From Genes to Genetic Diseases: What Kinds of Mutations Matter?

Determining How a Disease Gene Is Mutated

This science project focuses on the genetic disease cystic fibrosis, but you can do it with any genetic disease you would like to choose. See the Variations section and Table 4 for information on doing this science project with genetic diseases other than cystic fibrosis.

The first step of this project is to find more information about cystic fibrosis. Go to the Medline Plus Genetics site, which is an online tool and resource for the general public to identify and learn about genetic mutations responsible for genetic diseases.

1. On the Medline Plus Genetics website, click on "Genetic Conditions." In this section, you can find information on hundreds of genetic conditions listed alphabetically.
2. Click on the letter "C" and scroll down until you see "cystic fibrosis" and click on it. On this page, you can find the following:
   1. A general description of the disease.
   2. Information about how common the disease is.
   3. Information about the causes of the disease. This includes information about the gene(s) that are related to the disease.
   4. Information about how the disease is inherited.
   5. Links to additional information related to cystic fibrosis.
3. Read through the entry on the genetic disease, paying special attention to the genetic changes that cause the disease. What does the entry on cystic fibrosis tell you about its genetic causes? How are the chloride channels different in cystic fibrosis, and how does this affect the body as a whole?
4. Record in your notebook the following information:
   1. The symptoms and problems of the genetic disease.
   2. The name of the gene (or genes) that is mutated.

The next step is to find out more about the CFTR gene.

5. Go back to the Medline Plus Genetics main site and click on the "Genes" section. In the "Genes" section, you can look up specific genes and find a variety of information on them, including their effects on health and genetics and how they are grouped together.
6. Click on the letter "C" and scroll down until you see "CFTR" and click on it. On this page, you can find the following:
   1. The official name of the gene.
   2. The normal function of the gene.
   3. The health conditions related to genetic changes of the gene.
   4. How the gene has been changed to cause the genetic disease.
   5. The chromosome location of the gene.
   6. Links to additional information relating to the gene, as well as other genetics resources.
7. Read about the CFTR gene and how it is involved in cystic fibrosis. What chromosome is the CFTR gene located on? What amino acids in the CFTR gene are commonly mutated to cause cystic fibrosis?

From DNA to Amino Acids

Once you have gathered the information on cystic fibrosis and the CFTR gene, you can figure out if and how a simple mutation in the DNA ended up changing the amino acids that are made, as well as the function of the entire protein. You will use the National Center for Biotechnology Information (NCBI) Gene database to learn more about the gene sequence, gene alleles, and mutations of the CFTR gene. Before you continue, it might be helpful to familiarize yourself with the NCBI website by watching the NCBI tutorial below.

How to Use the NCBI’s Bioinformatics Tools and Databases

https://www.youtube.com/watch?v=QLcmEqBayr0

Table 3 has been partly filled in with information on non-pathogenic alleles of the CFTR gene. Alleles are alternative forms of a gene that occur through mutation of the DNA. Non-pathogenic alleles are alleles that have been shown to not cause cystic fibrosis. They are mutations that do not affect if a person gets the disease or not. This is the opposite of pathogenic alleles, which are alleles that are known to be harmful and actually cause the disease. In this part of the science project, you will be filling in the rest of Table 3 with additional information on these non-pathogenic alleles. You will also select ten pathogenic alleles and fill in their information in the remaining empty rows in Table 3.

1. Copy Table 3 into your lab notebook. You will pick ten pathogenic alleles for cystic fibrosis and fill in Table 3 accordingly.
2. Go to the NCBI Gene database website. At the top, enter the name of your gene of interest ("CFTR") and click "Search."
3. The resulting page, shown in Figure 2, may have a long list of related results. The top results are usually the most relevant ones. You are looking for the first entry that both starts with CFTR and includes the species name for humans (Homo sapiens). In Figure 2, this is the first result; click on it to proceed to the gene page.

Screenshot of search results on the ncbi.nlm.nih.gov website. Searching for the gene CFTR shows a list of results that provide a gene name, gene ID, description, location, aliases and a mendelian inheritance in man value (MIM).

Figure 2. When you enter the gene name CFTR, you will get many results on the NCBI Gene database. The gene name is given on the left, followed by its description (unabbreviated name) in the second column. The species name is given in brackets at the end of the description entry. Additional gene information, including the chromosome location, is given in the columns farther to the right. Pick the top gene result, (circled in red) for this project.

4. The gene page contains a large amount of information about the CFTR gene.
   1. Use the table of contents on the right side of the page to navigate to different information on the gene page. Table 1 gives an overview of the different types of information provided.
      
      

      Link Name	What Information It Provides
      Summary	Summary of the gene name and its known functions.
      Genomic context	A graphical representation of where the gene is located on the chromosome.
      Genomic regions, transcripts, and products	A graphical representation of different areas of the gene, including where known mutations are located.
      Expression	A graphical display of expression data for the gene in different tissue types.
      Bibliography	Scientific articles related to this gene.
      Phenotypes	Diseases and conditions related to mutations in this gene.
      Variation	Collection of known variants of this gene.
      Pathways from BioSystems	Metabolic pathways the gene is involved in.
      Interactions	Proteins known to interact with the protein made by this gene.
      General gene info	General information on the gene, including: Other animals this gene belongs to (under "Homology") Pathways that this gene is involved in (under "Pathways from BioSystems") The different functions the protein made from this gene has (under "Gene Ontology")
      General protein info	Names of the protein made from this gene.
      NCBI Reference sequences (RefSeq)	Links to where you can find the entire DNA sequence of this gene.
      Related sequences	Sequences closely related to this gene.
      Additional links	Links to more information on this gene and other genetic tools.

      Table 1. On the right side of the NCBI Gene page for a given gene, there is a list of links in the "Table of contents." This table shows what information these links will provide.
      
      
   2. Use the "Related information" section on the ride side of the page to navigate to additional NCBI pages with information on the gene and its role in human biology. Table 2 highlights some of the links that are particularly relevant to learning more about the gene's normal and disease functions.
      
      

      Link Name	What Information It Provides
      BioProjects	Chromosome and sequencing studies that have involved the gene.
      BioSystems	Bodily functions the gene may be involved in.
      Conserved Domains	Functional domains, which are DNA regions that form distinct protein structures that affect the overall function of the protein. Functional domains are shared, or "conserved," among different members of the same gene family.
      Full text in PMC	Scientific articles, with free access to full text, published on the gene.
      GEO Profiles	How much protein is made from this gene in different tissues and in scientific studies, referred to as the gene's expression profile.
      HomoloGene	A list of potential homologs of the gene (evolutionarily related genes in different animals)
      Nucleotide	Links to where you can find the DNA sequence of the gene.
      OMIM	Information about the gene on the OMIM database. The links here discuss the history and discovery of the gene, its function, how the disease manifests, and more.
      Protein	Links to where you can find the amino acid sequence of the protein the gene codes for.
      PubMed	Scientific articles published on the gene. Note: Some articles cannot be freely accessed.
      RefSeq Proteins	Amino acid sequence of the protein the gene codes for and additional gene information.
      RefSeq RNAs	mRNA and amino acid sequences that the gene (DNA) codes for.
      RefSeqGene	The genomic DNA sequence of the gene (includes introns and exons) and other information about the gene.
      SNP	Links to where you can find short genetic variations of the gene.
      SNP: GeneView	A list of short genetic variations of the gene and the functional amino acid changes they cause.
      Variation Viewer	A list of the short genetic variations of the gene with a lot of information about the variations, including what the DNA mutations are and which variations are pathogenic.

      Table 2. On the right side of the NCBI Gene page for a given gene, there is a list of links in the "Related information" section. This table shows what resources some of these links will provide.

Once you have gathered information on the CFTR gene, the next step is to find mutated versions of the gene that cause cystic fibrosis.

5. On the page for the CFTR gene, scroll down through the "Related information" section on the right until you see the "Variation Viewer" link. Click on this link.
6. A gene can have many different alleles, or alternative forms that occur through mutation of the DNA. Each row of data on this page, shown in Figure 3, lists a different allele for the gene you just searched for.
   1. On the left side of the page you can choose different options to filter the data. Click on "Pathogenic" (circled in blue in Figure 3) to sort the alleles according to this criterion. Here are the different clinical interpretations for alleles:
      1. "Likely pathogenic:" Alleles that are thought to be likely to cause disease, but are not proven.
      2. "Pathogenic:" Alleles that have been proven to cause disease.
      3. Alleles for which the "Clinical interpretation" column is blank. There is "no data" for these alleles. These still could be pathogenic.
      
      
      

      Screenshot of an allele chart on the ncbi.nlm.nih.gov website. The variation viewer window displays an allele chart for a given gene at the top of the page. At the bottom-left of the page filters can be applied to the chart to find specific alleles, such as ones that could be potentially pathogenic. Directly underneath the chart is a list of variants of alleles that display the variation type and location.

      
      Figure 3. Clicking on "Variation Viewer" takes you to a table listing different alleles, or alternative forms that occur through mutation of the DNA, for your gene. Each row is a different allele of the gene. You can filter these alleles by their "Most severe clinical significance" (circled in blue), sort by "Variant type" (circled in green), or find more information about them by clicking on their "Variant ID" (circled in red).
      
      
   2. After filtering by "Most severe clinical significance," you should get a list of variants labeled only "Pathogenic" as their "Most severe clinical interpretation."
   3. You also want to filter the alleles according to their variant type. Check the box that says "Single nucleotide variant" (which means this variant type has a single nucleotide change), circled green in Figure 3.
   4. Once you have applied all your filter criteria (variant type, clinical significance, etc.), click on the arrow to the left of the variant ID (circled in yellow in Figure 3 and Figure 4) to open a drop-down window that provides more information on this specific gene variant. Here you will find more allele information, such as the "Transcript change," which lists what the DNA mutation is (circled in green in Figure 4) or the "Protein change" that result from the mutation (circled in red in Figure 4).
7. From the resulting list, pick ten single nucleotide variant alleles that have "Pathogenic" as their "Most severe clinical interpretation." In your notebook, fill out Table 3 with the following information on the 10 pathogenic alleles you selected:
   1. The "Transcript change" which lists what the DNA mutation is. You get this information from the drop down table once you click on the arrow (circled yellow in Figure 4) to the left of the variant ID (circled blue in Figure 4). Put this information in the "Transcript mutation" column in Table 3.
   2. The "Variant ID". This is an identifier for this allele.
   
   
   

   Screenshot of a list of variations in the variation viewer on the ncbi.nlm.nih.gov website. Variations in the variation viewer each have a small arrow to the left of each entry on the list. Clicking on the arrow of a specific allele variation shows additional information such as the transcript changes and protein changes in the variation.

   
   Figure 4. Clicking on the small arrow (circled in yellow) to the left of the variant ID (circled in blue), pulls up more allele information, such as the "Transcript change" (circled in green) or "Protein change" (circled in red).
   
   
8. For each selected allele, click on its "Variant ID" link (circled in blue in Figure 4), to go to a new page with information on that specific allele. This information is part of the SNP Database.
   1. For each allele page, go to the "Variant Details" tab and scroll down to the "Gene" section shown in Figure 5.
   2. Look where the changed "Amino acid [Codon]" is listed (circled yellow in Figure 5). There should be an amino acid mutation that matches the "Protein change" information that was listed with this allele on the previous page, which is circled in red in Figure 4.
      1. For example, the CFTR allele listed in Figure 4 had a protein mutation of "Met1Val" This means that the first amino acid in the protein has been changed from Methionine (abbreviated Met or M) to Valine (abbreviated Val or V). This matches the amino acid change listed as "M [Met] > V [Val]" in the "Amino acid [Codon]" column.
      2. Record the corresponding "Amino acid [Codon]" change for each of the ten alleles in your notebook. Put this in the "Codon Sequence Change (DNA)" column of Table 3. This is the DNA nucleotide that has changed.
   
   
   

   Screenshot of a of a specific variant page on the ncbi.nlm.nih.gov website. The page displays information on that specific allele and includes the 'Amino acid [Codon]' change of the allele (circled in yellow) as well as the location of the mutation within the gene sequence.

   
   Figure 5. The variant ID page includes information such as the "Amino acid [Codon]" change of the allele (circled in yellow) and the location of the mutation within the gene sequence.
   
   

9. Convert the DNA sequence to mRNA, by changing every T to a U, and record this in your notebook in Table 3, under the "Codon Sequence Change (mRNA)" column.
   1. For example, GAG would still be GAG, and GTG would convert to GUG.
   2. Be sure to do this for the non-pathogenic alleles as well.
10. To determine if and how the DNA change caused a change in the amino acid the gene makes, look at Figure 6.
    1. Record the amino acid sequence change in Table 3 under the "Amino Acid Sequence Change" column.
       1. Be sure to do this for the non-pathogenic alleles as well.
       2. This entry should match the "Amino acid [Codon]" change that you observed in step 8b.
    2. What DNA mutations would not have caused a change in the amino acid that is made?
       1. Do any of the non-pathogenic alleles have such a DNA mutation?
    3. On the gene SNP page, scroll down to "Genomic regions, trancripts, and products."
       1. You will see part of the sequence of the CFTR gene. The locations of many different gene mutations are shown underneath the sequence, each of which is linked to a different gene variant. You should be able to pick out where the mutated nucleotide of your selected gene variant is as the mutated base is highlighted in blue (Figure 5).
       2. What would have happened if the mutated nucleotide had instead been one of the nucleotides immediately adjacent to the actual mutated nucleotide?

A DNA sequence is altered to change the nucleotide T to a U and a table is used to convert the altered DNA sequences into set of 3 mRNA nucleotides called codons which code for a specific amino acid.

Figure 6. A DNA sequence is converted into mRNA, and every three mRNA nucleotides (called "codons") code for a certain amino acid. This figure shows what mRNA codons code for what amino acids. A protein is made up of a sequence of several specific amino acids. Consequently, a gene's mutated DNA can ultimately change the function of the protein that results (image courtesy of Schering-Plough).

Testing How the Mutation Matters

You now know which amino acids are mutated in the genetic disease you're interested in, and how they became that way, but why is this mutation so damaging to the normal structure of the protein?

1. For each allele, look at the "Amino Acid Sequence Change" you entered in Table 3.
2. Look at Table 4 to determine how the normal amino acid and the mutant amino acid are different from each other.
3. Record in your notebook in Table 3, under the column labeled "Effect," what the differences are between the normal and mutant amino acids.
   1. Be sure to do this for the non-pathogenic alleles as well.
   2. How do you think these differences affect how the protein as a whole functions?
   3. Why do you think the non-pathogenic alleles do not disrupt the protein's function?