Biventricular outflow tract obstruction due to hypertrophy related to compound heterozygous variants in LZTR1

Noonan syndrome is caused by genetic defects in the RAS/mitogen-activated protein kinase pathway and is an important cause of phenocopy of HCM, accounting for up to 12.5% for paediatric patients who was initially diagnosed as HCM.1 Compared with sarcomeric HCM, HCM in Noonan syndrome shows increased prevalence and severity of left ventricular outflow tract obstruction, higher rates of hospitalization for heart failure or need for septal myectomy during childhood.2 Biventricular outflow obstruction and specific electrocardiographic features may present useful clinical clues for differentiating between Noonan Syndrome-related hypertrophic cardiomyopathy and sarcomeric HCM.3, 4 Noonan syndrome is caused by mutation in more than 10 genes. Recently, LZTR1 has been reported to be associated with Noonan syndrome.5-10 Although Noonan syndrome is mainly transmitted as a dominant trait, an autosomal recessive form of disease has recently been associated with compound heterozygous LZTR1 variants.6, 7, 9 A 14-year-old male patient, who was found with a cardiac murmur at physical examination at 1 year old, presented to our institution for exertional chest tightness and dizziness. He was born from healthy non-consanguineous parents. At examination, the patient had normal stature compared to peers. He had characteristic facial traits of ocular hypertelorism and epicanthal folds. No intellectual disability was found. Grade III systolic murmur was noted at the right second-to-third intercostal space and at the left sternum third intercostal space. Electrocardiography showed extreme left axis deviation, small left precordial R-wave in V5 and V6 and large right precordial S-waves (Figure 1A). Cardiac ultrasound examination revealed asymmetrical hypertrophy with major involvement of basal interventricular septum which cause obstruction in the left and right ventricular outflow tracts (LVOT, RVOT). Systolic anterior motion (SAM) of the mitral valve with moderate regurgitation was also detected (Figure 1B). Continuous-wave Doppler imaging showed a calculated peak gradient of 45 mmHg across the LVOT and 99 mmHg across RVOT at rest (Figure 1C,D). Cardiac magnetic resonance imaging (MRI) yielded evidence of myocardial asymmetrical hypertrophy, similar to transthoracic echocardiography results (Figure 1E). Additional LVOT and ROVT view of cine MRI showed LVOT and RVOT obstruction. Late gadolinium-enhanced (LGE) showed patchy delayed enhancement within the hypertrophic myocardium (Figure 1F,G). Genetic testing of the patient was performed using whole exome sequencing, which identified two likely pathogenic variants of LZTR1 gene, NM_006767.4: c.1549del, p.E517Rfs*39 and NM_006767.4: c.1735G>A, p.V579M. Subsequent validation via Sanger sequencing corroborated these findings in the proband. Cascade testing of first-degree relatives for these two likely pathogenic variants found each variant being transmitted to the patient by the asymptomatic heterozygous parents (Figure 2). Frameshift variant c.1549del (p.Glu517Argfs*39) altered the open reading frame of the gene, leading to changes in protein function. Missense variant c.1735G>A (p.Val579Met) is located in the BACK1 domain, where other pathogenic missense mutations have been identified. This variant has been reported in trans with a pathogenic variant in NS patients where autosomal recessive inheritance was considered.9, 11 The structural change caused by mutant protein is predicted by Alphafold2 (Figure S1–S3). In silico analysis, multiple lines of computational evidence (SIFT, Mutation Taster, Polyphen-2, DANN, and FATHMM predictor) support the variant c.1735G>A (p.Val579Met) as damaging. Both variants are not found in the reference population of the 1000 Genomes (1000G), and the frequencies in the Exome Aggregation Consortium (ExAC) and the Genome Aggregation Database (gnomAD) are extremely low. According to The American College of Medical Genetics and Genomics (ACMG) criteria, c.1549del (p.E517Rfs*39) is classified as 'likely pathogenic' (PVS1 and PM2) and c.1735G>A (p.Val579Met) is classified as likely pathogenic (PM2, PM3, PP3, and PP4).12 Family gene screen identified that the two mutations were inherited from normal parents. This result suggested that this case is an autosomal recessive Noonan syndrome associated with compound heterozygous LZTR1 variants. NS is a multisystemic genetic disorder characterized by a heterogeneous phenotype. NS typically manifests with a constellation of features including short stature, congenital cardiac anomalies, distinctive craniofacial dysmorphology, and an increased risk for certain benign or malignant neoplasms, notably leukaemia and various solid tumours. Cardiac involvement in Noonan syndrome is prevalent, with congenital heart disease (CHD) being a common finding. Pulmonary valve stenosis and myocardial hypertrophy (usually described as 'HCM' in previous literatre) are among the most frequently observed cardiac defects. HCM, in particular, is documented in approximately 20–30% of Noonan syndrome cases, often manifesting early in life with a mean age of onset around 6 months. In comparative studies, children with Noonan syndrome-related HCM exhibit a greater incidence and severity of LVOT obstruction compared to their age-matched counterparts with idiopathic or familial/sarcomeric HCM.13-15 Moreover, these children face higher rates of hospitalization due to heart failure and a more pressing need for interventional procedures, such as septal myectomy, during their childhood.16 Notably, the presence of biventricular outflow tract obstruction in these patients may serve as a significant clinical indicator or 'red flag' for the diagnosis of NS. This hallmark feature underscores the necessity for a thorough evaluation, as it may guide clinicians towards an early diagnosis of NS.4 To date, 15 genes have been identified in association with NS. Recently, variants in the LZTR1 gene were reported to be pathogenic for NS.5 LZTR1, situated on chromosome 22q11.2, encodes a protein that is a member of the BTB-Kelch superfamily. This protein localizes to the Golgi apparatus and plays a role in the process of ubiquitination. Mutations in LZTR1 contribute to the pathophysiology of NS by disrupting the regulation of RAS ubiquitination, subsequently amplifying RAS-MAPK signalling pathways.17 HCM phenocopy has been reported in approximately 48.3–71.4% of individuals with NS related to LZTR1 mutations. To date, there is a paucity of literature specifically addressing HCM in the context of LZTR1, with only few studies documenting such cases (Table 1).6-9, 18, 19 Among the 16 documented cases, 9 were male. The majority of these patients were diagnosed HCM shortly after birth, and 10 exhibited additional congenital heart defects. These cases demonstrate a heterogeneity in modes of inheritance; 3 cases exhibited autosomal dominant patterns, while 12 cases were autosomal recessive inheritance. NS, traditionally recognized as an autosomal dominant disorder, may also exhibit autosomal recessive inheritance patterns when associated with mutations in the LZTR1 gene. This dual inheritance potential is a distinguishing characteristic of LZTR1, with implications for its clinical management.6, 7, 9, 10 In the case study we report, the patient was a compound herterozygous of LZTR1, inherited biallelic LZTR1 variants from his unaffected parents, each contributing one variant allele. The location of the mutation sites within the LZTR1 gene appears to be a critical determinant of the inheritance pattern: missense variants located between codons 119 and 287 in the protein's Kelch domain are predominantly associated with the dominant form of Noonan syndrome, while variants implicated in recessive cases are dispersed throughout the gene.6 Functional studies have showed recessive NS-causing mutations can affect LZTR1 function through impairing protein synthesis, affecting protein stability or causing aberrant subcelluar localization.10 Previous studies have identified electrocardiographic features that can be of use in differentiating sarcomeric hypertrophic cardiomyopathy from NS-related hypertrophic cardiomyopathy. Typical NS-related electrocardiographic features are a negative aVF, left axis deviation and abnormal R/S ratio in precordial leads. Especially, an extreme QRS axis in the north-west was considered as 'red flag' for NS-related hypertrophic cardiomyopathy.3 In conclusion, we report a case of autosomal recessive NS related HCM phenocopy, which is attributed to compound heterozygous variants in the LZTR1. The presence of biventricular outflow tract obstruction and marked left axis deviation strongly implicates NS as the aetiology of myocardial hypertrophy. Although infrequent, our case, in conjunction with prior reports, contributes to the recognition of an autosomal recessive inheritance pattern for NS, specifically associated with pathogenic variants of LZTR1. This report expands our understanding of the phenotypic spectrum of Noonan syndrome. The authors declare no conflicts of interest. This work was supported by grants from the National Nature Science Foundation of China Youth Project (81600359) and the Nature Science Foundation of Hunan Province Youth Project (2023JJ40855). Figure S1. Predicted structure of wild type and mutated LZTR1 by Alphafold2. (A) Complex molecular dynamics RMSD value plot. (B-D) the predicted secondary structure of wild type LZTR1 and mutated LZTR1. Figure S2. Structural comparison of wild type and mutated LZTR1. (A) Composite structure diagram of LZTR1(WT) and LZTR1(E517Rfs*39) revealed the structural morphology of LZTR1(E517Rfs*39) is completely different from that of the wild-type system. (B) Compared to the wild-type LZTR1 protein, the LZTR1(V579M) mutation maintains the overall structural conformation of the wild-type protein with minor differences. Figure S3. Structural changes Caused by V579M mutation. Amino acid sticks picture near the mutation point of WT (A) and V579M (C) showed that in LZTR1(WT), amino acid V579 formed hydrophobic interactions with surrounding amino acids V574-F601-L576-V581-L580-V611. However, in the mutated protein LZTR1(V579M), due to the larger side chain of M, M579 forms a more compact binding with surrounding amino acids V574-F601-L576-V581-L580-V611. Cavity map of amino acids near the mutation site of WT (B) and V597M (D) revealed the structural conformation of V597M undergoes certain changes: the spatial bubbles (gaps) within the neighbouring protein are filled, and the compactness of the amino acid binding is enhanced. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.