Tyrosyl phosphorylation of PZR promotes hypertrophic cardiomyopathy in PTPN11-associated Noonan syndrome with multiple lentigines.

Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder that presents with cardio-cutaneous-craniofacial defects. Hypertrophic cardiomyopathy (HCM) represents the major life-threatening presentation in NSML. Mutations in the PTPN11 gene that encodes for the protein tyrosine phosphatase (PTP), SHP2, represents the predominant cause of HCM in NSML. NSML-associated PTPN11 mutations render SHP2 catalytically inactive with an "open" conformation. NSML-associated PTPN11 mutations cause hypertyrosyl phosphorylation of the transmembrane glycoprotein, protein zero-related (PZR), resulting in increased SHP2 binding. Here we show that NSML mice harboring a tyrosyl phosphorylation-defective mutant of PZR (NSML/PZRY242F) that is defective for SHP2 binding fail to develop HCM. Enhanced AKT/S6 kinase signaling in heart lysates of NSML mice was reversed in NSML/PZRY242F mice, demonstrating that PZR/SHP2 interactions promote aberrant AKT/S6 kinase activity in NSML. Enhanced PZR tyrosyl phosphorylation in the hearts of NSML mice was found to drive myocardial fibrosis by engaging an Src/NF-κB pathway, resulting in increased activation of IL-6. Increased expression of IL-6 in the hearts of NSML mice was reversed in NSML/PZRY242F mice, and PZRY242F mutant fibroblasts were defective for IL-6 secretion and STAT3-mediated fibrogenesis. These results demonstrate that NSML-associated PTPN11 mutations that induce PZR hypertyrosyl phosphorylation trigger pathophysiological signaling that promotes HCM and cardiac fibrosis.

Genetic landscape of RASopathies in Chinese: Three decades' experience in Hong Kong.

RASopathies are a group of genetic disorders due to dysregulation of the RAS-MAPK signaling pathway, which is important in regulating cell growth, proliferation, and differentiation. These include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), cardiofaciocutaneous (CFC) syndrome, and Costello syndrome (CS), clinical manifestations include growth retardation, developmental delay, cardiac defects, and specific dysmorphic features. There were abundant publications describing the genotype and phenotype from the Western populations. However, detailed study of RASopathies in Chinese population is lacking. We present here the largest cohort of RASopathies ever reported in Chinese populations, detailing the mutation spectrum and clinical phenotypes of these patients. The Clinical Genetic Service, Department of Health, and Queen Mary Hospital are tertiary referral centers for genetic disorders in Hong Kong. We retrospectively reviewed all the genetically confirmed cases of RASopathies, including NS, NSML, CFC syndrome, and CS, over the past 29 years (from 1989 to 2017). Analyses of the mutation spectrum and clinical phenotypes were performed. One hundred and ninety-one ethnic Chinese patients with genetically confirmed RASopathies were identified, including 148 patients with NS, 23 NSML, 12 CFC syndrome, and eight CS. We found a lower incidence of hypertrophic cardiomyopathy in individuals with NSML (27.3%), and NS caused by RAF1 mutations (62.5%). Another significant finding was for those NS patients with myeloproliferative disorder, the mutations fall within Exon 3 of PTPN11 but not only restricted to the well-known hotspots, that is, p.Asp61 and p.Thr731, which suggested that re-evaluation of the current tumor surveillance recommendation maybe warranted.

Generation of an induced pluripotent stem cell line (TRNDi003-A) from a Noonan syndrome with multiple lentigines (NSML) patient carrying a p.Q510P mutation in the PTPN11 gene.

Noonan syndrome with multiple lentigines (NSML), formerly known as LEOPARD Syndrome, is a rare autosomal dominant disorder. Approximately 90% of NSML cases are caused by missense mutations in the PTPN11 gene which encodes the protein tyrosine phosphatase SHP2. A human induced pluripotent stem cell (iPSC) line was generated using peripheral blood mononuclear cells (PBMCs) from a patient with NSML that carries a gene mutation of p.Q510P on the PTPN11 gene using non-integrating Sendai virus technique. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development to treat NSML.

Heterozygous deletion of AKT1 rescues cardiac contractility, but not hypertrophy, in a mouse model of Noonan Syndrome with Multiple Lentigines.

Noonan Syndrome with Multiple Lentigines (NSML) is associated with congenital heart disease in form of pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). Genetically, NSML is primarily caused by mutations in the non-receptor protein tyrosine phosphatase SHP2. Importantly, certain SHP2 mutations such as Q510E can cause a particularly severe form of HCM with heart failure in infancy. Due to lack of insight into the underlying pathomechanisms, an effective custom-tailored therapy to prevent heart failure in these patients has not yet been found. SHP2 regulates numerous signaling cascades governing cell growth, differentiation, and survival. Experimental models have shown that NSML mutations in SHP2 cause dysregulation of downstream signaling, in particular involving the protein kinase AKT. AKT, and especially the isoform AKT1, has been shown to be a major regulator of cardiac hypertrophy. We therefore hypothesized that hyperactivation of AKT1 is required for the development of Q510E-SHP2-induced HCM. We previously generated a transgenic mouse model of NSML-associated HCM induced by Q510E-SHP2 expression in cardiomyocytes starting before birth. Mice display neonatal-onset HCM with initially preserved contractile function followed by functional decline around 2months of age. As a proof-of-principle study, our current goal was to establish to which extent a genetic reduction in AKT1 rescues the Q510E-SHP2-induced cardiac phenotype in vivo. AKT1 deletion mice were crossed with Q510E-SHP2 transgenic mice and the resulting compound mutant offspring analyzed. Homozygous deletion of AKT1 greatly reduced viability in our NSML mouse model, whereas heterozygous deletion of AKT1 in combination with Q510E-SHP2 expression was well tolerated. Despite normalization of pro-hypertrophic signaling downstream of AKT, heterozygous deletion of AKT1 did not ameliorate cardiac hypertrophy induced by Q510E-SHP2. However, the functional decline caused by Q510E-SHP2 expression was effectively prevented by reducing AKT1 protein. This demonstrates that AKT1 plays an important role in the underlying pathomechanism. Furthermore, the functional rescue was associated with an increase in the capillary-to-cardiomyocyte ratio and normalization of capillary density per tissue area in the compound mutant offspring. We therefore speculate that limited oxygen supply to the hypertrophied cardiomyocytes may contribute to the functional decline observed in our mouse model of NSML-associated HCM.

Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome.

Dilated cardiomyopathy (DCM) is a life-threatening disorder whose genetic basis is heterogeneous and mostly unknown. Five Arab Christian infants, aged 4-30 months from four families, were diagnosed with DCM associated with mild skin, teeth, and hair abnormalities. All passed away before age 3. A homozygous sequence variation creating a premature stop codon at PPP1R13L encoding the iASPP protein was identified in three infants and in the mother of the other two. Patients' fibroblasts and PPP1R13L-knocked down human fibroblasts presented higher expression levels of pro-inflammatory cytokine genes in response to lipopolysaccharide, as well as Ppp1r13l-knocked down murine cardiomyocytes and hearts of Ppp1r13l-deficient mice. The hypersensitivity to lipopolysaccharide was NF-κB-dependent, and its inducible binding activity to promoters of pro-inflammatory cytokine genes was elevated in patients' fibroblasts. RNA sequencing of Ppp1r13l-knocked down murine cardiomyocytes and of hearts derived from different stages of DCM development in Ppp1r13l-deficient mice revealed the crucial role of iASPP in dampening cardiac inflammatory response. Our results determined PPP1R13L as the gene underlying a novel autosomal-recessive cardio-cutaneous syndrome in humans and strongly suggest that the fatal DCM during infancy is a consequence of failure to regulate transcriptional pathways necessary for tuning cardiac threshold response to common inflammatory stressors.

Molecular screening strategies for NF1-like syndromes with café-au-lait macules (Review).

Multiple café-au-lait macules (CALM) are usually associated with neurofibromatosis type 1 (NF1), one of the most common hereditary disorders. However, a group of genetic disorders presenting with CALM have mutations that are involved in human skin pigmentation regulation signaling pathways, including KIT ligand/KIT proto­oncogene receptor tyrosine kinase and Ras/mitogen­activated protein kinase. These disorders, which include Legius syndrome, Noonan syndrome with multiple lentigines or LEOPARD syndrome, and familial progressive hyperpigmentation) are difficult to distinguish from NF1 at early stages, using skin appearance alone. Furthermore, certain syndromes are clinically overlapping and molecular testing is a vital diagnostic method. The present review aims to provide an overview of these 'NF1­like' inherited diseases and recommend a cost­effective strategy for making a clear diagnosis among these diseases with an ambiguous borderline.

Pathogenesis of multiple lentigines in LEOPARD syndrome with PTPN11 gene mutation.

LEOPARD syndrome (LS) is an autosomal dominant condition with multiple anomalies, including multiple lentigines. LS is caused by mutations in PTPN11, encoding the protein tyrosine phosphatase, SHP-2. We report here 2 unrelated Japanese cases of LS with different PTPN11 mutations (p.Y279C and p.T468P). To elucidate the pathogenesis of multiple lentigines in LS, ultrastructural and immunohistochemical analyses of lentigines and non-lesional skin were performed. Numerous mature giant melanosomes in melanocytes and keratinocytes were observed in lentigines. In addition, the levels of expression of endothelin-1 (ET-1), phosphorylated Akt, mTOR and STAT3 in the epidermis in lentigines were significantly elevated compared with non-lesional skin. In in vitro assays, melanin synthesis in human melanoma cells expressing SHP-2 with LS-associated mutations was higher than in cells expressing normal SHP-2, suggesting that LS-associated SHP-2 mutations might enhance melanin synthesis in melanocytes, and that the activation of Akt/mTOR signalling may contribute to this process.

Elevated Ca2+ transients and increased myofibrillar power generation cause cardiac hypercontractility in a model of Noonan syndrome with multiple lentigines.

Noonan syndrome with multiple lentigines (NSML) is primarily caused by mutations in the nonreceptor protein tyrosine phosphatase SHP2 and associated with congenital heart disease in the form of pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). Our goal was to elucidate the cellular mechanisms underlying the development of HCM caused by the Q510E mutation in SHP2. NSML patients carrying this mutation suffer from a particularly severe form of HCM. Drawing parallels to other, more common forms of HCM, we hypothesized that altered Ca(2+) homeostasis and/or sarcomeric mechanical properties play key roles in the pathomechanism. We used transgenic mice with cardiomyocyte-specific expression of Q510E-SHP2 starting before birth. Mice develop neonatal onset HCM with increased ejection fraction and fractional shortening at 4-6 wk of age. To assess Ca(2+) handling, isolated cardiomyocytes were loaded with fluo-4. Q510E-SHP2 expression increased Ca(2+) transient amplitudes during excitation-contraction coupling and increased sarcoplasmic reticulum Ca(2+) content concurrent with increased expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase. In skinned cardiomyocyte preparations from Q510E-SHP2 mice, force-velocity relationships and power-load curves were shifted upward. The peak power-generating capacity was increased approximately twofold. Transmission electron microscopy revealed that the relative intracellular area occupied by sarcomeres was increased in Q510E-SHP2 cardiomyocytes. Triton X-100-based myofiber purification showed that Q510E-SHP2 increased the amount of sarcomeric proteins assembled into myofibers. In summary, Q510E-SHP2 expression leads to enhanced contractile performance early in disease progression by augmenting intracellular Ca(2+) cycling and increasing the number of power-generating sarcomeres. This gives important new insights into the cellular pathomechanisms of Q510E-SHP2-associated HCM.

[Cardiofaciocutaneous syndrome, a Noonan syndrome related disorder: clinical and molecular findings in 11 patients]. / Síndrome cardiofaciocutáneo, un trastorno relacionado con el síndrome de Noonan: hallazgos clínicos y moleculares en 11 pacientes.

OBJECTIVES: To describe 11 patients with cardiofaciocutaneous syndrome (CFC) and compare them with 130 patients with other RAS-MAPK syndromes (111 Noonan syndrome patients [NS] and 19 patients with LEOPARD syndrome). PATIENTS AND METHODS: Clinical data from patients submitted for genetic analysis were collected. Bidirectional sequencing analysis of PTPN11, SOS1, RAF1, BRAF, and MAP2K1 focused on exons carrying recurrent mutations, and of all KRAS exons were performed. RESULTS: Six different mutations in BRAF were identified in 9 patients, as well as 2 MAP2K1 mutations. Short stature, developmental delay, language difficulties and ectodermal anomalies were more frequent in CFC patients when compared with other neuro-cardio-faciocutaneous syndromes (P<.05). In at least 2 cases molecular testing helped reconsider the diagnosis. DISCUSSION: CFC patients showed a rather severe phenotype but at least one patient with BRAF mutation showed no developmental delay, which illustrates the variability of the phenotypic spectrum caused by BRAF mutations. Molecular genetic testing is a valuable tool for differential diagnosis of CFC and NS related disorders.

Insights into desmosome biology from inherited human skin disease and cardiocutaneous syndromes.

The importance of desmosomes in tissue homeostasis is highlighted by natural and engineered mutations in desmosomal genes, which compromise the skin or heart and in some instances both. Desmosomal gene mutations account for 45-50% of cases of arrhythmogenic right ventricular cardiomyopathy, and are mutated in an array of other disorders such as striate palmoplantar keratoderma, hypotrichosis with or without skin vesicles and lethal acantholytic epidermolysis bullosa. Recently, we reported loss-of-function mutations in the human ADAM17 gene, encoding for the 'sheddase' ADAM17, a transmembrane protein which cleaves extracellular domains of substrate proteins including TNF-α, growth factors and desmoglein (DSG) 2. Patients present with cardiomyopathy and an inflammatory skin and bowel syndrome with defective DSG processing. In contrast, the dominantly inherited tylosis with oesophageal cancer appears to result from gain-of-function in ADAM17 due to increased processing via iRHOM2. This review discusses the heterogeneity of mutations in desmosomes and their regulatory proteins.

LEOPARD syndrome: maxillofacial care.

This article reports a case of a boy with LEOPARD syndrome with unusual mandibular osteolytic osteoclastic-like lesions and eruption disorder. The patient was referred to our department for bilateral facial swelling: systemic examinations, diagnosis, and dental and maxillofacial care are reported.

Structural insights into Noonan/LEOPARD syndrome-related mutants of protein-tyrosine phosphatase SHP2 (PTPN11).

BACKGROUND: The ubiquitous non-receptor protein tyrosine phosphatase SHP2 (encoded by PTPN11) plays a key role in RAS/ERK signaling downstream of most, if not all growth factors, cytokines and integrins, although its major substrates remain controversial. Mutations in PTPN11 lead to several distinct human diseases. Germ-line PTPN11 mutations cause about 50% of Noonan Syndrome (NS), which is among the most common autosomal dominant disorders. LEOPARD Syndrome (LS) is an acronym for its major syndromic manifestations: multiple Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth, and sensorineural Deafness. Frequently, LS patients have hypertrophic cardiomyopathy, and they might also have an increased risk of neuroblastoma (NS) and acute myeloid leukemia (AML). Consistent with the distinct pathogenesis of NS and LS, different types of PTPN11 mutations cause these disorders. RESULTS: Although multiple studies have reported the biochemical and biological consequences of NS- and LS-associated PTPN11 mutations, their structural consequences have not been analyzed fully. Here we report the crystal structures of WT SHP2 and five NS/LS-associated SHP2 mutants. These findings enable direct structural comparisons of the local conformational changes caused by each mutation. CONCLUSIONS: Our structural analysis agrees with, and provides additional mechanistic insight into, the previously reported catalytic properties of these mutants. The results of our research provide new information regarding the structure-function relationship of this medically important target, and should serve as a solid foundation for structure-based drug discovery programs.

When rare illuminates common: how cardiocutaneous syndromes transformed our perspective on arrhythmogenic cardiomyopathy.

The classic cardiocutaneous syndromes of Naxos and Carvajal are rare. The myocardial disorder integral to their pathology - arrhythmogenic cardiomyopathy - is arguably not uncommon, with a prevalence of up to 1 in 1,000 despite almost certain under-recognition. Yet the study of cardiocutaneous syndromes has been integral to evolution of the contemporary perspective of arrhythmogenic cardiomyopathy - its clinical course, disease spectrum, genetics, and cellular and molecular mechanisms. Here we discuss how recognition of the association of hair and skin abnormalities with underlying heart disease transformed our conception of a little-understood but important cause of sudden cardiac death.

Clinical and molecular analysis of RASopathies in a group of Turkish patients.

The 'RASopathies' are a group of disorders sharing many clinical features and a common pathophysiology. In this study, we aimed to clinically evaluate a group of Turkish patients and elucidate the underlying genetic etiology. Thirty-one patients with a clinical diagnosis of one of the RASopathy syndromes were included in the study. Of these, 26 (83.8%) had a clinical diagnosis of Noonan syndrome, whereas 5 had a clinical diagnosis of either Costello, LEOPARD or cardio-facio-cutaneous syndromes. Twenty of 31 (64.5%) patients were found to be mutation positive. Mutations in PTPN11, SOS1 and SHOC2 genes were detected in patients with Noonan syndrome (57.6%). Mutations in MEK1, PTPN11, BRAF and HRAS genes were detected in the remaining. Pulmonary stenosis was the most common (61.5%) cardiac anomaly. Among Noonan syndrome patients with a confirmed mutation, mild intellectual disability tended to be more common in patients with PTPN11 mutation than in those with SOS1 mutation. Hematologic evaluation revealed coagulation defects in three Noonan syndrome patients with a mutation. This is currently the largest clinical and molecular study in Turkish RASopathy patients. Our findings indicate that molecular epidemiology and genotype-phenotype correlations in RASopathies are relatively independent from the ethnic population background.

High-purity enrichment of functional cardiovascular cells from human iPS cells.

AIMS: A variety of human inherited heart diseases affect the normal functions of cardiomyocytes (CMs), endothelial cells (ECs), or smooth muscle cells (SMCs). To study human heart disease and generate cardiac cells for basic and translational research, an efficient strategy is needed for production of cardiac lineages from human stem cells. In the present study, a highly reproducible method was developed that can simultaneously enrich a large number of CMs and cardiac SMCs and ECs from human induced pluripotent stem (iPS) cells with high purity. METHODS AND RESULTS: Human multipotent cardiovascular progenitor cells were generated from human iPS cells, followed by selective differentiation of the multipotent cardiovascular progenitor cells into CMs, ECs, and SMCs. With further fluorescence-activated cell sorting, each of the three cardiovascular cell types could be enriched with high purity (>90%). These enriched cardiovascular cells exhibited specific gene expression signatures and normal functions when assayed both in vitro and in vivo. Moreover, CMs purified from iPS cells derived from a patient with LEOPARD syndrome, a disease characterized by cardiac hypertrophy, showed the expected up-regulated expression of genes associated with cardiac hypertrophy. CONCLUSIONS: Overall, our technical advance provides the means for generating a renewable resource of pure human cardiovascular cells that can be used to dissect the mechanisms of human inherited heart disease and for the future development of drug and cell therapeutics for heart disease.

Implantable cardioverter defibrillator for progressive hypertrophic cardiomyopathy in a patient with LEOPARD syndrome and a novel PTPN11 mutation Gln510His.

LEOPARD syndrome (LS), generally caused by heterozygous mutations in the PTPN11 gene, is a rare autosomal-dominant multiple congenital anomaly condition, characterized by skin, facial, and cardiac abnormalities. Prognosis appears to be related to the type of structural, myocardial, and arrhythmogenic cardiac disease, especially hypertrophic cardiomyopathy (HCM). We report on a woman with LS and a novel Gln510His mutation in PTPN11, who had progressive HCM with congestive heart failure and nonsustained ventricular tachycardia, successfully treated with implantable cardioverter defibrillator (ICD). Comparing our patient to the literature suggests that specific mutations at codon 510 in PTPN11 (Gln510Glu, Gln510His, but not Gln510Pro) might be a predictor of fatal cardiac events in LS. Molecular risk stratification and careful evaluations for an indication of ICD implantation are likely to be beneficial in managing patients with LS and HCM.