Genetic confirmation of a patient’s diagnosis is important, as it is needed for proper disease management, family planning, and participation in most clinical trials. Additionally, some treatments being developed for genetic diseases are specific to particular types of mutations (e.g. Ataluren works specifically on nonsense mutations), so it’s important to know the exact mutations each patient has in order to determine which patients are candidates for which treatments.
Diagnosis of genetic diseases requires confirming that a person suspected of having the disease carries causative mutations. Standard genetic sequencing looks for variants in exons (regions of DNA that determine the amino acid sequence of a particular protein), and the exons are separated by non-coding regions called introns which are cut or spliced out when the DNA is made into RNA. The final protein is made from the RNA.
Standard genetic sequencing, however, overlooks some types of mutations. One hard-to-detect type of mutation are variants in introns, especially those that are found deep within the intron. In most cases, mutations located in introns prevent the RNA from being spliced correctly and lead to the correct protein not being made. Because introns are often very large and have many harmless variations, it’s difficult to find deep intronic variations which cause disease by directly sequencing DNA. Sequencing a person’s RNA to see if the splicing is correct can help find these deep intronic mutations.
The Jain Foundation
has a diagnostic program to identify and genetically confirm patients who have dysferlinopathy (muscular dystrophy caused by mutations in the dysferlin gene, identified clinically as Limb Girdle Muscular Dystrophy 2B or Miyoshi Myopathy). Dysferlinopathy has a recessive inheritance, meaning that patients having the disease must have two mutations, one in each copy of the dysferlin gene. Among patients suspected of having dysferlinopathy based on clinical symptoms and protein deficiency, a significant number (10-20%) only have one mutation identified using standard genetic sequencing. Since these patients are not making dysferlin protein (which can be evaluated on a blood test), the most likely explanation is that they have a second mutation undetectable by conventional sequencing techniques, such as a deep intronic mutation.
A few deep intronic mutations in dysferlin that affect splicing have already been identified. These include one in intron 44 (Dominov et al. 2014, Annals of Clinical and Translational Neurology) and one in intron 50 (personal communication – Dr. Janice Dominov, University of Massachusetts). After the discovery of these two deep intronic variants, the Jain Foundation went back through the patients registered in the Dysferlin Registry and evaluated the DNA of those individuals who didn’t have 2 identified mutations in the dysferlin gene. On retesting, 6 out of 47 individuals tested had one of the two deep intronic variants and received a complete diagnosis. These results suggested that there must be additional deep intronic dysferlin mutations still to be identified.
The Jain Foundation, in collaboration with Dr. Madhuri Hegde at Emory University, has begun a program to sequence the RNA from patients who have dysferlinopathy based on clinical symptoms and reduced or absent dysferlin protein, but who do not have two identified mutations in the dysferlin gene. To do this, RNA from cells that make dysferlin are needed. Fortunately, dysferlin is made in white blood cells, so the RNA analysis can be done using blood rather than requiring a muscle biopsy.
With the new RNA sequencing program, the Jain Foundation is trying to identify novel deep intronic variants not identified using standard genetic sequencing techniques. This work has an impact on the patients in the Dysferlin Registry as well as the strength of dysferlin diagnostic testing around the world. Our goal is that as such deep intronic mutations are identified, sequencing of the relevant intronic regions of dysferlin can be added to standard next-generation sequencing panels in order to provide a more complete definitive diagnosis for all patients suspected of having a dysferlinopathy.
If you are interested in finding out more about the Jain Foundation’s diagnostic program, RNA sequencing program or have a patient who could benefit from these types of analysis please contact the organization at firstname.lastname@example.org