FAQ: Proteins, DNA, and mutations

Click on the questions below to see the answers.

What are proteins?

Proteins are large molecules that make up cells in the human body. There are two basic types of proteins. Some form the structure of cells – you can think of these proteins as building blocks that are put together to make cells. Others carry out certain functions within the cell – you can think of these proteins as small machines inside your body. The dysferlin protein is one of these machines, and scientists think that its job is to help fix any holes in the membrane (the outer wall) of the cell.

 

All proteins, both building blocks and machines, are actually long chains that are folded up into three-dimensional shapes. Each protein chain is made up of connected links called amino acids. There are 20 different kinds of amino acids, each with a slightly different shape. The different kinds of amino acids are strung together in a specific sequence to form a protein chain. The exact sequence of these amino acids in the protein is very important for the protein to fold up correctly and to carry out its proper function in the cell.

What is DNA and what is a gene?

DNA (deoxyribonucleic acid) is the information storage system of the body. DNA is a code that contains instructions telling the cell how to make all of its proteins. There is a separate DNA code (a gene) corresponding to each protein that is made by the cell. For example, instructions for how to make the dysferlin protein are written down in DNA code in the dysferlin gene.

 

Like proteins, DNA molecules are also long chains. But DNA chains don’t act as machines, they just store instructions for making the protein chains. Each protein is a chain of amino acids, and each DNA molecule is a chain of connected links called nucleotides. There are four different nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C). Each set of three nucleotides is code for a specific amino acid. For example, if the DNA has the nucleotides adenine, then thymine, then guanine in a row (ATG), that is code for an amino acid called Methionine.

What is the relationship between a gene and a protein?

The terminology can be somewhat confusing. Dysferlin is a protein, and "the dysferlin gene" means "the gene which contains the instructions for producing the dysferlin protein." Each gene tells the cell how to put together the building blocks for one specific protein. However, the gene (DNA) sits inside a different compartment of the cell (the nucleus) from the location of the cellular machines that make proteins (ribosomes). Therefore, the gene must first make a copy of itself (called messenger RNA - mRNA), which is smaller and more portable than DNA and is able to leave the nucleus to reach the ribosomes. A ribosome then reads each set of three nucleotides in the mRNA code and converts the instructions into a chain of amino acids that attach together to form a protein. The mRNA also tells the ribosome where to start the protein and when the protein is finished; namely, when it should stop attaching new amino acids to the protein. Because the nucleotides are read in groups of three, it is important for the ribosome to know how to group the nucleotides. If the nucleotides are grouped incorrectly, the ribosome will choose the wrong amino acids and the protein will not function. Usually, when a protein is not properly produced, it is because there is some mutation in the gene which contains its instructions.

What is a mutation and what types of mutations might a patient have?

The DNA that makes up the gene that encodes a protein sometimes has mistakes, called mutations, which cause defects in proteins. For example, if there are mutations in the dysferlin gene, then the dysferlin protein will not be made correctly. Each individual patient has one or two specific mutations, which can be found by gene mutation analysis (gene sequencing). Defects in a protein can be mild or severe depending on the type of mutation:

 

  • A MISSENSE mutation causes just one of the amino acids in the protein to be wrong.
    This mutation is a mistake in one DNA nucleotide in a gene. That one nucleotide is part of a set of three nucleotides that code for a specific amino acid. When a ribosome reads this particular set of three nucleotides, one of them is wrong and the ribosome selects the wrong amino acid for the protein. Missense mutations can range from very mild to severe, depending on where in the protein the affected amino acid is located and how important the affected amino acid is to the protein’s function.

 

  • A SPLICE-SITE mutation causes a sizeable section of the protein to be missing.
    This mutation is a mistake in the DNA code that leads to a whole section of the protein being left out. In the case of dysferlin, this type of mutation usually means that 1-4% of the protein’s amino acids are missing. Splice-site mutations can range from moderate to severe, depending on which section of the protein is missing.

 

  • A FRAMESHIFT mutation causes all of the amino acids in the protein, after a certain point, to be wrong.
    This mutation is an insertion or deletion of one or more nucleotides in the DNA. Because a ribosome makes the protein by reading sets of three nucleotides, inserting or deleting a nucleotide means that the ribosome can no longer correctly group the sets of three. Every set of three after the insertion/deletion is incorrectly grouped, so the ribosomes pick the wrong amino acid for every set of three after this point in the protein. By analogy, imagine that each word in a sentence stole the first letter of the next word (THE RED CAR would become HER EDC AR) - the words would no longer be read correctly. This is a severe defect because much of the protein is wrong and the protein cannot function correctly.

 

  • A NONSENSE or STOP mutation causes the protein chain to stop prematurely.
    This mutation is a mistake in the DNA code that tells the ribosomes to stop attaching new amino acids to the protein while the protein is still incomplete. The ribosomes stop too early and never even make part of the protein. This is a severe defect because much of the protein is missing, so the protein cannot function correctly. It may be possible to correct this type of mutation, however, by using a molecular signal to tell the ribosomes not to stop making the protein. Therapies based on this strategy are currently in development.
What are the benefits of identifying the gene mutations causing my disease (i.e. dysferlin gene mutational analysis)?

Getting a dysferlin gene mutational analysis only requires a small blood sample from a patient. Genetic analysis is only recommended after an initial diagnosis of dysferlin deficiency has already been made by either looking for the amount of dysferlin protein in a muscle biopsy or a blood monocyte dysferlin assay.

 

There are two main reasons for getting dysferlin gene mutation analysis (gene sequencing) of your two copies of the dysferlin gene:

 

1.  

The gold-standard for diagnosis of genetic disorders is the identification of the defect (mutation) in the gene causing the disease. There are several forms of muscular dystrophy and LGMDs with very similar clinical symptoms, and analysis at the genetic level is the only way to definitively confirm your diagnosis of LGMD2B. A deficiency of dysferlin protein seen in a biopsy or a blood monocyte dysferlin assay points towards dysferlinopathy, but only the identification of specific mutations in the dysferlin gene will confirm that diagnosis.

 

 
2.   If you know the nature and position of your dysferlin mutations you may be able to benefit from future therapies that target specific mutations. Examples of two such therapies currently in development are given below:

 

  • Stop-codon readthrough: This therapy is currently in clinical trials for Duchenne Muscular dystrophy and several other non-muscle diseases and will potentially benefit those patients who have a specific kind of mutation, called a “nonsense” or “stop” mutation (see explanation for a stop mutation).

 

  • Exon skipping: This therapy is in clinical trials for another type of muscular dystrophy and could potentially benefit a subset of patients with mutations in specific areas of the dysferlin gene.
What is the difference between a genotype and a phenotype?

A genotype refers to the genetic characteristics of an organism. A phenotype refers to the physical characteristics. For example, having blue eyes (an autosomal recessive trait) is a phenotype; lacking the gene for brown eyes is a genotype. Dysferlin deficiency will only cause muscle weakness (phenotype) if a person has two defective copies of the dysferlin gene (genotype). The genotype of two defective dysferlin genes is associated with two different phenotypes, the symptoms of LGMD2B or of Miyoshi Myopathy.