Exploring the role of LZTR1 in an autosomal recessive form of Noonan Syndrome by genome-edited induced pluripotent stem cells

Abstract

Noonan syndrome is a multisystemic developmental disorder and is characterized by variable symptoms such as facial dysmorphisms, short stature, webbing of the neck and mild intellectual disability. The most severe clinical feature of this disease is an early onset and often life-threatening hypertrophic cardiomyopathy. Noonan syndrome belongs to the class of RASopathies, which share increased activity of the RAS-MAPK pathway as a common feature. In a previous study, we presented a family with two children that were diagnosed with a rare recessive form of Noonan syndrome at 6 months and 3 years of age. Whole-exome sequencing revealed a biallelic loss of LZTR1, a gene whose functions were first described in 2018 as a mediator for RAS ubiquitination and degradation. Within recent years, LZTR1 emerged as an etiologic factor in Noonan syndrome and dysfunctions are associated with a hyperactivation of the RAS-MAPK signaling pathway. Interestingly, this disorder caused by LZTR1 dysfunction can be inherited in an autosomal dominant or an autosomal recessive manner, depending on the position of the mutation. However, most studies that investigated functions of LZTR1 and its connection to Noonan syndrome were conducted in HEK293T cells or murine systems that have limited capacities to model human cardiac diseases. Thus, many open questions mainly regarding cardiac related fields remained unsolved. To further elucidate the effects of LZTR1 loss in a cardiac related system, we generated LZTR1-deficient iPSC-CMs via CRISPR/Cas9, mimicking both the maternal and paternal mutations of the familial case of Noonan syndrome. We were able to demonstrate that biallelic but not monoallelic LZTR1 loss increased RAS protein abundance in iPSC-CMs. Additionally, we showed that overexpression of full-length but not truncated LZTR1 in HEK293T cells reduced RAS protein levels with pronounced affinity for the MRAS isoform. Overexpression of full-length LZTR1 in iPSC-CMs showed a clearly spotted, vesicular like structure and high degree of co-localization with different RAS isoforms and CUL3, a key player in the ubiquitin-proteasome machinery. Strikingly, the subcellular localization of a truncated LZTR1 variant, resembling the paternal mutation from the familial Noonan syndrome case, showed homogenous cytosolic distribution. This led to a new proposed model for LZTR1 function, explaining the variable symptoms of the disease with different available level of functional LZTR1 proteins. Although the hypertrophic cardiac phenotype of Noonan syndrome patients was not reproducible in LZTR1-deficient iPSC-CMs, we observed an abnormal calcium handling, allowing first insights into the impact of LZTR1 loss in a physiological, cardiac related cell system. In conclusion, the findings from this thesis further fortify the role of LZTR1 as a negative regulator of the RAS-MAPK signaling pathway by RAS degradation.