Presentation Title

Using iPSC Derived Cardiomyocytes to Evaluate the Pathogenicity of a LAMP2B Variant in Causing Danon Disease

Faculty Mentor

Jessica Wang

Start Date

23-11-2019 10:00 AM

End Date

23-11-2019 10:45 AM

Location

65

Session

poster 3

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Danon disease is an X-linked dominant disorder that is characterized by cardiomyopathy, myopathy and cognitive impairment. Danon disease is caused by a deficiency in lysosome-associated membrane protein-2B (LAMP2B) and can result in either the dilated or hypertrophic cardiomyopathy phenotypes. Recently, two patients with hypertrophic cardiomyopathy were found with a LAMP2B V397I gene mutation. A cardiac tissue sample obtained from one of the patients who underwent myectomy demonstrated autophagic dysfunction, suggestive of Danon disease. To test whether the LAMP2B V3971 mutation causes autophagic dysfunction in cardiac tissue leading to the development of Danon disease, CRISPR/cas9 and induced pluripotent stem cells (iPSC) were utilized. CRISPR/cas9 genome editing began in HEK cells, a very stable cell line. CRISPR RNA guides were designed and inserted into PX459 plasmid containing the cas9 protein. Transformation of the HEK cells allowed for the discovery of working RNA guides which will be implemented in iPSCs to introduce the V3971 mutation. After cell lines with the target mutation are established, iPSCs will be differentiated into cardiomyocytes. These iPSC-derived cardiomyocytes will be examined for dysfunction in autophagic flux using established markers which will help determine if the LAMP2B V397I variant is causal to the disease.

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Nov 23rd, 10:00 AM Nov 23rd, 10:45 AM

Using iPSC Derived Cardiomyocytes to Evaluate the Pathogenicity of a LAMP2B Variant in Causing Danon Disease

65

Danon disease is an X-linked dominant disorder that is characterized by cardiomyopathy, myopathy and cognitive impairment. Danon disease is caused by a deficiency in lysosome-associated membrane protein-2B (LAMP2B) and can result in either the dilated or hypertrophic cardiomyopathy phenotypes. Recently, two patients with hypertrophic cardiomyopathy were found with a LAMP2B V397I gene mutation. A cardiac tissue sample obtained from one of the patients who underwent myectomy demonstrated autophagic dysfunction, suggestive of Danon disease. To test whether the LAMP2B V3971 mutation causes autophagic dysfunction in cardiac tissue leading to the development of Danon disease, CRISPR/cas9 and induced pluripotent stem cells (iPSC) were utilized. CRISPR/cas9 genome editing began in HEK cells, a very stable cell line. CRISPR RNA guides were designed and inserted into PX459 plasmid containing the cas9 protein. Transformation of the HEK cells allowed for the discovery of working RNA guides which will be implemented in iPSCs to introduce the V3971 mutation. After cell lines with the target mutation are established, iPSCs will be differentiated into cardiomyocytes. These iPSC-derived cardiomyocytes will be examined for dysfunction in autophagic flux using established markers which will help determine if the LAMP2B V397I variant is causal to the disease.