In coordination with the New York City Medical Examiner's Office, we received the sequence of a mutated SCN5A gene that was found in a five-week-old girl who died in her sleep. SCN5A codes for the voltage-gated cardiac sodium channel alpha subunit (Nav1.5) and is responsible for the fast depolarization in phase zero of the cardiac action potential. The mutations that were present in the girl's SCN5A gene were a missense mutation, Q1832E, and a truncation mutation, R1944X. In order to gain an understanding of the conditions that led to the patient's death, we carried out a functional analysis on the mutant channels and measured how their properties differed from wild type Nav1.5 properties.
For our functional analysis we carried out mutagenesis reactions to produce three experimental constructs in order to examine independent effects of Q1832E or R1944X, and to examine their interaction (mutant Nav1.5 that contains both Q1832E or R1944X; as was found in the genetic screen). These constructs were transfected into HEK 293 cells and studied using the patch clamp analysis using the whole cell configuration. Experiments were carried out to test the Nav1.5 current voltage relationships, the recovery from inactivation properties, and steady state inactivation properties.
The data demonstrated that each of the three constructs resulted in a significantly reduced current density when compared to wild type Nav1.5 currents. The gating properties of the mutant channels were similar to those of wild type Nav1.5, though Nav1.5-R1944X did show a statistically significant slower recovery from inactivation than the wild type channel. Though more experimentation is needed to determine the mechanism behind the reduced current in the mutant channels, our data shows that each of the mutations is sufficient to produce a severely dysfunctional channel and this is likely the cause of the patient's death.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/15047 |
| Date | 22 January 2016 |
| Creators | Morganstein, Jace Grant |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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