Abstract Details

Title High-throughput Variant Resolution Utilizing Deep Mutational Scanning in Sarcoglycanopathies

Topic Neuromuscular and Clinical Neurophysiology (EMG)

Presentation(s) P13 - Poster Session 13 (8:00 AM-9:00 AM)

Poster/Presentation
Number 11-004

Background Limb-girdle muscular dystrophies R3 and R4 are due to recessive mutations in SGCA and SGCB. Genetic variation identified within genetic test reports leads to diagnostic uncertainty. Only 14% of missense variants in SGCA and SGCB have been asserted as either benign or pathogenic; with the vast majority being "variants of unknown significance (VUS)." VUS in LGMD genes leads to an under recognition of these diseases that have treatments within the pre-clinical pipeline.

Objective To classify genetic variants in the limb girdle muscular dystrophy genes α-sarcoglycan (SGCA) and β-sarcoglycan (SGCB) as benign or pathogenic.

Design/Methods

Cells expressing α- β-, γ- and δ-sarcoglycan (ABGD cellls) were generated. Flow cytometry using an α-sarcoglycan antibody detects a membrane associated sarcoglycan complex. A lentiviral mutational library expressing all possible coding variants in SGCA was generated. A similar library expressing coding variants in SGCB and SGCG is under development. Next generation sequencing of lentiviral expressing cells will identify genetic variants within flow sorted cells.

Results

Cell sorting of ABDG cells transduced with lentivirus expressing wild-type or SGCA variants distinguished between benign and pathogenic variants. Transduction of a reported VUS, SGCA-R221P, aligned with pathogenic variants and secondary assays found that this variant failed to localize to the sarcolemma of muscle. In addition, skeletal muscle from a patient carrying this variant had an absence of α-sarcoglycan immunostaining. ABDG cells transduced with a lentiviral SGCA deep mutational library, sorted and sequenced are currently being analyzed. A catalog of SGCA variants that affect membrane localization and sarcoglycan complex formation will be forthcoming.

Conclusions Cell-based functional assays to define benign or deleterious variants in LGMD genes are time consuming. High throughput screens using mutational libraries allows for multiplexed localization of missense variants in LGMD associated proteins. These studies will facilitate the classification of VUS into the categories of benign or pathogenic ultimately improving patient care.

Authors/Disclosures
Conrad Weihl, MD, PhD (Washington University in St. Louis) PRESENTER	Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Casma therapeutics. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Abata. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Acceleron. Dr. Weihl has received personal compensation in the range of $5,000-$9,999 for serving as an Expert Witness for Wilson Elser Moskowitz Edelman & Dicker LLP.
	No disclosure on file
	No disclosure on file
	No disclosure on file
	No disclosure on file
Katy Eichinger, PhD, PT, DPT, NCS (University of Rochester)	Dr. Eichinger has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Fulcrum. Dr. Eichinger has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Avidity. Dr. Eichinger has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for DyneTherapeutic. Dr. Eichinger has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Roche. Dr. Eichinger has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for FSHD Society. The institution of Dr. Eichinger has received research support from Charcot Marie Tooth Association.
Nicholas E. Johnson, MD, FAAN (Virginia Commonwealth University)	Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Sarepta Therapuetics. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Dyne Therapeutics. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Vertex Pharma. Dr. Johnson has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for ML BIo . Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Avidity. Dr. Johnson has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Arthex. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Juvena. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Entrada. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Rgenta. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Angle Therapeutics. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving as a Consultant for AskBio. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Biogen. Dr. Johnson has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Fulcrum. Dr. Johnson has stock in Angle Therapeutics. Dr. Johnson has stock in Juvena Therapeutics. The institution of Dr. Johnson has received research support from Novartis. The institution of Dr. Johnson has received research support from Takeda . The institution of Dr. Johnson has received research support from AMO Pharma. The institution of Dr. Johnson has received research support from Sarepta. The institution of Dr. Johnson has received research support from Dyne Therapeutics. The institution of Dr. Johnson has received research support from AskBio. The institution of Dr. Johnson has received research support from Pepgen. The institution of Dr. Johnson has received research support from ML Bio. The institution of Dr. Johnson has received research support from Vertex. The institution of Dr. Johnson has received research support from Arthex. Dr. Johnson has received intellectual property interests from a discovery or technology relating to health care.
Gabriel Haller, PhD (Washington university)	Dr. Haller has nothing to disclose.