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OBJECTIVES/GOALS: In a familial case where 10 of 17 members inherited EA/LVNC in an autosomal dominant pattern, we discovered a novel, damaging missense variant in the gene KLHL26 that segregates with disease and comprises an altered electrostatic surface profile, likely decoupling the CUL3-interactome. We hypothesize that this KLHL26 variant is etiologic of EA/LVNC. METHODS/STUDY POPULATION: We differentiated a family trio (a heart-healthy daughter and EA/LVNC-affected mother and daughter) of induced pluripotent stem cells into cardiomyocytes (iPSC-CMs) in a blinded manner on three iPSC clones per subject. Using flow cytometry, immunofluorescence, and biomechanical, electrophysiological, and automated contraction methods, we investigated iPSC-CM differentiation efficiency between D10-20, contractility analysis and cell cycle regulation at D20, and sarcomere organization at D60. We further conducted differential analyses following label-free protein and RNA-Seq quantification at D20. Via CRISPR-Cas9 gene editing, we plan to characterize KLHL26 variant-specific iPSC-CM alterations and connect findings to discoveries from patient-specific studies. RESULTS/ANTICIPATED RESULTS: All iPSC lines differentiated into CMs with an increased percentage of cTnT+ cells in the affected daughter line. In comparison to the unaffected, affected iPSC-CMs had fewer contractions per minute and altered calcium transients, mainly a higher amount of total calcium release, faster rate of rise and faster rate of fall. The affected daughter line further had shorter shortening and relaxation times, higher proliferation, lower apoptosis, and a smaller cell surface area per cardiac nucleus. The affected mother line trended in a similar direction to the affected daughter line. There were no gross differences in sarcomere organization between the lines. We also discovered differential expression of candidate proteins such as kinase VRK1 and collagen COL5A1 from proteomic profiling. DISCUSSION/SIGNIFICANCE: These discoveries suggest that EA/LVNC characteristics or pathogenesis may result from decreased contractile ability, altered calcium transients, and cell cycle dysregulation. Through the KLHL26 variant correction and introduction in the daughter lines, we will build upon this understanding to inform exploration of critical clinical targets.
ABSTRACT IMPACT: This work represents a novel way in which genetic information can be used to improve clinical decision making as it pertains to both treatment and management of congenital heart disease. OBJECTIVES/GOALS: Our lab found that MYH6 variants are both enriched in hypoplastic left heart syndrome (HLHS) and associated with decreased cardiac transplant-free survival. To elucidate the mechanisms of MYH6 variant pathogenicity, we are assessing their impact on atrial function during HLHS development and progression. METHODS/STUDY POPULATION: We are using 2D speckle-based tracking to retrospectively evaluate echocardiograms (echos) from 51 HLHS patients, 17 with MYH6 variants and 34 matched controls. Atrial function will be assessed by myocardial strain and strain rate at seven time points, beginning at the time of the patients’ earliest prenatal echo, and ending with their last available echo before death or cardiac transplant. Early left atrial function will examine the role of MYH6 variants in the development of HLHS in vivo, while longitudinal right atrial function will be assessed in order to look for differences that could be contributing to the decreased transplant-free survival seen in MYH6 variant carriers. RESULTS/ANTICIPATED RESULTS: We hypothesize that MYH6 variants cause HLHS by impairing early left atrial (LA) contractility, resulting in altered left ventricular hemodynamics and consequent hypoplasia. We therefore expect to find diminished prenatal LA function in HLHS patients with MYH6 variants. We also hypothesize that MYH6 variants continue to impair right atrial (RA) function in surgically-reconstructed HLHS hearts, necessitating earlier transplantation. Accordingly, we expect variant carriers to exhibit lower RA function at birth versus controls. We expect differences between groups to persist over time, and possibly increase in magnitude. In HLHS patients with MYH6 variants, we anticipate declining RA function will precede right ventricular function and therefore be an early indicator of transplant need. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study represents a novel way in which genetic information can inform clinical decision-making. Identifying MYH6 variants as an early cause of HLHS offers chances for intervention. Understanding long-term effects of MYH6 on right atrial function in HLHS may aid in cardiac transplant risk stratification, thus improving patient outcomes.
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