LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome

Abstract

Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. However, the underling disease mechanisms of LZTR1 missense variants driving the cardiac pathology are poorly understood. Hence, therapeutic options for Noonan syndrome patients are limited. In this study, we investigated the mechanistic consequences of a novel homozygous causative variant LZTR1L580P by using patient-specific and CRISPR/Cas9-corrected iPSC-cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction of protein complexes uncovered a unique LZTR1L580P-specific disease mechanism provoking the cardiac hypertrophy. The homozygous variant was predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymer chains. The altered polymerization resulted in dysfunction of the LZTR1-cullin 3 ubiquitin ligase complexes and subsequently, in accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Importantly, uni- or biallelic genetic correction of the LZTR1L580P missense variant rescued the molecular and cellular disease-associated phenotype, providing proof-of-concept for CRISPR-based gene therapies.

Preclinical evaluation of CRISPR-based therapies for Noonan syndrome caused by deep-intronic LZTR1 variants

“…Using monoclonal iPSC-CMs harboring the top indel variants, correction of pathological splicing, restoration of LZTR1 function, and normalization of RAS protein accumulation to WT levels were observed for the del9 and insA indel variants. Importantly, and in line with previous reports by our group and others, 7 , 44 , 46 one functional LZTR1 allele was sufficient to regulate the pool of RAS GTPases. In contrast, analysis of the indel variant insT (the top variant induced by guide RNA A in iPSC-CMs) failed to disrupt the cryptic donor splice site and to rescue LZTR1 function, highlighting the significance of cell type-specific screens for CRISPR-based gene therapies.…”

“… 40 c.1739T>C p.L580P recessive p.L580P not detected not detected not detected – Busley et al. 44 c.2062C>G p.R688G recessive c.1943-256C>T not detected not detected not detected – Johnston et al. 6 c.2069 + 2T>C recessive p.P701H not detected not detected not detected – Umeki et al.…”

“…Recent findings by our group and others demonstrated that LZTR1 deficiency resulted in strong accumulation of the RAS GTPase protein pool in cardiomyocytes. 7 , 44 , 46 Accordingly, patient-specific, CRISPR-edited, as well as WT iPSC lines were differentiated into functional ventricular-like iPSC-CMs in feeder-free culture conditions, 47 and were subjected to molecular phenotyping on day 30 of differentiation ( Figure 2 A). Immunocytochemical staining of myocardium-specific proteins revealed a well-organized sarcomeric structure in the patient-specific iPSC-CMs as well as the edited iPSC-CMs with a pronounced striated expression of α-actinin and ventricular-specific MLC2V ( Figure 2 B).…”