations help to dislodge mucus, which you can then cough out. 3. Antibiotic treatment à since cystic fibrosis patients have lots of mucus in their lungs, this is a breeding ground for bacterial infections. By using oral, inhaled, or injected antibiotics, the harmful bacteria in the lungs can be killed to prevent infections But what if These current methods treat the symptoms of cystic fibrosis, but what if we could treat the cause of the disease (the mutated gene)? We could do this by adding a normal cftr gene to make the normal CFTR protein and CURE cystic fibrosis. How could we do this? By using gene therapy!!! What does gene therapy mean? What does gene therapy mean? [collect ideas and suggestions] The American Society for Gene and Cell Therapy say that gene therapy is the introduction or alteration of genetic material within a cell or organism with the intention of curing or treating a disease. This means you re adding or changing DNA to try to fix a disease. What does gene therapy mean? (diagram) On the level of a cell, this means sending a piece of DNA into a cell using a vehicle (vector) Releasing the DNA from the vehicle, then getting that DNA to the nucleus In the nucleus, the DNA can be transcribed to RNA Then the RNA can be translated to make the protein you want and treat the disease! How can we deliver genes? How do we get these genes into cells and into a person?

14 There are a number of different ways that are used. Naked DNA could be delivered with a gene gun (high pressure delivery The DNA could be put into a virus [center]. A viruses natural job is to deliver genetic material into cells! And maybe you would inject specifically into the tissue you want (such as the brain) Or A man- made material (polymer) could be used to deliver the DNA perhaps to the whole body (systemically) Another option is to remove some of the cells you want, introduce the DNA, and them put them back into the body [second slide] Or any of these methods could be recombined with different delivery routes. Gene therapy can be used for many types of disease Many of these delivery methods have been used in clinical trials to treat people. Clinical trials are where small numbers of people volunteer to test a new treatment. So, what kind of disease can you treat? Gene therapy approaches aren t just for genetic disease (monogenic diseases in the graph). Can also be used to treat more complex disease such as cancer or heart disease. In this case, instead of inserting a normal copy of a mutated gene, you might give a gene to make a protein that is helpful for treating the disease. For instance, for heart failure, you might use a gene that makes the heart pump more strongly. Many gene therapy clinical trials are in the USA! Where do these clinical trials happen? All around the world, but over half have been in the United States! Example of successful gene therapy Gene therapy clinical trials have recently shown increasing success. One example is for Leber s congenital amaurosis or LCA. This genetic disease is an inherited form of blindness that leads to poor vision at birth that continues to worsen as time goes on. For people with LCA, seeing in low light is especially difficult. The gene therapy approach to treat this disease is to inject the eye with a virus carrying a good copy of the mutated gene. After this treatment people can see better! Maze performance of a LCA gene therapy patient These are videos from the LCA clinical trial. [videos are at 3X speed] The LCA patient in the videos will try to complete the maze by following the arrows and avoiding the obstacles.

15 Before treatment [left], the patient goes immediately off track and has to be redirected; then the patient hits many obstacles before completing the maze. After treatment [right], the patient moves confidently through the maze even in much lower lighting and avoids all of the obstacles. Gene therapy works!! What ethical questions does gene therapy raise? At the UNC Gene Therapy Center, many researchers are working to develop new gene therapy techniques and approaches. A gene therapy product meant to treat certain forms of cancer was approved for use in China in A gene therapy for a rare genetic disorder was approved by the European Union in November of This is the first gene therapy approved in the West. The company is European and so applied in the EU first but will apply for approval in the USA, Canada, and elsewhere in the near future. [Glybera by uniqure] Given that gene therapy is now becoming commercially available, what sort of possible ethical dilemmas are raised by gene therapy? [collect questions from the class, there are many possibilities] Although there are many possible questions, some that are important to consider are: [pros and cons given to help structure discussion if necessary] o If you re using a virus to deliver a gene, are there parts of the virus that might make people sick? Cons: The immune response might respond to a virus or virus could be caused to replicate under very rare conditions; integrating viruses (retroviruses) can new mutations and even cancer Pros: Viruses are very efficient gene delivery vectors and genes important for virus replicating or for causing disease are generally removed; if you consider DNA vaccines to be gene therapy, then immune responses might be beneficial Past issues include a cystic fibrosis patient dying from an immune response in the early 90 s and leukemia caused by a retrovirus approach for severe combined immunodeficiency (Possible question: if the patients that immunodeficiency would have died years before they got leukemia, is this an acceptable risk? The viral vectors used are now much better understood and most have shown no issues many clinical trials. Viral vectors are widely accepted in the gene therapy field.

16 o If you re treating a genetic disease, do you want people to be able to pass the treatment to their children or is this too risky? Cons: Possibility of bad possibilities being passed on, a non- replicating (non- integrating vector) would be diluted during cell division and lost before development is done, integrating vectors could cause mutagenesis to the whole body of a child, regulators (FDA) would definitely not allow this Pros: a person and all of their descendants would be cured of the disease Interesting issue to consider: many of the most common genetic disorders have or historically had benefits in the heterozygote called heterozygote advantage (i.e. carrying the sickle cell allele can be protective against malaria, carrying the CF help protect against cholera or tuberculosis (researchers are not sure)) o What is the cutoff for a disease that is serious enough to treat with gene therapy? Gene therapy is causing a permanent change to the way that peoples body works that generally cannot be reversed if something was to go wrong. This leads to the question of how serious a problem has to be to treat people with gene therapy. Currently most, but not all, diseases being investigated are lethal. Many others lead to serious debilitation and to loss function of an organ if not treated. For regulatory purposes, diseases treated in adults are easier to approach. o How much will gene therapy cost? Will it be available to everyone who needs it? The common worry would be that gene therapy would be expensive and so not be available to everyone. However, for disease such as hemophilia (inability to stop bleeding) treating by traditional methods can be very expensive (hundreds of thousand dollars a year) and technically difficult (injections requiring refrigeration). Therefore, gene therapy can be a cheaper alternative to traditional treatment and can be more accessible in third world countries. Indeed, the fact that only one treatment per person might be required and that the numbers of patients might be relatively small can make developmental causes hard to support for companies. This is where universities, federal (NIH) funding, and foundation funding is really useful! Gene Therapy for Cystic Fibrosis

17 So returning to cystic fibrosis you could imagine a gene therapy strategy where you use a virus to deliver a good copy of the cftr gene to airway cells. This virus would bring the gene into the cells were it could be used to make normal CFTR protein and cure cystic fibrosis. Many gene therapy approaches for cystic fibrosis have not been successful in their early clinical trials. But a promising trial was started in 2012 that will test a cystic fibrosis treatment in about 130 patients. This approach uses a man- made compound to deliver a version of the cftr gene that has been optimized for maximum efficiency (expression). They will deliver the therapy through the airway repeatedly to try to improve efficacy. [The natural defense on the lungs that stop you from getting sick all the time make this tissue harder to introduce genes to, so gene therapy for CF is not as advanced as some other genes; there are 20 gene therapy approaches being tested in the US in their final stage before approval (stage III)] What did we learn today? Our physical characteristics, from our appearance to disease susceptibility, can be inherited genetically from our parents. Cystic Fibrosis is a disease caused by a mutation in a single gene. In the future, gene therapy may be used to treat many diseases, including cystic fibrosis. Vocabulary List Silent mutation a change in DNA that does not result in a change in amino acid Point mutation a change in DNA that results in a change in amino acid Bronchodilator an oral, injectable, or inhalable drug used to open up (dilate) airways Gene therapy method to treat the cause of genetic diseases by adding normal DNA to cells (usually via a viral vector) to allow expression of normal, functional protein