The pathogenesis of Noonan syndrome is modulated by NOC2L, a novel interactor of LZTR1 leading to impaired p53 signalling

The pathogenesis of Noonan syndrome is modulated by NOC2L, a novel interactor of LZTR1 leading to impaired p53 signalling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The pathogenesis of Noonan syndrome is modulated by NOC2L, a novel interactor of LZTR1 leading to impaired p53 signalling Sumana Chatterjee, Miho Ishida, Débora R. Bertola, Juliana Chizo Agwu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6280572/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction : Monoallelic dominant negative LZTR1 gene variants have been implicated as a cause of NS due to hyperactivation of the canonical RAS-MAPK signalling pathway. Missense LZTR1 variants have been associated with defective ubiquitination theoretically leading to increased Ras substrate availability and altered p53 signalling. We investigated the role of LZTR1 in this pathway. Methods : Single nucleotide substitutions were created by mutagenesis of an N-terminal MYC tagged- LZTR1 cDNA. WT and variant constructs were expressed in mammalian cells and lysates prepared for phosphoproteomic analysis and immunoblotting. Analysis of transcriptomic data was conducted using Ingenuity Pathway Analysis. Significant phospho-peptides, protein-protein interactions and pathways of interest were probed using western blotting, immunofluorescence, nanoluciferase assays and in silico prediction tools. Results : Two heterozygous LZTR1 variants, which segregated with short stature and features of growth hormone insensitivity (p.K156E, p.G248R), were expressed in a mammalian cell line. Both variants were thermodynamically stable and associated with elevated cytoplasmic levels of pan-Ras. Phosphoproteomic assays revealed upregulation of the histone acetyltransferase inhibitor, NOC2L (NOC2 Like Nucleolar Associated Transcriptional Repressor), in both variants. This finding, consistent upon immunoblotting and immunofluorescence, was associated with impaired acetylation of p53, with reduced levels of acetylated lysine residue 382 in both mutants. Furthermore, Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1), major effectors of the DNA damage response (DDR), were preferentially activated in LZTR1 variants. Despite an apparent activation of the DDR and diminished p53 activity, levels of LC3 and phosphorylated p70 S6 kinase were increased. In silico structure modelling suggested that LZTR1 interacts with NOC2L via the central part of the protein and this interaction was validated by nanoluciferase assays and disrupted in both LZTR1 variants. Conclusion : NOC2L and p53 form a complex which dictates p53 activation. We demonstrate a previously unknown interaction between NOC2L and LZTR1 and hypothesise that LZTR1 acts as a binding factor modulating the activity of this complex. As NOC2L negatively regulates p53, upregulation of this protein would lead to p53-mediated transcription inhibition. LZTR1 attenuation due to genetic mutations associated with NS, potentiate NOC2L activity leading to reduced apoptosis and a compensatory increase in autophagy. Given its potential role in the multisystem pathogenesis of NS, NOC2L may represent a novel therapeutic target however, additional work is needed to further characterise its organ-specific effects. Noonan syndrome RAS/MAPK LZTR1 NOC2L Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Noonan syndrome (NS) (OMIM 163950) is a dominantly inherited multi-system disorder with an estimated incidence of 1/1000–2500 live births 1 , 2 . It is defined by a characteristic phenotypic triad of short stature, distinctive facial features (short/webbed neck, low-set posteriorly rotated ears, ocular hypertelorism, ptosis, down slanting palpebral fissures) and congenital heart defects (pulmonary valve stenosis and hypertrophic cardiomyopathy in 60 and 20% cases, respectively). Other associated phenotypic features include cryptorchidism in males, skeletal abnormalities (pectus deformities and scoliosis), developmental delay and a predisposition to myeloproliferative disorders. Up to 80% of patients have postnatal proportionate short stature 3 . NS is a clinical diagnosis and establishing the diagnosis can be very difficult, especially in adulthood, given variable phenotypic expression. Variants in more than 20 genes have been associated with pathogenesis of NS ( PTPN11, SOS1, SOS2, KRAS, NRAS, RIT1, RRAS, RASA1, RASA2, MRAS, RAF1, BRAF, MAP2K1, MAP3K8, SPRY1, MYST4, A2ML1, SPRED2, CBL and LZTR1 ) and account for 75–80% cases 4,5 . All causative genes for NS, except LZTR1 , encode components or regulators of the well-studied RAS mitogen-activated protein kinase (MAPK) signalling pathway. LZTR1 , located on chromosome 22q11.21, was initially described as a putative transcriptional regulator acting as an adaptor protein for CUL3 ubiquitin ligase complexes 6 . In humans, LZTR1 mutations were first associated with DiGeorge syndrome (22q11.2 deletion syndrome) whilst somatic variants with loss of heterozygosity at 22q11 were identified in glioblastoma multiforme, a malignant central nervous system tumour 7 . Subsequently, several studies demonstrated an association between heterozygous missense LZTR1 variants and NS. To date, over 70 cases of NS with pathogenic LZTR1 variants have been described 8 – 13 . However, biallelic recessive variants have also been identified in several extensive kindreds 8 , 12 . The majority of heterozygous NS-causing variants are missense and map to the Kelch domains of the protein whilst biallelic variants may be frameshift, nonsense, missense, or splice-site and are distributed across all functional domains. Dominantly inherited LZTR1 variants do not significantly impair protein stability and subcellular localization but do enhance stimulus-dependent RAS-MAPK signalling 14 . Contrastingly, recessive variants are thermodynamically unstable and do not impact MAPK signalling 14 . The distribution pattern of dominant and recessive NS-causing LZTR1 mutations and their differential impact on MAPK signalling supports a dominant negative role for the former and a loss-of-function effect for the latter. LZTR1 has been shown to enable ubiquitination of RAS proteins via its complex with cullin3 and its loss negatively impacts RAS ubiquitination 15 . Impaired ubiquitination thereby increases the pool of RAS proteins and augments MAPK pathway activity. We investigated two naturally occurring heterozygous LZTR1 variants identified in two NS patients, both with characteristic facial features of NS, cardiac abnormalities, short stature and features of Growth Hormone Insensitivity. Interestingly, our studies have identified a previously undescribed interaction between LZTR1 and NOC2L. NOC2 like nucleolar associated transcriptional repressor (NOC2L) was recently identified as a novel inhibitor of histone acetyltransferase (INHAT), thereby inhibiting histone acetylation 16 , 17 . Further, studies have also shown that NOC2L is recruited by P53 to inhibit histone acetylation in P53-targeted genes 18 . NOC2L overexpression can therefore lead to inhibition of p53-mediated transcription, thereby impairing the process of apoptosis and cell cycle arrest. The identification of an underlying genetic mutation in NS provides a definitive diagnosis and has important clinical and therapeutic implications for patients. Results Clinical phenotypes of the two individuals harbouring LZTR1 variants Both patients (Patients 1 and patient 2) had classical facial features of NS (hypertelorism with down-slanting palpebral fissures, ptosis, low-set posteriorly rotated ears and webbed neck) (Table 1 ) and cardiac pathology; pulmonary artery stenosis (Patient 1) and pulmonary valve stenosis with an affiliated atrial septal defect (Patient 2). Table 1 Clinical details of patients with LZTR1 variants at diagnosis. Patient LZTR1 variant Age at genetic diagnosis/Sex/ Ethnicity Height (SDS) NS Features Birth-weight SDS Height velocity cm/year GH peak (mcg/L) Type of GH stimulation test and age IGF-1 ng/ml (SDS) IGFBP 3 mg/L (SDS) Segregation 1 c.466A > G; p.K156E 4.0 years, M, British 85 cm (-2.3) Classical facies*, webbed neck, Pulmonary artery stenosis, right undescended testis, left inguinal hernia, developmental delay -1.6 3.6 10.6 Arginine, 3.5 years 19.8 (-2.3) 0.8 (-3.0) Adopted 2^ c.742G > A; p.G248R 11.4 years, F, Brazilian 131.5 cm (-2.1) Classical facies*, webbed neck, pectus deformity, Pulmonary valve stenosis, Atrial Septal Defect, prominent corneal nerves -2.9 5.0 11.8 Clonidine, 5.6 years 35 (-2.2) 2.4 (-1.2) Mother and maternal grandfather ^Variant previously reported; Age and Height SDS are at presentation; M, male; F, female; *Classical facies, hypertelorism with down-slanting palpebral fissures, ptosis and low-set posteriorly rotated ears; GH, Growth Hormone; IGFBP3, IGF-binding protein 3; ND, no data; N/D, not done; N/A, not applicable. Patient 1, a 4-year-old British-Caucasian male, presented with: short stature (height SDS − 2.3), brachycephaly, bitemporal narrowing, minor degree of fifth finger clinodactyly, a single palmar crease on the left hand, tapering fingers, convex fingernails and widely-spaced nipples. Additional features included right unilateral cryptorchidism, left inguinal hernia and developmental delay. The patient was adopted hence genetic mapping was not possible. Biochemically, the patient had features of GHI insensitivity (GHI) with a peak GH of 10.56 mcg/L (GHRH-Arginine provocation testing) and IGF-1 and IGFBP3 deficiencies (-2.3 and − 3.0 SDS, respectively). Patient 2, an 11.4-year-old Brazilian girl with short stature (height − 2.1 SDS) and a history of congenital heart disease, presented in early childhood with classic features of NS and superimposed pectus excavatum, lacrimal duct obstruction and prominent corneal nerves. Growth hormone provocation (Clonidine) elicited a peak of 11.8 mcg/L in tandem with persistently low IGF-1 (-2.2 SDS) levels. Interestingly, the patient’s mother and maternal grandfather also had clinical features of NS and short stature (height − 2.5 and − 2.9 SDS respectively). The patient was given recombinant human Growth Hormone (rhGH) for several years however, the response was suboptimal with annual height velocities fluctuating between 5.0 and 7.0 cm/year and a final adult height of -2.6 SDS (age 18 years). Evaluation of pathogenicity of variants using in silico prediction tools Next generation sequencing revealed single missense LZTR1 variants harboured by each patient (Patient 1- c.466A > G ; p.K156E and Patient 2 - c.742G > A ; p.G248R). Segregation analysis revealed maternal inheritance of the p.G248R variant, which was highly penetrant and present in the proband’s affected mother and maternal grandfather. Both variants were rare; the p.K156E variant was novel and p.G248R had a MAF of 0.000003718 in the Genome Aggregation Database (gnomAD) with 6 reported heterozygotes classified as likely pathogenic based on ClinVar entries related to underlying Rasopathy (Table 2 ) 9 , 10 , 13 , 19 , 20 . The p.K156E variant was predicted benign by PolyPhen2 but deemed deleterious and disease causing by SIFT and Mutation Taster, respectively. The pathogenicity of this variant according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP)’s standards and guidelines for the interpretation of sequence variants was deemed to be likely pathogenic (PM2, PM1_Moderate, PP3 and PS3). The p.G248R variant was predicted deleterious across all 3 computational platforms and likely pathogenic according to concurrent ClinVar entries. Table 2 In silico predictions of the LZTR1 variants. LZTR1 variant GnomAD frequency SIFT PolyPhen2 CADD Mutation Taster c.466A > G ; p.K156E Not reported 0 (deleterious) 0.301 (Benign) 25.3 Disease causing, amino acid sequence changed, protein features might be affected ^ c.742G > A ; p.G248R 0.000003718 0.001 (deleterious) 1.0 (Damaging) 28.9 Disease causing, amino acid sequence changed, protein features might be affected For amino acid substitutions: Using SIFT a score of < 0.05 is predicted deleterious conversely using PolyPhen2, scores closer to 1 are more likely to be damaging with variants within the range 0.85-1.0 more confidently predicted to be damaging. Mutation Taster provided automatic assignation of variants as disease causing or as a polymorphism. ^ Variant previously reported. LZTR1 variants are thermally stable and associated with increased pan-RAS levels We assessed expression of both LZTR1 variants following transient transfection into an in vitro HEK 293T reconstitution system. Both variants were well-expressed upon immunoblotting, with levels demonstrably comparable to WT-LZTR1 (Fig. 1 A). Furthermore, both variants localised to the nucleus similar to WT-LZTR1 (Fig. 1 B) however, pan-Ras levels were markedly increased for both variants as evident on confocal microscopy (Fig. 1 C). Phosphoproteomics We conducted phosphoproteomic analysis of LZTR1 mutants to identify unique transcriptomic signatures associated with LZTR1 variation. Kinase activity estimation (KSEA) demonstrated significant enrichment of Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1), major effectors of the DNA damage response (DDR), which were both preferentially activated in LZTR1 variants (Fig. 1 D). Ingenuity pathway analysis and filtering of molecules based on p-value significance revealed a target histone acetyltransferase inhibitor, NOC2L (NOC2 Like Nucleolar Associated Transcriptional Repressor), upregulated in both variants (Fig. 1 E). Top networks and toxicity lists enriched in this dataset included cell morphology, cellular function and maintenance/cancer as well as cell death and cardiac dysfunction (Fig. 1 F). Protein interactome analysis of NOC2L and other differentially expressed targets revealed a complex network involving Chromodomain-helicase-DNA-binding protein 4 (CHD4) and Histone H2B, nucleosome and chromatin remodelling factors that suggest NOC2L may exert its effects via transcriptional regulation of several cofactors (Fig. 2 A). NOC2L localises to the nucleolus Immunoblotting of lysates expressing WT-LZTR1 and variant constructs confirmed the phosphoproteomic findings, demonstrating robust detection of NOC2L in both variants when compared to wild-type (Fig. 2 B). Immunofluorescence similarly highlighted an abundance of NOC2L for both variants, further denoting its nucleolar localisation (Fig. 2 C). NOC2L upregulation attenuates acetylation and expressivity of p53 Previous work has intimated that physiological levels of NOC2L inhibit acetylation of p53-targeted gene promoters and inadvertently p53 itself 18 (Fig. 3 A). Hence upregulation of NOC2L should theoretically demonstrate opposing functions. We probed acetylation of p53 lysine residue 382, critical for activation of p53 activity, via immunoblotting of WT-LZTR1 and variant expressing lysates. Levels of acetylated lysine 382 were reduced for both variants when compared to wild type (Fig. 3 B) and were concordant with reduced global levels of p53 as visualised by confocal microscopy (Fig. 3 C). The DNA Damage Response is activated downstream of LZTR1 variation The DNA damage response (DDR) involves several effectors such as Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1) that culminate in p53 activation or increased transcription of Rad51 to initiate homologous recombination (Fig. 3 D). Global levels of ATM and CHK1 appeared enhanced in both LZTR1 variants (Fig. 3 E) whilst two major substrates representative of their kinase activity, Rad50 and Adducin (ADD1/2), were preferentially phosphorylated in both variants, via residues Serine 635 and Serine 713/726 respectively (Fig. 3 F). Furthermore, increased levels of Rad51 were noted upon immunofluorescence of variant constructs relative to WT-LZTR1, when expressed in mammalian cells (Fig. 4 A). Attenuated p53 activity leads to compensatory activation of autophagy signalling The reduction in p53 expression was further associated with equivocal activity of apoptosis regulator, Cathepsin D in WT-LZTR1 and variant constructs as evidenced by commensurate expression of pro-and pre-procathepsin D levels (Fig. 4 B). This apparent impasse in apoptotic activation despite a relative increase in the DDR was associated with increased markers of autophagy. Immunoblotting of WT-LZTR1 and variant constructs revealed increased phosphorylation of the threonine 389 residue of p70 S6kinase in variant constructs when compared to wild type (Fig. 4 C). Phosphorylation of this residue is critical for kinase activation and reflective of increased autophagic flux (Fig. 4 C). This effect correlated to increased LC3 levels in LZTR1 variants detected upon confocal microscopy (Fig. 4 D). LZTR1 directly interacts with NOC2L In silico quaternary structure prediction using MultiFOLD2 demonstrated confidently modelled interactions between LZTR1 and NOC2L (Fig. 5 A). The models showed different relative binding orientations for the LZTR1 mutants versus wildtype, suggesting alternative binding modes with NOC2L (Fig. 5 B and C). Although the mutated residues in the models are not located within the binding interfaces, they may have downstream conformational effects. The model quality scores were marginally reduced for the p.G248R mutant, indicating a lower confidence in the interaction (Fig. 5 B and C). To validate possible predicted protein-protein interaction between both key targets, Nanoluc Binary technology (NanoBit) was utilised. Complementation assays using Reporter-tagged target proteins, demonstrated robust interaction between LZTR1 and NOC2L as evidenced by detectable luminescence (Fig. 5 D). Furthermore, this interaction with wild type NOC2L was disrupted for both LZTR1 variants with the greatest effect on p.G248R (Fig. 5 D). Discussion LZTR1, an adaptor protein for the CUL3 ubiquitin ligase complex, is implicated in NS yet its mechanism of action is not fully understood. LZTR1 variants causing autosomal dominant NS likely inhibit binding to either RAS and/or other target substrates, thereby impairing their ubiquitination and degradation. Our missense variants (p.K1563E and p.G248R), are stably expressed and consistent with previously published work, associated with enhanced RAS activity 21 . The homogeneous nature of dominant NS-causing LZTR1 variants (i.e. missense changes) and their clustering within the Kelch repeats strongly suggest a specific disruptive effect of variants on domain function and protein-protein interaction(s). Interestingly, we have identified NOC2L as a novel interactor of LZTR1 and whose levels appear to be enhanced in the setting of LZTR1 variation. Phosphoproteomic analysis of LZTR1 variants revealed specific phosphopeptide signatures indicative of upregulation of the DNA damage response (DDR). This was consistent with previous data demonstrating activation of the DDR and chromosomal instability in association with germline LZTR1 variants 22 , 23 . Key kinases involved in this pathway (ATM, CHK1) were upregulated with increased phosphorylation of respective target substrates, Rad50 and Adducin1/2 demonstrated by immunoblotting. A further marker of DNA damage and inducer of homologous recombination, Rad51 was shown to be increased in both LZTR1 variants when compared to WT. It is entirely possible that LZTR1 variation may trigger genotoxic stress and an overall propensity to chronic DNA damage and increased risk of tumorigenesis 24 . We also identified NOC2L upregulation which was concordant in both LZTR1 variants. NOC2 like nucleolar associated transcriptional repressor (NOC2L) is an endogenous inhibitor of histone acetylation and negatively regulates p53 activity 25 . p53 directly interacts with NOC2L, facilitating its recruitment to inhibit transcriptional activation of p53-target genes; in the process, p53 is itself modulated by NOC2L, which inhibits its acetylation and subsequent activity 25 . When compared to WT-LZTR1, both variants had attenuated acetylation of Lysine residue 382 and globally reduced levels of p53. The implication that apoptosis induction was impaired, despite activation of the DDR, was supported by relatively unaltered levels of apoptotic marker Cathepsin D across both LZTR1 mutants. This apparent blockade in apoptosis was compounded by an enhanced autophagic response in both LZTR1 variants, which demonstrated heightened LC3 levels and robust phosphorylation of threonine residue 389 of p70 S6Kinase, a downstream effector of mTOR whose activation correlates to an increase in autophagy. Previous work has demonstrated that transgenic expression of the human NS-associated PTPN11 variant ( c.236A > G , p.G79R) in murine endocardial cushions phenocopied the human cardiac valvulopathy resulting in increased cell proliferation and enlarged cushions due to reduced apoptosis 26 . This suggests that multi-organ pathology in NS may be related to attenuated apoptotic signalling. Our findings highlight an increase in autophagy markers either as a compensatory response to diminished p53 activity or reflective of a general disruption in cellular homeostasis 27 . Disequilibrium between both processes has been linked to a propensity to tumorigenesis, an established risk for patients with NS 28 , 29 . Under normal physiological conditions, NOC2L is involved in embryogenesis, lymphopoiesis and epidermal development likely mediated via its interaction with the closely related proteins, p53 and p63 25 . It is also implicated in carcinogenesis due to its diametrically opposing effect on p53 tumour suppressive activity 16 , 25 . Interestingly, Chinton et al . reported five individuals with NS, harbouring the LZTR1 variant ( c.742G > A ; p.G248R) seen in patient 2, one of whom developed acute lymphoblastic leukaemia (ALL) 13 . We report an upregulation of NOC2L in association with dominantly inherited missense LZTR1 variants. Furthermore, we demonstrated a direct interaction between both proteins using in silico predictive tools and in vitro nanoluciferase technology; an interaction disrupted upon LZTR1 variation. We hypothesise that wild type LZTR1 may ubiquitinate NOC2L and regulate its activity; an effect lost due to LZTR1 mutations leading to NOC2L hyperactivation. Given its involvement in cell cycle regulation, NOC2L may be a potential regulator of organogenesis downstream of or in tandem with LZTR1, although further work is needed to characterise this effect. There have been sporadic reports of affiliated growth hormone deficiency 8 , 11 , 12 , however, to date, growth hormone insensitivity associated with LZTR1 variants has not been reported and no underlying mechanism has been elucidated. Our patients both demonstrated biochemical evidence of IGF-1 deficiency associated with sufficient GH levels and short stature (height SDS <-2). However, short stature in NS often exhibits incomplete penetrance 9 , 10 , 13 , 19 , 20 . Therefore, growth dysregulation might be influenced by other factors including environmental factors, genetic modifiers or novel interacting partners such as NOC2L that modulate affiliated signalling pathways. Therefore, further in vitro and in vivo data are required to delineate these associations and identify common transcriptomic links affiliated with NOC2L that may be implicated in multi-system pathology. Human Growth hormone (hGH) is the only licenced treatment for growth failure associated with NS however, as demonstrated in Patient 2, therapy can often be suboptimal. However, agents that target the RAS/MAPK pathway such as the MEK inhibitor, Trametinib has been used with some success in treating NS patients with non-malignant pathology such as severe hypertrophic cardiomyopathy, heart failure and lymphatic anomalies 30 – 36 . Ongoing clinical trials are currently evaluating the impact of the C-type natriuretic peptide (CNP) analogue, Vosoritide on patients with wide ranging genetic causes of short stature including Rasopathies 37 . Thus, a deeper understanding of the molecular mechanisms underlying the growth failure in NS may lead to the identification of newer drug targets or justification for those currently under investigation. Given its overarching multi-system pathology, the health burden of NS is significant and associated with high morbidity 38 . Hence, uncovering the mechanistic pathways involved in disease propagation is integral to evolving patient care. We propose a new disease model for LZTR1-mediated NS in which LZTR1 is postulated to form an interacting partner of NOC2L, modulating its ubiquitination and hence stability. Missense variation in LZTR1 disrupts this interaction leading to upregulation of NOC2L activity and repression of p53 transcription. This triggers a cascade that culminates in an apoptosis blockade and compensatory autophagy induction that perpetuates a state of chronic DNA damage. Currently, no inhibitors of NOC2L exist, but this may be an apt therapeutic target that warrants exploration should future work uncover a broadly similar role for NOC2L in other genetic causes of NS. Materials and Methods Ethical approval Informed written consents for publication of clinical details, including indirect identifiers, were obtained from human research participants and their guardians. Participants consented to dissemination of anonymised clinical data in an open-access journal and were not compensated for their involvement. The study was approved by the Health Research Authority, East of England-Cambridge East Research Ethics Committee (REC reference 17/EE/0178). Genetic sequencing Whole exome sequencing (WES) was conducted using the Agilent SureSelect all exon V4 capture and paired-end (2x100) sequencing on an Illumina HiSeq 2000 at Otogenetics (Norcross, GA, USA). Common variants were filtered out by excluding those with an allele frequency of ≥0.1% in the 1000 genomes, ExAC and the NHLBI exomes. Variants predicted damaging by SIFT (http://sift.jcvi.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) or Mutation Taster (https://www.mutationtaster.org/) were explored further. Generation of LZTR1 variant constructs The wild type LZTR1 mammalian expression cDNA clone (pCDNA-MYC-Hist-LZTR1) was kindly donated by Dr. Antonio Lavarone at the Institute for Cancer Genetics, Columbia University Medical Centre, New York, USA. Our variants of interest: c.466A>G , p.K156E and c.742G>A , p.G248R were generated by Site-Directed Mutagenesis (QuikChange II XL SDM Kit, Agilent Technologies) as per manufacturer’s instructions. All constructs were verified by Sanger sequencing (primer sequences available on request). Cell lines and transfection protocols Human embryonic kidney (HEK 293T, ATCC® CRL-3216TM) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma D5648) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin at 37°C in 5% CO 2 . Transfection of cells was achieved using Lipofectamine™ 3000 according to manufacturer’s instructions. Immunoblotting Protein lysates were quantified using a Bradford protein assay (Bio-Rad), denatured by the addition of laemmli SDS sample buffer 6X (Thermo Scientific) and boiled for 5 minutes at 98°C. 20-30μg of protein were loaded into wells of 4 to 12% (Bis-Tris) 1.0 mm pre-cast gels (Invitrogen) prior to electrophoretic separation using MOPS SDS running buffer. Semi-dry protein transfer to nitrocellulose membrane was achieved using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad) at 15V for 1 hour. The membrane was blocked with 5% fat free milk in TBS/0.1% Tween-20 for 1 hour at room temperature. Primary antibody was added at a concentration of 1:1000 and anti-β-actin (1:10,000) used as a housekeeping control. Primary antibody incubation in blocking buffer or 5% Bovine serum albumin (BSA) in TBS/0.1% Tween-20 was performed overnight at 4°C on gentle agitation. The membrane was then washed for 10 minutes (3X) with Tris Buffered saline-Tween-20 (TBST). Secondary anti-mouse, anti-rabbit or anti-goat antibodies were added at a concentration of 1:5000 to antibody dilution buffer and at 37°C for 60 minutes. The membrane was washed three times (10 minutes each) with TBST and visualized with the LI-COR Image Studio software. Phosphoproteomics LZTR1 WT, variant (p.K156E and p.G248R) and empty vector constructs were transiently transfected into HEK 293T cells using Lipofectamine™ 3000 according to manufacturer’s instructions. After 24 hours, cells were lysed in urea buffer and homogenised by sonication. The insoluble material was removed by centrifugation and protein in the cell extracts was quantified. 250 µg of protein was reduced and digested with trypsin. Peptide solutions were desalted with Oasis cartridges and phosphopeptides were enriched using TiO2 as previously reported 39–41 . Phosphopeptide pellets were re-suspended in reconstitution buffer (20 fmol/µl enolase in 3% ACN, 0.1% TFA) and loaded onto an Orbitrap Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) as previously described 39–41 . Differences in phosphorylation patterns between WT, LZTR1 variant and empty vector constructs were reported as fold over WT and statistical significance for those changes assessed using unpaired two-tailed t-tests. Further analysis of transcriptomic data was conducted using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com). Immunofluorescence Mammalian cells seeded on glass coverslips (24 well plates) were transfected with WT and LZTR1 plasmid constructs. After 48 hours, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were washed three times in PBS and permeabilized with 0.5% Triton X-100 in PBS for 10 minutes. After further PBS washes, coverslips were incubated in Blocking buffer (1X PBS / 5% goat serum / 0.3% Triton™ X-100) at room temperature for 60 min. Primary antibody (mouse anti-myc, rabbit anti-Pan-RAS, rabbit anti-NOC2L, mouse anti-p53, mouse anti-CHK1, rabbit anti-ATM, rabbit anti-LC3B) reconstituted in dilution Buffer (1X PBS / 1% BSA / 0.3% Triton™ X-100 buffer) was added to cells and left at 4 °C overnight with gentle agitation. Cells were washed and incubated in appropriate fluorescent secondary antibody at room temperature for 60 minutes (protected from light). Coverslips were stained with DAPI and mounted on microscope slides. Protein Structure Modelling The latest version of the MultiFOLD server 42 (https://www.reading.ac.uk/bioinf/MultiFOLD/) was used to model the quaternary structures from the wildtype sequences of LZTR1 with NOC2L as well as each of the LZTR1 variants with NOC2L. Each of the modelled complexes was visualised using PyMOL and coloured by chain identifier. The NOC2L chains from all models were superposed using the align command to orientate the modelled complexes in the same frame of reference. The disordered N and C termini on NOC2L were identified and removed from the visualization for clarity. The locations of the mutated residues were identified and highlighted. Finally, the models were raytraced and exported as high-quality images. NanoBiT complementation assays Binary protein interactions were assessed with NanoBiT complementation assays (as previously described) 43 using NOC2L WT and LZTR1 WT/variant plasmids N terminally fused with NanoBiT fragments (SmBiT and LgBiT). HEK 293T cells (1×10 5 cells/well) were seeded in clear bottomed 96-well white plates, and plasmids were reverse-transfected using Lipofectamine™ 3000 according to the manufacturer’s instructions. DNA concentrations were optimised and determined to be 200 ng per well; 100 ng SmBiT-NOC2L and 100 ng LgBiT-LZTR1. After 48 hours post-transfection, cell culture medium was replaced with 100 µL NanoBiT assay buffer (pH 7.4, HBSS 1X, HEPES 24 mM, NaHCO3 3.96 mM, CaCl2 1.3 mM, MgSO4 1 mM, BSA 0.1%) per well and left for 1 h at 37 °C in 5% CO 2 . Subsequently, six (6) baseline luminescence readings were recorded using the CLARIOstar Multimode Plate Reader (BMG Labtech) followed by addition of 25 µl/well of Furimazine (Nanolight Technology) prepared in a 1:20 dilution using assay buffer. Luminescence readings were resumed and continued for 1 hour. Resource Identification Initiative Mouse Anti-Myc tag Monoclonal Antibody, Unconjugated, Clone 9E10 (Abcam Cat# ab32, RRID:AB_303599), Pan Ras Monoclonal Antibody (Ras10) (Thermo Fisher Scientific Cat# MA1-012, RRID:AB_2536664), Rabbit polyclonal NOC2L antibody (Proteintech Cat# 28509-1-AP, RRID:AB_2881160), Human p53 (acetyl K382) antibody (Abcam Cat# ab75754, RRID:AB_1310532), Rabbit monoclonal ATM antibody [Y170] (Abcam Cat# ab32420, RRID:AB_725574), Mouse monoclonal p53 (1C12) antibody (Cell Signaling Technology Cat# 2524, RRID:AB_331743), Mouse monoclonal Chk1 (2G1D5) antibody (Cell Signaling Technology Cat# 2360, RRID:AB_2080320), Rabbit Phospho-Rad50 (Ser635) Antibody (Cell Signaling Technology Cat# 14223, RRID:AB_2798430), Rabbit Phospho-ADD1/ADD2 (Ser726, Ser713) Polyclonal Antibody (Thermo Fisher Scientific Cat# PA5-40276, RRID:AB_2608892), Rabbit monoclonal anti-Rad51 antibody (Abcam Cat# ab133534, RRID:AB_2722613), Rabbit polyclonal Cathepsin D antibody (Abcam Cat# ab72915, RRID:AB_2040714), Rabbit polyclonal p70 S6 Kinase Antibody (Cell Signaling Technology Cat# 9202, RRID:AB_331676), Mouse monoclonal Phospho-p70 S6 Kinase (Thr389) (1A5) (Cell Signaling Technology Cat# 9206, RRID:AB_2285392), Rabbit monoclonal LC3B (D11) antibody (Cell Signaling Technology Cat# 3868, RRID:AB_2137707), Rabbit anti-GAPDH antibody (ab9485, RRID:AB_307275), Mouse anti-beta Actin monoclonal antibody (ab6276, RRID:AB_2223210), IRDye® 800CW Goat anti-Mouse IgG (RRID:AB_10793856), IRDye® 800CW Goat anti-Rabbit IgG (RRID:AB_10796098), IRDye® 680RD Goat anti-Mouse IgG (RRID:AB_2651128), IRDye® 680RD Goat anti- Rabbit IgG (RRID:AB_2721181) Declarations Conflict of Interest The authors declare that this study was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest. Author Contributions HLS, CGM and AVM conceptualized the study and AVM supervised the experimental work. SC, MI and AVM performed the experimental work, conducted data acquisition and analysis. DB, JCA and SC collated the clinical data and phenotyped participants. DB, MI and AVM performed genetic analysis and interpretation. SC and AVM performed analysis of phosphoproteomic data. LJM carried out the in-silico protein structure modelling work. SC and AVM generated the initial manuscript. All authors contributed to critical appraisal and final draft of the manuscript. Funding This work was supported by an NIHR Advanced fellowship NIHR300098 awarded to HLS and a Barts Charity Seed Grant (MEAG2C4R) awarded to AVM and HLS. SC was funded by William Harvey Limited sponsored Clinical Research and Barts Charity (MRC0220) Fellowships. CGM was funded by Action Medical Research (GN 2272) and BTLC (GN 417/2238 and MGU0551). LJM was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) [BB/T018496/1]. Acknowledgments We would like to thank Professor Pedro R. Cutillas and Dr. Vinothini Rajeeve at Barts Cancer Institute for conducting mass spectrometry (MS)-based proteomics. References Roberts, A.E., Allanson, J.E., Tartaglia, M., and Gelb, B.D. (2013). Noonan syndrome. Lancet 381 , 333–342. https://doi.org/10.1016/S0140-6736(12)61023-X . Tartaglia, M., Gelb, B.D., and Zenker, M. (2011). Noonan syndrome and clinically related disorders. Best Pract Res Clin Endocrinol Metab 25 , 161–179. https://doi.org/10.1016/j.beem.2010.09.002 . Tartaglia, M., Gelb, B.D., and Zenker, M. (2011). Noonan syndrome and clinically related disorders. Best Pract Res Clin Endocrinol Metab 25 , 161–179. https://doi.org/10.1016/j.beem.2010.09.002 . Tajan, M., Paccoud, R., Branka, S., Edouard, T., and Yart, A. (2018). The RASopathy Family: Consequences of Germline Activation of the RAS/MAPK Pathway. 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Prediction of protein structures, functions and interactions using the IntFOLD7, MultiFOLD and ModFOLDdock servers. Nucleic Acids Res 51 , W274–W280. https://doi.org/10.1093/nar/gkad297 . Maharaj, A.V., Ishida, M., Rybak, A., Elfeky, R., Andrews, A., Joshi, A., Elmslie, F., Joensuu, A., Kantojärvi, K., Jia, R.Y., et al. (2024). QSOX2 Deficiency-induced short stature, gastrointestinal dysmotility and immune dysfunction. Nat Commun 15 , 8420. https://doi.org/10.1038/s41467-024-52587-w . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6280572","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432303727,"identity":"445dfce4-10c2-48c7-a539-b130826ad5ce","order_by":0,"name":"Sumana Chatterjee","email":"","orcid":"","institution":"William Harvey Research Institute, QMUL","correspondingAuthor":false,"prefix":"","firstName":"Sumana","middleName":"","lastName":"Chatterjee","suffix":""},{"id":432303728,"identity":"e0a5a4e5-ee93-43f3-9bb7-dbcc78ad4d00","order_by":1,"name":"Miho Ishida","email":"","orcid":"","institution":"William Harvey Research Institute, QMUL","correspondingAuthor":false,"prefix":"","firstName":"Miho","middleName":"","lastName":"Ishida","suffix":""},{"id":432303729,"identity":"d1c926ab-f137-406d-b8c7-67e78a2b69fc","order_by":2,"name":"Débora R. 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McGuffin","email":"","orcid":"","institution":"University of Reading","correspondingAuthor":false,"prefix":"","firstName":"Liam","middleName":"J.","lastName":"McGuffin","suffix":""},{"id":432303733,"identity":"977f27c1-e715-43b2-abd0-aaa7414d89a0","order_by":6,"name":"Helen L. Storr","email":"","orcid":"","institution":"William Harvey Research Institute, QMUL","correspondingAuthor":false,"prefix":"","firstName":"Helen","middleName":"L.","lastName":"Storr","suffix":""},{"id":432303735,"identity":"9f0eded7-73de-455a-9a5e-8df2b33bed28","order_by":7,"name":"Avinaash V. Maharaj","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYPCCAyAi8QGQkEESIKwl2QBI8JCkhU2CKC38DTwGTDdq7sibtx94VvE25zAPA/vhB8w8Z3BrkTjAY8Ccc+yZ4ZwzCWk3524DauFJM2DmuYHPSWwJzDlshxlnMCSk3eYFaWHIYWDm+YBbhzxYy7/D9jP4H6QVg7Xwv8GvxeAA8wHm3LbDiTMkEtKYwVokQLbgcZjhYeYDh3P7DifPkHiQLDl3WzoPm8Qzg4Nz8Hhf7nhj4+Ocb4dtZ/DnJH54u81ajp8/+eGDN8fweJ8ZHgc8CeBIYWMgJiIhgP0ALB5HwSgYBaNgFKAAAIW1UPSwCUYOAAAAAElFTkSuQmCC","orcid":"","institution":"William Harvey Research Institute, QMUL","correspondingAuthor":true,"prefix":"","firstName":"Avinaash","middleName":"V.","lastName":"Maharaj","suffix":""}],"badges":[],"createdAt":"2025-03-21 23:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6280572/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6280572/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79165833,"identity":"6c8ba57f-d2cf-469a-9d3a-54b5eaf01cd0","added_by":"auto","created_at":"2025-03-25 08:31:12","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":674273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterisation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLZTR1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e variants and phosphoproteomic analysis. \u003c/strong\u003e(A) Immunoblotting showed stable expression levels of LZTR1 variants, p.K156E and p.G248R, analogous to Wild type-LZTR1. LZTR1 expression localised to the nucleus and was broadly similar under all conditions (B). (C) Immunofluorescence highlighted increased pan-RAS levels for both variants detected on confocal microscopy. (D) Kinase activity estimation (KSEA) of two key variants, p.K156E and p.G248R, showed significant enrichment of DNA damage response (DDR) related-effectors, Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1). (E) Pathway analysis based on p-value significance revealed a target histone acetyltransferase inhibitor, NOC2L (NOC2 Like Nucleolar Associated Transcriptional Repressor), upregulated in both variants. (F) Top networks and toxicity lists enriched in this dataset included cell morphology, cellular function and maintenance/cancer as well as cell death and cardiac dysfunction.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6280572/v1/2e64c4fd99115934e266c5c9.jpeg"},{"id":79165058,"identity":"d496a6af-5ee1-4034-bb0f-6568598e9c70","added_by":"auto","created_at":"2025-03-25 08:23:12","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":585108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNOC2L is a novel target related to LZTR1 variation. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eProtein interactome analysis of NOC2L and other differentially expressed targets using Ingenuity Pathway Analysis revealed multiple interactions with chromatin remodelling factors. (B, C) Increased levels of NOC2L were seen in both LZTR1 variants both by western blotting and immunofluorescence.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6280572/v1/f654efaf3d06bad75b591b5d.jpeg"},{"id":79165055,"identity":"d1960493-3832-485e-bf0a-290ae6732e1d","added_by":"auto","created_at":"2025-03-25 08:23:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":231779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNOC2L upregulation dampens p53 activity. \u003c/strong\u003e(A) In normal physiological states, NOC2L regulates the activity of p53 target genes and the acetylation and transcription of p53 itself. (B) For both LZTR1 variants, acetylation of the critical Lysine residue 382 of p53 was reduced when compared to WT-LZTR1. (C) Global levels of p53 as detected on confocal microscopy were also reduced for both variants. (D). The DNA damage response involves several critical effectors such at ATM, CHK1 and Rad51 that induce p53-mediated apoptosis or DNA repair in response to chronic stress. (E) In LZTR1 variants, the kinases ATM and CHK1 were increased in both variants as probed by immunofluorescence. (F) Activity of both kinases ATM and CHK1 were probed by phosphorylation of two key substrates (Rad50, serine 635) and (Adducin1/2, serine 713/726) respectively. Levels of these phosphorylated substrates were increased for both LZTR1 variants. Figures 3A and 3D were created in BioRender (Maharaj, A. (2025) \u003ca href=\"https://biorender.com/p19j619\"\u003ehttps://BioRender.com/p19j619\u003c/a\u003e, Maharaj, A. (2025 \u003ca href=\"https://biorender.com/j82t350\"\u003ehttps://BioRender.com/j82t350\u003c/a\u003e)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6280572/v1/369ac19c85a568d85f75f019.jpeg"},{"id":79165065,"identity":"d1f72f3e-d5b0-4a7d-ade1-ea746b062bd5","added_by":"auto","created_at":"2025-03-25 08:23:13","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":540646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLZTR1 variants are associated with homeostatic disturbances between apoptosis and autophagy.\u003c/strong\u003e (A) Expression levels of Rad51, a protein responsible for DNA repair, were increased for both LZTR1 variants. (B) Despite this relative increase in DNA damage markers, apoptotic signalling remained static as evidenced by equivocal levels of Cathepsin D for both WT-LZTR1 and mutant constructs. (C) Phosphorylated levels of downstream MTOR effector and marker of autophagy, p70 S6kinase, were increased for both LZTR1 variants. This correlated to a similar increase in LC3 levels detected upon immunofluorescence (D).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6280572/v1/560820b9e6e29446523035d2.jpeg"},{"id":79165068,"identity":"69b27106-e52b-44f9-8de0-51268f1309bc","added_by":"auto","created_at":"2025-03-25 08:23:13","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":818236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLZTR1 directly interacts with NOC2L. \u003c/strong\u003eMultiFOLD2 models showing interactions between LZTR1 and NOC2L. NOC2L in cyan is kept in the same frame of reference and LZTR1 variants are shown in green. The disordered N and C termini on NOC2L have been removed for clarity.\u003cstrong\u003e \u003c/strong\u003e(A) Wildtype LZTR1 with NOC2L (plDDT=0.90, pTM=0.59). (B) LZTR1 mutant p.K156E with NOC2L (plDDT=0.90, pTM=0.59) (C) LZTR1 mutant p.G248R with NOC2L (plDDT=0.89 pTM=0.58). (D) NanoBit complementation assays demonstrated blunted interaction between NOC2L and LZTR1 variants p.K156E and p.G248R when compared to WT-LZTR1 (p \u0026lt; 0.0001). Ordinary one-way-ANOVA was used for statistical analysis with multiple testing corrections performed using Sidak’s test. Data are presented as the mean ± SD of three repeated measurements (3 independent replicates).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6280572/v1/c46f2d37464004aef56eebae.jpeg"},{"id":80133671,"identity":"9155792e-b210-4cf5-b1d3-306e0537f372","added_by":"auto","created_at":"2025-04-08 09:38:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3961242,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6280572/v1/197e0ace-d3dc-4647-9cfa-e4138beaa6b4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The pathogenesis of Noonan syndrome is modulated by NOC2L, a novel interactor of LZTR1 leading to impaired p53 signalling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNoonan syndrome (NS) (OMIM 163950) is a dominantly inherited multi-system disorder with an estimated incidence of 1/1000\u0026ndash;2500 live births \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It is defined by a characteristic phenotypic triad of short stature, distinctive facial features (short/webbed neck, low-set posteriorly rotated ears, ocular hypertelorism, ptosis, down slanting palpebral fissures) and congenital heart defects (pulmonary valve stenosis and hypertrophic cardiomyopathy in 60 and 20% cases, respectively). Other associated phenotypic features include cryptorchidism in males, skeletal abnormalities (pectus deformities and scoliosis), developmental delay and a predisposition to myeloproliferative disorders. Up to 80% of patients have postnatal proportionate short stature\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNS is a clinical diagnosis and establishing the diagnosis can be very difficult, especially in adulthood, given variable phenotypic expression. Variants in more than 20 genes have been associated with pathogenesis of NS (\u003cem\u003ePTPN11, SOS1, SOS2, KRAS, NRAS, RIT1, RRAS, RASA1, RASA2, MRAS, RAF1, BRAF, MAP2K1, MAP3K8, SPRY1, MYST4, A2ML1, SPRED2, CBL and LZTR1\u003c/em\u003e) and account for 75\u0026ndash;80% cases\u003csup\u003e4,5\u003c/sup\u003e. All causative genes for NS, except \u003cem\u003eLZTR1\u003c/em\u003e, encode components or regulators of the well-studied RAS mitogen-activated protein kinase (MAPK) signalling pathway.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLZTR1\u003c/em\u003e, located on chromosome 22q11.21, was initially described as a putative transcriptional regulator acting as an adaptor protein for CUL3 ubiquitin ligase complexes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In humans, \u003cem\u003eLZTR1\u003c/em\u003e mutations were first associated with DiGeorge syndrome (22q11.2 deletion syndrome) whilst somatic variants with loss of heterozygosity at 22q11 were identified in glioblastoma multiforme, a malignant central nervous system tumour\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Subsequently, several studies demonstrated an association between heterozygous missense \u003cem\u003eLZTR1\u003c/em\u003e variants and NS. To date, over 70 cases of NS with pathogenic \u003cem\u003eLZTR1\u003c/em\u003e variants have been described\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, biallelic recessive variants have also been identified in several extensive kindreds\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The majority of heterozygous NS-causing variants are missense and map to the Kelch domains of the protein whilst biallelic variants may be frameshift, nonsense, missense, or splice-site and are distributed across all functional domains.\u003c/p\u003e \u003cp\u003eDominantly inherited \u003cem\u003eLZTR1\u003c/em\u003e variants do not significantly impair protein stability and subcellular localization but do enhance stimulus-dependent RAS-MAPK signalling\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Contrastingly, recessive variants are thermodynamically unstable and do not impact MAPK signalling\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The distribution pattern of dominant and recessive NS-causing \u003cem\u003eLZTR1\u003c/em\u003e mutations and their differential impact on MAPK signalling supports a dominant negative role for the former and a loss-of-function effect for the latter.\u003c/p\u003e \u003cp\u003eLZTR1 has been shown to enable ubiquitination of RAS proteins via its complex with cullin3 and its loss negatively impacts RAS ubiquitination\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Impaired ubiquitination thereby increases the pool of RAS proteins and augments MAPK pathway activity.\u003c/p\u003e \u003cp\u003eWe investigated two naturally occurring heterozygous \u003cem\u003eLZTR1\u003c/em\u003e variants identified in two NS patients, both with characteristic facial features of NS, cardiac abnormalities, short stature and features of Growth Hormone Insensitivity. Interestingly, our studies have identified a previously undescribed interaction between LZTR1 and NOC2L. NOC2 like nucleolar associated transcriptional repressor (NOC2L) was recently identified as a novel inhibitor of histone acetyltransferase (INHAT), thereby inhibiting histone acetylation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Further, studies have also shown that NOC2L is recruited by P53 to inhibit histone acetylation in P53-targeted genes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. NOC2L overexpression can therefore lead to inhibition of p53-mediated transcription, thereby impairing the process of apoptosis and cell cycle arrest. The identification of an underlying genetic mutation in NS provides a definitive diagnosis and has important clinical and therapeutic implications for patients.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eClinical phenotypes of the two individuals harbouring\u003c/b\u003e \u003cb\u003eLZTR1\u003c/b\u003e \u003cb\u003evariants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBoth patients (Patients 1 and patient 2) had classical facial features of NS (hypertelorism with down-slanting palpebral fissures, ptosis, low-set posteriorly rotated ears and webbed neck) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and cardiac pathology; pulmonary artery stenosis (Patient 1) and pulmonary valve stenosis with an affiliated atrial septal defect (Patient 2).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical details of patients with \u003cem\u003eLZTR1\u003c/em\u003e variants at diagnosis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLZTR1\u003c/em\u003e variant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge at genetic diagnosis/Sex/\u003c/p\u003e \u003cp\u003eEthnicity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeight (SDS)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS Features\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBirth-weight SDS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHeight velocity\u003c/p\u003e \u003cp\u003ecm/year\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGH peak (mcg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eType of GH stimulation test and age\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eIGF-1 ng/ml\u003c/p\u003e \u003cp\u003e(SDS)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIGFBP 3\u003c/p\u003e \u003cp\u003emg/L\u003c/p\u003e \u003cp\u003e(SDS)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eSegregation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.466A\u0026thinsp;\u0026gt;\u0026thinsp;G; p.K156E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.0 years, M, British\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85 cm\u003c/p\u003e \u003cp\u003e(-2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClassical facies*, webbed neck, Pulmonary artery stenosis, right undescended testis, left inguinal hernia, developmental delay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eArginine,\u003c/p\u003e \u003cp\u003e3.5 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e19.8\u003c/p\u003e \u003cp\u003e(-2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003cp\u003e(-3.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eAdopted\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2^\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.742G\u0026thinsp;\u0026gt;\u0026thinsp;A; p.G248R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.4 years, F, Brazilian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e131.5 cm\u003c/p\u003e \u003cp\u003e(-2.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClassical facies*, webbed neck, pectus deformity, Pulmonary valve stenosis, Atrial Septal Defect, prominent corneal nerves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eClonidine,\u003c/p\u003e \u003cp\u003e5.6 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e35\u003c/p\u003e \u003cp\u003e(-2.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003cp\u003e(-1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eMother and maternal grandfather\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e^Variant previously reported; Age and Height SDS are at presentation; M, male; F, female; *Classical facies, hypertelorism with down-slanting palpebral fissures, ptosis and low-set posteriorly rotated ears; GH, Growth Hormone; IGFBP3, IGF-binding protein 3; ND, no data; N/D, not done; N/A, not applicable.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePatient 1, a 4-year-old British-Caucasian male, presented with: short stature (height SDS \u0026minus;\u0026thinsp;2.3), brachycephaly, bitemporal narrowing, minor degree of fifth finger clinodactyly, a single palmar crease on the left hand, tapering fingers, convex fingernails and widely-spaced nipples. Additional features included right unilateral cryptorchidism, left inguinal hernia and developmental delay. The patient was adopted hence genetic mapping was not possible. Biochemically, the patient had features of GHI insensitivity (GHI) with a peak GH of 10.56 mcg/L (GHRH-Arginine provocation testing) and IGF-1 and IGFBP3 deficiencies (-2.3 and \u0026minus;\u0026thinsp;3.0 SDS, respectively).\u003c/p\u003e \u003cp\u003ePatient 2, an 11.4-year-old Brazilian girl with short stature (height \u0026minus;\u0026thinsp;2.1 SDS) and a history of congenital heart disease, presented in early childhood with classic features of NS and superimposed pectus excavatum, lacrimal duct obstruction and prominent corneal nerves. Growth hormone provocation (Clonidine) elicited a peak of 11.8 mcg/L in tandem with persistently low IGF-1 (-2.2 SDS) levels. Interestingly, the patient\u0026rsquo;s mother and maternal grandfather also had clinical features of NS and short stature (height \u0026minus;\u0026thinsp;2.5 and \u0026minus;\u0026thinsp;2.9 SDS respectively). The patient was given recombinant human Growth Hormone (rhGH) for several years however, the response was suboptimal with annual height velocities fluctuating between 5.0 and 7.0 cm/year and a final adult height of -2.6 SDS (age 18 years).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of pathogenicity of variants using\u003c/b\u003e \u003cb\u003ein silico\u003c/b\u003e \u003cb\u003eprediction tools\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext generation sequencing revealed single missense \u003cem\u003eLZTR1\u003c/em\u003e variants harboured by each patient (Patient 1- \u003cem\u003ec.466A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/em\u003e; p.K156E and Patient 2 - \u003cem\u003ec.742G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e; p.G248R). Segregation analysis revealed maternal inheritance of the p.G248R variant, which was highly penetrant and present in the proband\u0026rsquo;s affected mother and maternal grandfather. Both variants were rare; the p.K156E variant was novel and p.G248R had a MAF of 0.000003718 in the Genome Aggregation Database (gnomAD) with 6 reported heterozygotes classified as likely pathogenic based on ClinVar entries related to underlying Rasopathy (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The p.K156E variant was predicted benign by PolyPhen2 but deemed deleterious and disease causing by SIFT and Mutation Taster, respectively. The pathogenicity of this variant according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP)\u0026rsquo;s standards and guidelines for the interpretation of sequence variants was deemed to be likely pathogenic (PM2, PM1_Moderate, PP3 and PS3). The p.G248R variant was predicted deleterious across all 3 computational platforms and likely pathogenic according to concurrent ClinVar entries.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e predictions of the \u003cem\u003eLZTR1\u003c/em\u003e variants.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLZTR1\u003c/em\u003e variant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGnomAD frequency\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSIFT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolyPhen2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCADD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMutation Taster\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ec.466A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/em\u003e; p.K156E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNot reported\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e(deleterious)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.301 (Benign)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDisease causing, amino acid sequence changed, protein features might be affected\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e^\u003c/b\u003e\u003cem\u003ec.742G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e; p.G248R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.000003718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001 (deleterious)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0 (Damaging)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDisease causing, amino acid sequence changed, protein features might be affected\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eFor amino acid substitutions: Using SIFT a score of \u0026lt;\u0026thinsp;0.05 is predicted deleterious conversely using PolyPhen2, scores closer to 1 are more likely to be damaging with variants within the range 0.85-1.0 more confidently predicted to be damaging. Mutation Taster provided automatic assignation of variants as disease causing or as a polymorphism. ^ Variant previously reported.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLZTR1\u003c/b\u003e \u003cb\u003evariants are thermally stable and associated with increased pan-RAS levels\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe assessed expression of both \u003cem\u003eLZTR1\u003c/em\u003e variants following transient transfection into an \u003cem\u003ein vitro\u003c/em\u003e HEK 293T reconstitution system. Both variants were well-expressed upon immunoblotting, with levels demonstrably comparable to WT-LZTR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, both variants localised to the nucleus similar to WT-LZTR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) however, pan-Ras levels were markedly increased for both variants as evident on confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhosphoproteomics\u003c/h2\u003e \u003cp\u003eWe conducted phosphoproteomic analysis of LZTR1 mutants to identify unique transcriptomic signatures associated with LZTR1 variation. Kinase activity estimation (KSEA) demonstrated significant enrichment of Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1), major effectors of the DNA damage response (DDR), which were both preferentially activated in LZTR1 variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Ingenuity pathway analysis and filtering of molecules based on p-value significance revealed a target histone acetyltransferase inhibitor, NOC2L (NOC2 Like Nucleolar Associated Transcriptional Repressor), upregulated in both variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Top networks and toxicity lists enriched in this dataset included cell morphology, cellular function and maintenance/cancer as well as cell death and cardiac dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Protein interactome analysis of NOC2L and other differentially expressed targets revealed a complex network involving Chromodomain-helicase-DNA-binding protein 4 (CHD4) and Histone H2B, nucleosome and chromatin remodelling factors that suggest NOC2L may exert its effects via transcriptional regulation of several cofactors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNOC2L localises to the nucleolus\u003c/h3\u003e\n\u003cp\u003eImmunoblotting of lysates expressing WT-LZTR1 and variant constructs confirmed the phosphoproteomic findings, demonstrating robust detection of NOC2L in both variants when compared to wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Immunofluorescence similarly highlighted an abundance of NOC2L for both variants, further denoting its nucleolar localisation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003eNOC2L upregulation attenuates acetylation and expressivity of p53\u003c/h3\u003e\n\u003cp\u003ePrevious work has intimated that physiological levels of NOC2L inhibit acetylation of p53-targeted gene promoters and inadvertently p53 itself\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Hence upregulation of NOC2L should theoretically demonstrate opposing functions. We probed acetylation of p53 lysine residue 382, critical for activation of p53 activity, via immunoblotting of WT-LZTR1 and variant expressing lysates. Levels of acetylated lysine 382 were reduced for both variants when compared to wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and were concordant with reduced global levels of p53 as visualised by confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003eThe DNA Damage Response is activated downstream of LZTR1 variation\u003c/h3\u003e\n\u003cp\u003eThe DNA damage response (DDR) involves several effectors such as Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1) that culminate in p53 activation or increased transcription of Rad51 to initiate homologous recombination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Global levels of ATM and CHK1 appeared enhanced in both LZTR1 variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) whilst two major substrates representative of their kinase activity, Rad50 and Adducin (ADD1/2), were preferentially phosphorylated in both variants, via residues Serine 635 and Serine 713/726 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Furthermore, increased levels of Rad51 were noted upon immunofluorescence of variant constructs relative to WT-LZTR1, when expressed in mammalian cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n\u003ch3\u003eAttenuated p53 activity leads to compensatory activation of autophagy signalling\u003c/h3\u003e\n\u003cp\u003eThe reduction in p53 expression was further associated with equivocal activity of apoptosis regulator, Cathepsin D in WT-LZTR1 and variant constructs as evidenced by commensurate expression of pro-and pre-procathepsin D levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This apparent impasse in apoptotic activation despite a relative increase in the DDR was associated with increased markers of autophagy. Immunoblotting of WT-LZTR1 and variant constructs revealed increased phosphorylation of the threonine 389 residue of p70 S6kinase in variant constructs when compared to wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Phosphorylation of this residue is critical for kinase activation and reflective of increased autophagic flux (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This effect correlated to increased LC3 levels in LZTR1 variants detected upon confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLZTR1 directly interacts with NOC2L\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e quaternary structure prediction using MultiFOLD2 demonstrated confidently modelled interactions between LZTR1 and NOC2L (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The models showed different relative binding orientations for the LZTR1 mutants versus wildtype, suggesting alternative binding modes with NOC2L (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C). Although the mutated residues in the models are not located within the binding interfaces, they may have downstream conformational effects. The model quality scores were marginally reduced for the p.G248R mutant, indicating a lower confidence in the interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C). To validate possible predicted protein-protein interaction between both key targets, Nanoluc Binary technology (NanoBit) was utilised. Complementation assays using Reporter-tagged target proteins, demonstrated robust interaction between LZTR1 and NOC2L as evidenced by detectable luminescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, this interaction with wild type NOC2L was disrupted for both LZTR1 variants with the greatest effect on p.G248R (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLZTR1, an adaptor protein for the CUL3 ubiquitin ligase complex, is implicated in NS yet its mechanism of action is not fully understood. \u003cem\u003eLZTR1\u003c/em\u003e variants causing autosomal dominant NS likely inhibit binding to either RAS and/or other target substrates, thereby impairing their ubiquitination and degradation. Our missense variants (p.K1563E and p.G248R), are stably expressed and consistent with previously published work, associated with enhanced RAS activity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The homogeneous nature of dominant NS-causing \u003cem\u003eLZTR1\u003c/em\u003e variants (i.e. missense changes) and their clustering within the Kelch repeats strongly suggest a specific disruptive effect of variants on domain function and protein-protein interaction(s). Interestingly, we have identified NOC2L as a novel interactor of LZTR1 and whose levels appear to be enhanced in the setting of \u003cem\u003eLZTR1\u003c/em\u003e variation.\u003c/p\u003e \u003cp\u003ePhosphoproteomic analysis of LZTR1 variants revealed specific phosphopeptide signatures indicative of upregulation of the DNA damage response (DDR). This was consistent with previous data demonstrating activation of the DDR and chromosomal instability in association with germline \u003cem\u003eLZTR1\u003c/em\u003e variants\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Key kinases involved in this pathway (ATM, CHK1) were upregulated with increased phosphorylation of respective target substrates, Rad50 and Adducin1/2 demonstrated by immunoblotting. A further marker of DNA damage and inducer of homologous recombination, Rad51 was shown to be increased in both LZTR1 variants when compared to WT. It is entirely possible that \u003cem\u003eLZTR1\u003c/em\u003e variation may trigger genotoxic stress and an overall propensity to chronic DNA damage and increased risk of tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe also identified NOC2L upregulation which was concordant in both \u003cem\u003eLZTR1\u003c/em\u003e variants. NOC2 like nucleolar associated transcriptional repressor (NOC2L) is an endogenous inhibitor of histone acetylation and negatively regulates p53 activity\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. p53 directly interacts with NOC2L, facilitating its recruitment to inhibit transcriptional activation of p53-target genes; in the process, p53 is itself modulated by NOC2L, which inhibits its acetylation and subsequent activity\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. When compared to WT-LZTR1, both variants had attenuated acetylation of Lysine residue 382 and globally reduced levels of p53. The implication that apoptosis induction was impaired, despite activation of the DDR, was supported by relatively unaltered levels of apoptotic marker Cathepsin D across both LZTR1 mutants. This apparent blockade in apoptosis was compounded by an enhanced autophagic response in both LZTR1 variants, which demonstrated heightened LC3 levels and robust phosphorylation of threonine residue 389 of p70 S6Kinase, a downstream effector of mTOR whose activation correlates to an increase in autophagy. Previous work has demonstrated that transgenic expression of the human NS-associated \u003cem\u003ePTPN11\u003c/em\u003e variant (\u003cem\u003ec.236A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/em\u003e, p.G79R) in murine endocardial cushions phenocopied the human cardiac valvulopathy resulting in increased cell proliferation and enlarged cushions due to reduced apoptosis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This suggests that multi-organ pathology in NS may be related to attenuated apoptotic signalling. Our findings highlight an increase in autophagy markers either as a compensatory response to diminished p53 activity or reflective of a general disruption in cellular homeostasis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Disequilibrium between both processes has been linked to a propensity to tumorigenesis, an established risk for patients with NS\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnder normal physiological conditions, NOC2L is involved in embryogenesis, lymphopoiesis and epidermal development likely mediated via its interaction with the closely related proteins, p53 and p63\u003csup\u003e25\u003c/sup\u003e. It is also implicated in carcinogenesis due to its diametrically opposing effect on p53 tumour suppressive activity\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Interestingly, Chinton \u003cem\u003eet al\u003c/em\u003e. reported five individuals with NS, harbouring the \u003cem\u003eLZTR1\u003c/em\u003e variant (\u003cem\u003ec.742G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e; p.G248R) seen in patient 2, one of whom developed acute lymphoblastic leukaemia (ALL)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We report an upregulation of NOC2L in association with dominantly inherited missense \u003cem\u003eLZTR1\u003c/em\u003e variants. Furthermore, we demonstrated a direct interaction between both proteins using \u003cem\u003ein silico\u003c/em\u003e predictive tools and \u003cem\u003ein vitro\u003c/em\u003e nanoluciferase technology; an interaction disrupted upon \u003cem\u003eLZTR1\u003c/em\u003e variation. We hypothesise that wild type LZTR1 may ubiquitinate NOC2L and regulate its activity; an effect lost due to \u003cem\u003eLZTR1\u003c/em\u003e mutations leading to NOC2L hyperactivation. Given its involvement in cell cycle regulation, NOC2L may be a potential regulator of organogenesis downstream of or in tandem with LZTR1, although further work is needed to characterise this effect.\u003c/p\u003e \u003cp\u003eThere have been sporadic reports of affiliated growth hormone deficiency\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, however, to date, growth hormone insensitivity associated with \u003cem\u003eLZTR1\u003c/em\u003e variants has not been reported and no underlying mechanism has been elucidated. Our patients both demonstrated biochemical evidence of IGF-1 deficiency associated with sufficient GH levels and short stature (height SDS \u0026lt;-2). However, short stature in NS often exhibits incomplete penetrance\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, growth dysregulation might be influenced by other factors including environmental factors, genetic modifiers or novel interacting partners such as NOC2L that modulate affiliated signalling pathways. Therefore, further \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e data are required to delineate these associations and identify common transcriptomic links affiliated with NOC2L that may be implicated in multi-system pathology. Human Growth hormone (hGH) is the only licenced treatment for growth failure associated with NS however, as demonstrated in Patient 2, therapy can often be suboptimal. However, agents that target the RAS/MAPK pathway such as the MEK inhibitor, Trametinib has been used with some success in treating NS patients with non-malignant pathology such as severe hypertrophic cardiomyopathy, heart failure and lymphatic anomalies\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Ongoing clinical trials are currently evaluating the impact of the C-type natriuretic peptide (CNP) analogue, Vosoritide on patients with wide ranging genetic causes of short stature including Rasopathies\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Thus, a deeper understanding of the molecular mechanisms underlying the growth failure in NS may lead to the identification of newer drug targets or justification for those currently under investigation.\u003c/p\u003e \u003cp\u003eGiven its overarching multi-system pathology, the health burden of NS is significant and associated with high morbidity\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Hence, uncovering the mechanistic pathways involved in disease propagation is integral to evolving patient care. We propose a new disease model for LZTR1-mediated NS in which LZTR1 is postulated to form an interacting partner of NOC2L, modulating its ubiquitination and hence stability. Missense variation in \u003cem\u003eLZTR1\u003c/em\u003e disrupts this interaction leading to upregulation of NOC2L activity and repression of p53 transcription. This triggers a cascade that culminates in an apoptosis blockade and compensatory autophagy induction that perpetuates a state of chronic DNA damage. Currently, no inhibitors of NOC2L exist, but this may be an apt therapeutic target that warrants exploration should future work uncover a broadly similar role for NOC2L in other genetic causes of NS.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed written consents for publication of clinical details, including indirect identifiers, were obtained from human research participants and their guardians. Participants consented to dissemination of anonymised clinical data in an open-access journal and were not compensated for their involvement. The study was approved by the Health Research Authority, East of England-Cambridge East Research Ethics Committee (REC reference 17/EE/0178).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole exome sequencing (WES) was conducted using the Agilent SureSelect all exon V4 capture and paired-end (2x100) sequencing on an Illumina HiSeq 2000 at Otogenetics (Norcross, GA, USA). Common variants were filtered out by excluding those with an allele frequency of \u0026ge;0.1% in the 1000 genomes, ExAC and the NHLBI exomes. Variants predicted damaging by SIFT (http://sift.jcvi.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) or Mutation Taster (https://www.mutationtaster.org/) were explored further.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of \u003cem\u003eLZTR1\u0026nbsp;\u003c/em\u003evariant constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild type \u003cem\u003eLZTR1\u003c/em\u003e mammalian expression cDNA clone (pCDNA-MYC-Hist-LZTR1) was kindly donated by Dr. Antonio Lavarone at the Institute for Cancer Genetics, Columbia University Medical Centre, New York, USA. Our variants of interest: \u003cem\u003ec.466A\u0026gt;G\u003c/em\u003e, p.K156E and \u003cem\u003ec.742G\u0026gt;A\u003c/em\u003e, p.G248R were generated by Site-Directed Mutagenesis (QuikChange II XL SDM Kit, Agilent Technologies) as per manufacturer\u0026rsquo;s instructions. All constructs were verified by Sanger sequencing (primer sequences available on request).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and transfection protocols\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman embryonic kidney (HEK 293T, ATCC\u0026reg; CRL-3216TM) cells were cultured in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) high glucose (Sigma D5648) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Transfection of cells was achieved using Lipofectamine\u0026trade; 3000 according to manufacturer\u0026rsquo;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein lysates were quantified using a Bradford protein assay (Bio-Rad), denatured by the addition of laemmli SDS sample buffer 6X (Thermo Scientific) and boiled for 5 minutes at 98\u0026deg;C. 20-30\u0026mu;g of protein were loaded into wells of 4 to 12% (Bis-Tris) 1.0 mm pre-cast gels (Invitrogen) prior to electrophoretic separation using MOPS SDS running buffer. Semi-dry protein transfer to nitrocellulose membrane was achieved using a Trans-Blot\u0026reg; SD Semi-Dry Transfer Cell (Bio-Rad) at 15V for 1 hour. The membrane was blocked with 5% fat free milk in TBS/0.1% Tween-20 for 1 hour at room temperature. Primary antibody was added at a concentration of 1:1000 and anti-\u0026beta;-actin (1:10,000) used as a housekeeping control. Primary antibody incubation in blocking buffer or 5% Bovine serum albumin (BSA) in TBS/0.1% Tween-20 was performed overnight at 4\u0026deg;C on gentle agitation. The membrane was then washed for 10 minutes (3X) with Tris Buffered saline-Tween-20 (TBST). Secondary anti-mouse, anti-rabbit or anti-goat antibodies were added at a concentration of 1:5000 to antibody dilution buffer and at 37\u0026deg;C for 60 minutes. The membrane was washed three times (10 minutes each) with TBST and visualized with the LI-COR Image Studio software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhosphoproteomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLZTR1\u003c/em\u003e WT, variant (p.K156E and p.G248R) and empty vector constructs were transiently transfected into HEK 293T cells using Lipofectamine\u0026trade; 3000 according to manufacturer\u0026rsquo;s instructions. After 24 hours, cells were lysed in urea buffer and homogenised by sonication. The insoluble material was removed by centrifugation and protein in the cell extracts was quantified. 250\u0026thinsp;\u0026micro;g of protein was reduced and digested with trypsin. Peptide solutions were desalted with Oasis cartridges and phosphopeptides were enriched using TiO2 as previously reported\u003csup\u003e39\u0026ndash;41\u003c/sup\u003e. Phosphopeptide pellets were re-suspended in reconstitution buffer (20\u0026thinsp;fmol/\u0026micro;l enolase in 3% ACN, 0.1% TFA) and loaded onto an Orbitrap Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) as previously described\u003csup\u003e39\u0026ndash;41\u003c/sup\u003e. Differences in phosphorylation patterns between WT, \u003cem\u003eLZTR1\u003c/em\u003e variant and empty vector constructs were reported as fold over WT and statistical significance for those changes assessed using unpaired two-tailed t-tests. Further analysis of transcriptomic data was conducted using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMammalian cells seeded on glass coverslips (24 well plates) were transfected with WT and \u003cem\u003eLZTR1\u003c/em\u003e plasmid constructs. After 48 hours, cells were fixed with 4% paraformaldehyde for 15\u0026thinsp;minutes at room temperature. Cells were washed three times in PBS and permeabilized with 0.5% Triton X-100 in PBS for 10 minutes. After further PBS washes, coverslips were incubated in Blocking buffer (1X PBS / 5% goat serum / 0.3% Triton\u0026trade; X-100) at room temperature for 60\u0026thinsp;min. Primary antibody (mouse anti-myc, rabbit anti-Pan-RAS, rabbit anti-NOC2L, mouse anti-p53, mouse anti-CHK1, rabbit anti-ATM, rabbit anti-LC3B) reconstituted in dilution Buffer (1X PBS / 1% BSA / 0.3% Triton\u0026trade; X-100 buffer) was added to cells and left at 4\u0026thinsp;\u0026deg;C overnight with gentle agitation. Cells were washed and incubated in appropriate fluorescent secondary antibody at room temperature for 60\u0026thinsp;minutes (protected from light). Coverslips were stained with DAPI and mounted on microscope slides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein Structure Modelling\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe latest version of the MultiFOLD server\u003csup\u003e42\u003c/sup\u003e (https://www.reading.ac.uk/bioinf/MultiFOLD/) was used to model the quaternary structures from the wildtype sequences of LZTR1 with NOC2L as well as each of the LZTR1 variants with NOC2L. Each of the modelled complexes was visualised using PyMOL and coloured by chain identifier. The NOC2L chains from all models were superposed using the align command to orientate the modelled complexes in the same frame of reference. The disordered N and C termini on NOC2L were identified and removed from the visualization for clarity. The locations of the mutated residues were identified and highlighted. Finally, the models were raytraced and exported as high-quality images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanoBiT complementation assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBinary protein interactions were assessed with NanoBiT complementation assays (as previously described)\u003csup\u003e43\u003c/sup\u003e using NOC2L WT and LZTR1 WT/variant plasmids N terminally fused with NanoBiT fragments (SmBiT and LgBiT). HEK 293T cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded in clear bottomed 96-well white plates, and plasmids were reverse-transfected using Lipofectamine\u0026trade; 3000 according to the manufacturer\u0026rsquo;s instructions. DNA concentrations were optimised and determined to be 200\u0026thinsp;ng per well; 100\u0026thinsp;ng SmBiT-NOC2L and 100\u0026thinsp;ng LgBiT-LZTR1. After 48\u0026thinsp;hours post-transfection, cell culture medium was replaced with 100\u0026thinsp;\u0026micro;L NanoBiT assay buffer (pH 7.4, HBSS 1X, HEPES 24\u0026thinsp;mM, NaHCO3 3.96\u0026thinsp;mM, CaCl2 1.3\u0026thinsp;mM, MgSO4 1\u0026thinsp;mM, BSA 0.1%) per well and left for 1\u0026thinsp;h at 37\u0026thinsp;\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, six (6) baseline luminescence readings were recorded using the CLARIOstar Multimode Plate Reader (BMG Labtech) followed by addition of 25\u0026thinsp;\u0026micro;l/well of Furimazine (Nanolight Technology) prepared in a 1:20 dilution using assay buffer. Luminescence readings were resumed and continued for 1\u0026thinsp;hour.\u003c/p\u003e\n\u003ch2\u003eResource Identification Initiative\u003c/h2\u003e\n\u003cp\u003eMouse Anti-Myc tag Monoclonal Antibody, Unconjugated, Clone 9E10 (Abcam Cat# ab32, RRID:AB_303599), Pan Ras Monoclonal Antibody (Ras10) (Thermo Fisher Scientific Cat# MA1-012, RRID:AB_2536664), Rabbit polyclonal NOC2L antibody (Proteintech Cat# 28509-1-AP, RRID:AB_2881160), Human p53 (acetyl K382) antibody (Abcam Cat# ab75754, RRID:AB_1310532), Rabbit monoclonal ATM antibody [Y170] (Abcam Cat# ab32420, RRID:AB_725574), Mouse monoclonal \u0026nbsp;p53 (1C12) antibody (Cell Signaling Technology Cat# 2524, RRID:AB_331743), Mouse monoclonal Chk1 (2G1D5) antibody (Cell Signaling Technology Cat# 2360, RRID:AB_2080320), Rabbit Phospho-Rad50 (Ser635) Antibody (Cell Signaling Technology Cat# 14223, RRID:AB_2798430), Rabbit Phospho-ADD1/ADD2 (Ser726, Ser713) Polyclonal Antibody (Thermo Fisher Scientific Cat# PA5-40276, RRID:AB_2608892), Rabbit monoclonal anti-Rad51 antibody (Abcam Cat# ab133534, RRID:AB_2722613), Rabbit polyclonal Cathepsin D antibody (Abcam Cat# ab72915, RRID:AB_2040714), Rabbit polyclonal p70 S6 Kinase Antibody (Cell Signaling Technology Cat# 9202, RRID:AB_331676), Mouse monoclonal Phospho-p70 S6 Kinase (Thr389) (1A5) (Cell Signaling Technology Cat# 9206, RRID:AB_2285392), Rabbit monoclonal LC3B (D11) antibody (Cell Signaling Technology Cat# 3868, RRID:AB_2137707), Rabbit anti-GAPDH antibody (ab9485, RRID:AB_307275), Mouse anti-beta Actin monoclonal antibody (ab6276, RRID:AB_2223210), IRDye\u0026reg; 800CW Goat anti-Mouse IgG (RRID:AB_10793856), IRDye\u0026reg; 800CW Goat anti-Rabbit IgG (RRID:AB_10796098), IRDye\u0026reg; 680RD Goat anti-Mouse IgG (RRID:AB_2651128), IRDye\u0026reg; 680RD Goat anti- Rabbit IgG (RRID:AB_2721181)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this study was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eHLS, CGM and AVM conceptualized the study and AVM supervised the experimental work. SC, MI and AVM performed the experimental work, conducted data acquisition and analysis. DB, JCA and SC collated the clinical data and phenotyped participants. DB, MI and AVM performed genetic analysis and interpretation. SC and AVM performed analysis of phosphoproteomic data. LJM carried out the \u003cem\u003ein-silico\u003c/em\u003e protein structure modelling work. SC and AVM generated the initial manuscript. All authors contributed to critical appraisal and final draft of the manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by an NIHR Advanced fellowship NIHR300098 awarded to HLS and a Barts Charity Seed Grant (MEAG2C4R) awarded to AVM and HLS. SC was funded by William Harvey Limited sponsored Clinical Research and Barts Charity (MRC0220) Fellowships.\u0026nbsp;CGM was funded by Action Medical Research (GN 2272) and BTLC (GN 417/2238 and MGU0551). LJM was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) [BB/T018496/1].\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe would like to thank Professor Pedro R. Cutillas and Dr. Vinothini Rajeeve at Barts Cancer Institute for conducting mass spectrometry (MS)-based proteomics.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRoberts, A.E., Allanson, J.E., Tartaglia, M., and Gelb, B.D. (2013). Noonan syndrome. Lancet \u003cem\u003e381\u003c/em\u003e, 333\u0026ndash;342. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0140-6736(12)61023-X\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(12)61023-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTartaglia, M., Gelb, B.D., and Zenker, M. (2011). 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Nat Commun \u003cem\u003e15\u003c/em\u003e, 8420. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-52587-w\u003c/span\u003e\u003cspan address=\"10.1038/s41467-024-52587-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Noonan syndrome, RAS/MAPK, LZTR1, NOC2L","lastPublishedDoi":"10.21203/rs.3.rs-6280572/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6280572/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e: Monoallelic dominant negative \u003cem\u003eLZTR1\u003c/em\u003e gene variants have been implicated as a cause of NS due to hyperactivation of the canonical RAS-MAPK signalling pathway. Missense \u003cem\u003eLZTR1 \u003c/em\u003evariants have been associated with defective ubiquitination theoretically leading to increased Ras substrate availability and altered p53 signalling. We investigated the role of LZTR1 in this pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Single nucleotide substitutions were created by mutagenesis of an N-terminal MYC tagged-\u003cem\u003eLZTR1\u003c/em\u003e cDNA. WT and variant constructs were expressed in mammalian cells and lysates prepared for phosphoproteomic analysis and immunoblotting. Analysis of transcriptomic data was conducted using Ingenuity Pathway Analysis. Significant phospho-peptides, protein-protein interactions and pathways of interest were probed using western blotting, immunofluorescence, nanoluciferase assays and \u003cem\u003ein silico\u003c/em\u003e prediction tools.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Two heterozygous \u003cem\u003eLZTR1\u003c/em\u003e variants, which segregated with short stature and features of growth hormone insensitivity (p.K156E, p.G248R), were expressed in a mammalian cell line. Both variants were thermodynamically stable and associated with elevated cytoplasmic levels of pan-Ras. Phosphoproteomic assays revealed upregulation of the histone acetyltransferase inhibitor, NOC2L (NOC2 Like Nucleolar Associated Transcriptional Repressor), in both variants. This finding, consistent upon immunoblotting and immunofluorescence, was associated with impaired acetylation of p53, with reduced levels of acetylated lysine residue 382 in both mutants. Furthermore, Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1), major effectors of the DNA damage response (DDR), were preferentially activated in \u003cem\u003eLZTR1\u003c/em\u003e variants. Despite an apparent activation of the DDR and diminished p53 activity, levels of LC3 and phosphorylated p70 S6 kinase were increased. \u003cem\u003eIn silico\u003c/em\u003e structure modelling suggested that LZTR1 interacts with NOC2L via the central part of the protein and this interaction was validated by nanoluciferase assays and disrupted in both \u003cem\u003eLZTR1\u003c/em\u003e variants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: NOC2L and p53 form a complex which dictates p53 activation. We demonstrate a previously unknown interaction between NOC2L and LZTR1 and hypothesise that LZTR1 acts as a binding factor modulating the activity of this complex. As NOC2L negatively regulates p53, upregulation of this protein would lead to p53-mediated transcription inhibition. LZTR1 attenuation due to genetic mutations associated with NS, potentiate NOC2L activity leading to reduced apoptosis and a compensatory increase in autophagy. Given its potential role in the multisystem pathogenesis of NS, NOC2L may represent a novel therapeutic target however, additional work is needed to further characterise its organ-specific effects.\u003c/p\u003e","manuscriptTitle":"The pathogenesis of Noonan syndrome is modulated by NOC2L, a novel interactor of LZTR1 leading to impaired p53 signalling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-25 08:23:06","doi":"10.21203/rs.3.rs-6280572/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"008f2253-6f4f-4016-9efe-f84ab8b32b82","owner":[],"postedDate":"March 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-08T09:38:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-25 08:23:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6280572","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6280572","identity":"rs-6280572","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}